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Castro, D.

Publications and source records attributed to Castro, D..

12 recordsLinked to original sources

Comparative genomics of abiotic stress response in Norway spruce and Scots pine

Norway spruce (Picea abies) and Scots pine (Pinus sylvestris) are dominant boreal forest species with globally important contributions to carbon capture and storage and sustain an extensive forestry industry. Both species have adapted to cold and episodic drought, yet each occupies a distinct ecological niche. How conserved their stress responses are, which features are lineage-specific, and how far mechanisms known from herbaceous angiosperms apply, remain open questions of importance in the face of ongoing climate change. We profiled roots and needles of both species under drought and cold, combining differential expression with an orthology-aware comparative co-expression framework that places each gene on a conservation-divergence gradient. Differential expression was largely organ- and stress-specific, yet cross-species overlap at the orthogroup level was extensive and increased with stress intensity. Comparative co-expression recovered a further conserved regulatory backbone that per-timepoint differential expression did not resolve, enriched for abscisic-acid-centred signalling, oxidative and osmotic-stress responses and growth suppression, and containing canonical stress response transcription-factor families including NAC, WRKY and bZIP/ABF. The breadth of co-expression conservation for a gene was coupled to purifying selection on its coding sequence and to network connectivity. Additionally, segmental duplicates shared between the species were enriched among conserved drought circuits, whereas lineage-specific duplicates were enriched among genes lacking conserved co-expression. Regulatory conservation and genome architecture thus describe a single conservation-divergence axis, providing an evolution-anchored criterion that helps separate candidate core regulators (which are conserved, network-central and constrained) from reactive change that differential expression alone cannot resolve. Significance statementCold and drought are recurring threats to boreal forests, yet how conifers coordinate their responses, and how much of that response is evolutionarily conserved, has been difficult to establish. Comparing Norway spruce and Scots pine, two species separated by a deep evolutionary divergence, we show that the genes whose co-expression is most broadly conserved between the species are also those under the strongest purifying selection on their protein-coding sequences. As these two species diverged so long ago, this conserved regulatory core stands out as an evolutionary signal that simple comparisons of differentially expressed genes fail to capture. The approach provides an evolution-anchored way to distinguish genes central to the stress response from lineage-specific or reactive change.

plant biology↗

Learning shapes neural codes for sensory-motor integration in the tail of the striatum

Separating meaningful sensory stimuli from irrelevant ones requires learning sensorimotor associations, but how sensory-linked striatal circuits acquire and maintain these associations is unclear. We longitudinally imaged direct- and indirect-pathway (D1 and A2a) spiny projection neurons (SPNs) in the tail of the striatum (TS) as mice learned to push or pull a joystick in response to auditory cues in either a stimulus-response association (go/omit) task or a two-alternative forced choice (2AFC) task. Learning in both tasks increased the fraction and strength of task-modulated TS SPNs across the sound, action, and reward epochs, yet individual neuron selectivity often switched over days between behavioral epochs. In spite of individual neuron variability, population activity of direct and indirect pathways became aligned with characteristic behavioral features during learning: D1-SPNs dominated the action category, A2a-SPNs were biased toward the mixed category (multiple epochs), and both SPN types showed sound category specificity that depended on the sound-action association. Trial-wise modeling revealed a reweighting of behavioral predictors within the action window, with reward gaining and movement losing predictive weight. Learning the two-choice task led to a higher prevalence of association-preferring neurons and better behavioral decoding within the sound window than in the action/reward window, reflecting a task-dependent prioritization of sensory information. Association-preferring neurons also showed a stable local distance-similarity relationship, with nearby neurons more similar than distant neurons across learning. Together, our results support a population mechanism in TS during learning in which neurons from both direct and indirect pathways are recruited and take on distinct behavioral roles that vary with performance and task complexity.

neuroscience↗

Tree species shape ectomycorrhizal fungal traits and community composition through litter quality and resultant soil properties

Ectomycorrhizal (ECM) fungi are key components of the nitrogen (N) and carbon (C) cycles in temperate and boreal forests soils. However, it is still unclear how differences in soil properties driven by litter quality shape ECM fungal diversity and composition. To test this, we used a unique common garden experiment in Denmark which included three common European tree species, lime, beech, and Norway spruce, each associated with contrasting litter quality from fast-to slow-turnover respectively. We found that soil physicochemical properties varied significantly among tree species, with spruce soils having higher soil organic matter (SOM), phosphorus (P), inorganic N and lower pH, than beech and lime. A total of 1,248 ECM root tips were selected for Sanger sequencing and individually assessed for the presence of hyphae and/or rhizomorphs protruding from the fungal mantle. Sequencing revealed 133 fungal amplicon sequence variants (ASVs), clustered in 86 fungal operational taxonomic units (OTUs), from which 61 corresponded to ECM fungi. Compared to spruce, lime and beech supported a higher ECM fungal richness and diversity in lime and beech compared to spruce. The ECM fungal community composition differed significantly between broadleaved species and spruce, with Russula dominating in lime and beech. Regarding fungal traits, in addition to the direct observations of hyphae and rhizomorphs, species were also scored based on literature for exploration types and hyphae hydrophobicity. Tree species had a specific association with ECM fungal exploration types, with broadleaved species favoring contact and short-distance types and spruce associating with long-distance types. The results suggest that tree species via foliar litter quality have a direct impact on soil properties and indirect to the ECM fungal community, where broadleaved tree species with nutrient-rich and rapid decomposing litter support a more diverse ECM community with limited soil exploration capabilities, while spruce supports an ECM fungal community more adapted to soil exploration.

ecology↗

The rewiring of a terminal selector regulatory cascade generates convergent neuronal laterality

Neuronal identity is established and maintained by "terminal-selector" transcription factors, yet how these networks evolve remains unclear. We examined the specification of the chemosensory ASE and thermosensory AFD neurons in the nematode Pristionchus pacificus, a species that expresses the terminal-selector, Ppa-CHE-1, in both sensory neurons. To determine if the ASE neurons exhibits left-right laterality, we used HCR-FISH and transgenic reporters to discover 8 ASE left-right-specific and 3 AFD-specific receptor-type guanylyl-cyclases. Late embryos exhibit a multipotential state in which AFD precursors transiently co-express ASEL, ASER and AFD markers. A forward genetic screen for defects in ASER asymmetry identified a Ppa-DIE-1 homolog, whereas the maintenance of AFD neuronal identity requires another terminal-selector, Ppa-TTX-1, and CNG channels, Ppa-TAX-2/TAX-4. Defects in miRNA-processing convert ASEL to ASER fate while mutations in conserved regions in the 3' UTR of the cog-1 homolog reveal multiple miRNA binding sites that toggle between left/right ASE versus AFD identities. Together, these results demonstrate that P. pacificus deploys a convergent, likely miRNA-mediated regulatory repertoire to generate left/right neuronal asymmetry and highlight changes in the Ppa-cog-1 3 UTR as a key locus for rewiring selector networks.

developmental biology↗

13C tracing in synaptosomes reveals that SGLT2 inhibition with dapagliflozin prevents metabolic deficits in the 5X-FAD model of Alzheimer's Disease

Metabolic dysfunction is linked to several forms of age-related neurodegeneration including Alzheimers Disease (AD), and targeting brain energy metabolism is an increasingly attractive mode of therapeutic intervention. However, commonly used in vitro methods to identify specific metabolic pathways of interest in preclinical models of neurodegenerative disease have considerable limitations. They are prone to subselection of sample material, unable to identify cell type-specific effects, or cannot identify metabolic defects upstream of mitochondria. Here we address these challenges by validating a method for stable isotope tracing with isolated synaptic nerve terminals, or synaptosomes. We further applied this approach to study glucose metabolism in synaptosomes isolated from the 5X-FAD mouse model of AD treated with the antidiabetic sodium-glucose linked transporter-2 (SGLT-2) inhibitor Dapagliflozin. Treatment with Dapagliflozin preserved steady-state levels of synaptosomal metabolites and enrichment from labeled glucose into citrate that was reduced in the 5X-FAD model. These changes correlated with trends towards improved spatial working memory but not amyloid burden. The results highlight the utility of stable isotope tracing in synaptosomes to identify precise sites of metabolic dysfunction and mechanisms of action for metabolic drug candidates in preclinical models of neurodegeneration.

biochemistry↗

Enhancing lipid production in plant cells through high-throughput genome editing and phenotyping via a scalable automated pipeline

Plant bioengineering is a time-consuming and labor-intensive process, with no guarantee of achieving the desired trait. Here we report a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB). FAST-PB achieves gene cloning, genome editing, and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrate that FAST-PB can streamline the Golden Gate cloning process, with the capacity to construct 96 vectors in parallel. To prove the concept, using FAST-PB, we first found that PEG2050 significantly increases transfection efficiency by over 45%. To validate the pipeline, we established a reporter-gene-free method for CRISPR editing via mutation of HCF136, affecting cellular chlorophyll fluorescence. Next, we applied this pipeline for lipid production and found that diverse lipids were significantly enhanced up to sixfold through introducing multi-gene cassettes via CRISPR activation, and regenerated plant using this platform. Lastly, we harnessed FAST-PB to achieve high-throughput single-cell lipid profiling through the integration of MALDI-MS with the biofoundry, and differentiated engineered and unengineered cells using the single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering, and change what is possibly using single-cell metabolomics in plants.

synthetic biology↗

Bringing astrocytes into the spotlight of electrical brain stimulation

Astrocytes, primarily viewed only as supportive units, are now emerging as active players in the information processing of the brain. Accumulated evidence supports that the bidirectional communication between astrocytes and neurons maintains complex animal behaviours such as memory formation and decision-making. The lack of characterisation of astrocytic electrophysiology is, in our opinion, associated with the early idea of a passive electrical nature of astrocytes, in opposition to the electrically active neurons. A better understanding of the effect of electrical stimulation on astrocytes physiology and activity will greatly strengthen the current knowledge in neural biology. Here, we assessed if astrocytes may have a role in therapies based on electrical brain stimulation by being able to respond to the same electrical stimulus used to modulate neuronal activity. To do so, we took advantage of microelectrode arrays (MEAs) capability to simultaneously record and deliver extracellular electrical signals. Additionally, we synchronized the recording of electrophysiological data with the recording of calcium activity, a hallmark of astrocytic activity. Here, we show that astrocytes respond to electrical stimulation with the generation of strong membrane voltage oscillations and simultaneous production of calcium waves, demonstrating, unequivocally, that astrocytes respond to electrical stimulation in the same range as neurons do. Importantly, these responses are dependent on the stimuli amplitude. Furthermore, membrane voltage oscillations are significantly reduced in the absence of extracellular calcium, but not abolished, while calcium activity is not detected.

neuroscience↗

A novel closed-loop control algorithm to disrupt pathological neuronal oscillations - implementation and validation in vitro

Adaptive neuronal stimulation has a strong therapeutic potential for neurological disorders such as Parkinsons disease and epilepsy. However, standard stimulation protocols mostly rely on continuous open-loop stimulation. We implement here, for the first time in neuronal populations, two different Delayed Feedback Control (DFC) algorithms and assess their efficacy in disrupting unwanted neuronal oscillations. DFC is a well-established closed-loop control technique but its use in neuromodulation has been limited so far to models and computational studies. Leveraging on the high spatiotemporal monitoring capabilities of specialized in vitro platforms, we show that standard DFC in fact worsens the neuronal population oscillatory behaviour and promotes faster bursting, which was never reported in silico. Alternatively, we present adaptive DFC (aDFC) that monitors ongoing oscillation periodicity and self-tunes accordingly. aDFC disrupts collective neuronal oscillations and decreases network synchrony. Furthermore, we show that the intrinsic population dynamics have a strong impact in the susceptibility of networks to neuromodulation. Experimental data was complemented with computer simulations to show how this network controllability might be determined by specific network properties. Overall, these results support aDFC as a better candidate for therapeutic neurostimulation and provide new insights regarding the controllability of neuronal systems.

neuroscience↗

A memristor-based neuromodulation device for real-time monitoring and adaptive control of in vitro neuronal populations

Neurons are specialized cells in information transmission and information processing. Following this, many neurologic disorders are directly linked not to cellular viability/homeostasis issues but rather to specific anomalies in electrical activity dynamics. Acknowledging this fact, therapeutic strategies based on direct modulation of neuronal electrical activity have been producing remarkable results, with successful examples ranging from cochlear implants to deep brain stimulation. Development on these implantable devices are hindered, however, by important challenges: power requirements, size factor, signal transduction, and adaptability/computational capabilities. Memristors, nanoscale electronic components able to emulate natural synapses, provide unique properties to address these constraints and their use in neuroprosthetic devices is being actively explored. Here we demonstrate for the first time the use of memristive devices in a clinically relevant setting where communication between two neuronal populations is conditioned to specific activity patterns in the source population. In our approach, the memristor device performs a simple pattern detection computation and acts as a synapstor capable of reversible short-term plasticity. Using in vitro hippocampal neuronal cultures, we show real-time adaptive control with a high degree of reproducibility using our monitor-compute-actuate paradigm. We envision very similar systems being used for automatic detection and suppression of seizures in epileptic patients.

neuroscience↗

Interactions of SARS-CoV-2 envelope protein with amilorides correlate with antiviral activity

SARS-CoV-2 is the novel coronavirus that is the causative agent of COVID-19, a sometimes-lethal respiratory infection responsible for a world-wide pandemic. The envelope (E) protein, one of four structural proteins encoded in the viral genome, is a 75-residue integral membrane protein whose transmembrane domain exhibits ion channel activity and whose cytoplasmic domain participates in protein-protein interactions. These activities contribute to several aspects of the viral replication-cycle, including virion assembly, budding, release, and pathogenesis. Here, we describe the structure and dynamics of full-length SARS-CoV-2 E protein in hexadecylphosphocholine micelles by NMR spectroscopy. We also characterized its interactions with four putative ion channel inhibitors. The chemical shift index and dipolar wave plots establish that E protein consists of a long transmembrane helix (residues 8-43) and a short cytoplasmic helix (residues 53-60) connected by a complex linker that exhibits some internal mobility. The conformations of the N-terminal transmembrane domain and the C-terminal cytoplasmic domain are unaffected by truncation from the intact protein. The chemical shift perturbations of E protein spectra induced by the addition of the inhibitors demonstrate that the N-terminal region (residues 6-18) is the principal binding site. The binding affinity of the inhibitors to E protein in micelles correlates with their antiviral potency in Vero E6 cells: HMA {approx} EIPA > DMA >> Amiloride, suggesting that bulky hydrophobic groups in the 5 position of the amiloride pyrazine ring play essential roles in binding to E protein and in antiviral activity. An N15A mutation increased the production of virus-like particles, induced significant chemical shift changes from residues in the inhibitor binding site, and abolished HMA binding, suggesting that Asn15 plays a key role in maintaining the protein conformation near the binding site. These studies provide the foundation for complete structure determination of E protein and for structure-based drug discovery targeting this protein. Author SummaryThe novel coronavirus SARS-CoV-2, the causative agent of the world-wide pandemic of COVID-19, has become one of the greatest threats to human health. While rapid progress has been made in the development of vaccines, drug discovery has lagged, partly due to the lack of atomic-resolution structures of the free and drug-bound forms of the viral proteins. The SARS-CoV-2 envelope (E) protein, with its multiple activities that contribute to viral replication, is widely regarded as a potential target for COVID-19 treatment. As structural information is essential for drug discovery, we established an efficient sample preparation system for biochemical and structural studies of intact full-length SARS-CoV-2 E protein and characterized its structure and dynamics. We also characterized the interactions of amilorides with specific E protein residues and correlated this with their antiviral activity during viral replication. The binding affinity of the amilorides to E protein correlated with their antiviral potency, suggesting that E protein is indeed the likely target of their antiviral activity. We found that residue asparagine15 plays an important role in maintaining the conformation of the amiloride binding site, providing molecular guidance for the design of inhibitors targeting E protein.

biochemistry↗

Trackosome: a computational toolbox to study the spatiotemporal dynamics of centrosomes, nuclear envelope and cellular membrane

During the initial stages of mitosis, multiple mechanisms drive centrosome separation and positioning. How they are functionally coordinated to promote centrosome migration to opposite sides of the nucleus remains unclear. Imaging analysis software has been used to quantitatively study centrosome dynamics at this stage. However, available tracking tools are generic and not fine-tuned for the constrains and motion dynamics of centrosome pairs. Such generality limits the tracking performance and may require exhaustive optimization of parameters. Here, we present Trackosome, a freely available open-source computational tool to track the centrosomes and reconstruct the nuclear and cellular membranes, based on volumetric live-imaging data. The toolbox runs in MATLAB and provides a graphical user interface for easy and efficient access to the tracking and analysis algorithms. It outputs key metrics describing the spatiotemporal relations between centrosomes, nucleus and cellular membrane. Trackosome can also be used to measure the dynamic fluctuations of the nuclear envelope. A fine description of these fluctuations is important because they are correlated with the mechanical forces exerted on the nucleus by its adjacent cytoskeletal structures. Unlike previous algorithms based on circular/elliptical approximations of the nucleus, Trackosome measures membrane movement in a model-free condition, making it viable for irregularly shaped nuclei. Using Trackosome, we demonstrate significant correlations between the movements of the two centrosomes, and identify specific modes of oscillation of the nuclear envelope. Overall, Trackosome is a powerful tool to help unravel new elements in the spatiotemporal dynamics of subcellular structures.

cell biology↗