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Garcia, D.

Publications and source records attributed to Garcia, D..

25 records · Page 2Linked to original sources
bioRxiv2020

Genes with 5' terminal oligopyrimidine tracts preferentially escape global suppression of translation by the SARS-CoV-2 NSP1 protein

Viruses rely on the host translation machinery to synthesize their own proteins. Consequently, they have evolved varied mechanisms to co-opt host translation for their survival. SARS-CoV-2 relies on a non-structural protein, Nsp1, for shutting down host translation. However, it is currently unknown how viral proteins and host factors critical for viral replication can escape a global shutdown of host translation. Here, using a novel FACS-based assay called MeTAFlow, we report a dose-dependent reduction in both nascent protein synthesis and mRNA abundance in cells expressing Nsp1. We perform RNA-Seq and matched ribosome profiling experiments to identify gene-specific changes both at the mRNA expression and translation level. We discover a functionally-coherent subset of human genes are preferentially translated in the context of Nsp1 expression. These genes include the translation machinery components, RNA binding proteins, and others important for viral pathogenicity. Importantly, we uncovered a remarkable enrichment of 5' terminal oligo-pyrimidine (TOP) tracts among preferentially translated genes. Using reporter assays, we validated that 5 UTRs from TOP transcripts can drive preferential expression in the presence of NSP1. Finally, we found that LARP1, a key effector protein in the mTOR pathway may contribute to preferential translation of TOP transcripts in response to Nsp1 expression. Collectively, our study suggests fine tuning of host gene expression and translation by Nsp1 despite its global repressive effect on host protein synthesis.

molecular biology↗
bioRxiv2020

Parallel Movement-suppressing Striatal Output Pathways

The classic basal ganglia circuit model asserts a complete segregation of the two striatal output pathways. Empirical data argue that, in addition to indirect-pathway striatal projection neurons (iSPNs), direct-pathway striatal projection neurons (dSPNs) innervate the external globus pallidus (GPe). However, the functions of the latter were not known. In this study, we interrogated the organization principles of striatopallidal projections and their roles in full-body movement in mice (both males and females). In contrast to the canonical motor-promoting response of dSPNs in the dorsomedial striatum (DMSdSPNs), optogenetic stimulation of dSPNs in the dorsolateral striatum (DLSdSPNs) suppressed locomotion. Circuit analyses revealed that dSPNs selectively target Npas1+ neurons in the GPe. In a chronic 6-hydroxydopamine lesion model of Parkinsons disease, the dSPN-Npas1+ projection was dramatically strengthened. As DLSdSPN-Npas1+ projection suppresses movement, the enhancement of this projection represents a circuit mechanism for the hypokinetic symptoms of Parkinsons disease that has not been previously considered. In sum, our results suggest that dSPN input to the GPe is a critical circuit component that is involved in the regulation of movement in both healthy and parkinsonian states. Significance statementIn the classic basal ganglia model, the striatum is described as a divergent structure--it controls motor and adaptive functions through two segregated, opposing output streams. However, the experimental results that show the projection from direct-pathway neurons to the external pallidum have been largely ignored. Here, we showed that this striatopallidal sub-pathway targets a select subset of neurons in the external pallidum and is motor-suppressing. We found that this sub-pathway undergoes changes in a Parkinsons disease model. In particular, our results suggest that the increase in strength of this sub-pathway contributes to the slowness or reduced movements observed in Parkinsons disease.

neuroscience↗
bioRxiv2020

Capsid Proteins are Necessary for Replication of a Densovirus

AO_SCPLOWBSTRACTC_SCPLOWDespite tight genetic compression, viral genomes are often organized in functional gene clusters, a modular structure that might favor their evolvability. This has greatly facilitated biotechnological developments, such as the recombinant Adeno-Associated Virus (AAV) systems for gene therapy. Following this lead, we endeavored to engineer the related insect parvovirus Junonia coenia densovirus (JcDV) to create addressable vectors for insect pest biocontrol. To enable safer manipulation of capsid mutants, we translocated the non-structural (ns) gene cluster outside the viral genome. To our dismay, this yielded a virtually non-replicable clone. We linked the replication defect to an unexpected modularity breach, as ns translocation truncated the overlapping 3 UTR of the capsid transcript (vp). We found that native vp 3UTR is necessary to high VP production, but that decreased expression do not adversely impact the expression of NS proteins, which are known replication effectors. As nonsense vp mutations recapitulate the replication defect, VP proteins appear directly implicated in the replication process. Our findings suggest intricate replication-encapsidation couplings that favor maintenance of genetic integrity. We discuss possible connections with an intriguing cis-packaging phenomenon previously observed in parvoviruses, whereby capsids preferentially package the genome from which they were expressed. IO_SCPLOWMPORTANCEC_SCPLOWDensoviruses could be used as biological control agents to manage insect pests. Such applications require in depth biological understanding and associated molecular tools. However, the genomes of these viruses remain hard to manipulate due too poorly tractable secondary structures at their extremities. We devised a construction strategy that enable precise and efficient molecular modifications. Using this approach, we endeavored to create a split clone of the Junonia coenia densovirus (JcDV) that can be used to safely study the impact of capsid mutations on host specificity. Our original construct proved to be non-functional. Fixing this defect led us to uncover that capsid proteins and their correct expression are essential for continued rolling-hairpin replication. This points to an intriguing link between replication and packaging, which might be shared with related viruses. This serendipitous discovery illustrates the power of synthetic biology approaches to advance our knowledge of biological systems.

microbiology↗
bioRxiv2020

Landscape drivers and effectiveness of pest control by insectivorous birds in a landscape-dominant woody crop

Avian-mediated pest control is a significant ecosystem service with important economic implications. However, there is an overall paucity of experimental information about how landscape simplification affect its current level. Information on pest control by birds is missing in some permanent agroecosystems of worldwide importance, like olive orchards, that dominate vast areas in the Mediterranean region. We assess the effectiveness of insectivorous birds for controlling the two main pest insects in olive orchards and explore the effects of landscape complexity and distance to semi-natural patches on avian insectivore abundance and pest control. For this, we combine bird surveys with field experiments (branch exclusions and pest plasticine models) at the regional scale. Landscape heterogeneity increased the abundance and richness of insectivorous birds, which were also more abundant and diverse in semi-natural patches, compared to the farm olive matrix. Experiments evidenced that pest control by birds (measured as attack rates to plasticine models and pest damage) in the studied olive orchards is negligible, while pests were overall abundant and pest damage was high on most farms. This raises alarms about the status of avian pest control in this agroecosystem. Although landscape heterogeneity increased the abundance/richness of insectivorous birds, and favored some forest species, insectivorous bird abundance seems diluted in relation to prey availability in all landscapes. Thus, pest control by birds seems currently unsuccessful in olive orchards. Our results might be evidencing the loss of an ecosystem service due to a generalized massive decline of common and forest insectivorous birds. Key messageO_LIOlive orchards dominate extensive areas causing important landscape simplification. C_LIO_LIInsectivorous birds are more abundant in semi-natural patches within olive farms. C_LIO_LIField experiments show a low impact of birds on olive pests and damage. C_LIO_LIAvian-mediated pest biocontrol seems diluted by limited suitable habitat for birds. C_LIO_LIAgri-environmental measures should focus on increasing landscape complexity. C_LI

ecology↗
bioRxiv2020

DNA damage and macrophage infiltration in the ovaries of the long-lived GH deficient Ames Dwarf and the short-lived bGH transgenic mice

ObjectiveThe aim of the study was to evaluate the role of growth hormone (GH) in DNA damage, macrophage infiltration and the granulosa cells number of primordial and primary follicles. MethodsFor these experiments six groups of female mice were used. Four groups consisted of Ames dwarf (Prop-1df, df/df, n=12) and their normal littermates (N/df, n=12) mice, between sixteen and eighteen month-old, receiving GH (n=6 for df/df, and n=6 for N/df mice) or saline injections (n=6 for df/df, and n=6 for N/df mice). The other two groups consisted of ten to twelve-month-old bGH (n=6) and normal mice (N, n=6). Immunofluorescence for DNA damage (anti-{gamma}H2AX) and macrophage counting (anti-CD68) were performed. Granulosa cells of primordial and primary follicles were counted. ResultsFemale df/df mice had lower {gamma}H2AX foci intensity in in both oocytes and granulosa cells of primordial and primary follicles (p<0.05), indicating less DNA double strand breaks (DSBs). In addition, GH treatment increased DSBs in both df/df and N/df mice. Inversely, bGH mice had higher quantity of DSBs in both oocytes and granulosa cells of primordial and primary follicles (p<0.05). Df/df mice showed ovarian tissue with less macrophage infiltration than N/df mice (p<0.05) and GH treatment increased macrophage infiltration (p<0.05). On the other hand, bGH mice had ovarian tissue with more macrophage infiltration compared to normal mice (p<0.05). Df/df mice had less granulosa cells on primordial and primary follicles than N/df mice (p<0.05). GH treatment did not affect the granulosa cells number (p>0.05). However, bGH mice had an increased number of granulosa cells on primordial and primary follicles compared to normal mice (p<0.05). ConclusionThe current study points to the role of the GH/IGF-I axis in maintenance of oocyte DNA integrity and macrophage ovarian infiltration in mice.

physiology↗
bioRxiv2019

Npas1+-Nkx2.1+ Neurons Form A Unique Pallidal Neuron Subclass

Within the basal ganglia circuit, the external globus pallidus (GPe) is critically involved in motor control. Aside from Foxp2+ neurons and ChAT+ neurons that have been established as unique neuron types, there is little consensus on the classification of GPe neurons. Properties of the remaining neuron types are poorly-defined. In this study, we leverage new mouse lines, viral tools, and molecular markers to better define GPe neuron subtypes. We found that Sox6 represents a novel, defining marker for GPe neuron subtypes. Lhx6+ neurons that lack the expression of Sox6 were devoid of both parvalbumin and Npas1. This result confirms previous assertions of the existence of a unique Lhx6+ population. Neurons that arise from the Dbx1+ lineage were similarly abundant in the GPe and displayed a heterogeneous makeup. Importantly, tracing experiments revealed that Npas1+-Nkx2.1+ neurons represent the principal non-cholinergic, cortically-projecting neurons. In other words, they form the pallido-cortical arm of the cortico-pallido-cortical loop. Our data further described that pyramidal-tract neurons in the cortex collateralized within the GPe, forming a closed-loop system between the two brain structures. Overall, our findings reconcile some of the discrepancies that arose from differences in techniques or the reliance on pre-existing tools. While spatial distribution and electrophysiological properties of GPe neurons reaffirm the diversification of GPe subtypes, statistical analyses strongly support the notion that these neuron subtypes can be categorized under the two principal neuron classes--i.e., PV+ neurons and Npas1+ neurons.\n\nSignificance statementThe poor understanding of the neuronal composition in the GPe undermines our ability to interrogate its precise behavioral and disease involvements. In this study, twelve different genetic crosses were used, hundreds of neurons were electrophysiologically-characterized, and over 100,000 neurons were histologically- and/or anatomically-profiled. Our current study further establishes the segregation of GPe neuron classes and illustrates the complexity of GPe neurons in adult mice. Our results support the idea that Npas1+-Nkx2.1+ neurons are a distinct GPe neuron subclass. By providing a detailed analysis of the organization of the cortico-pallidal-cortical projection, our findings establish the cellular and circuit substrates that can be important for motor function and dysfunction.

neuroscience↗