Biology subjects

Cruz, R.

Publications and source records attributed to Cruz, R..

8 recordsLinked to original sources

bioRxiv2026

Dengue virus NS1 undergoes partial nuclear translocation to modulate host transcription and support viral replication

The dengue virus (DENV) non-structural protein 1 (NS1) is a glycoprotein highly conserved among mosquito-borne orthoflaviviruses. NS1 is typically localized in the lumen of the endoplasmic reticulum, where it forms part of the replication complexes, and is also exposed at the plasma membrane. In addition, NS1 is secreted as a lipoprotein. Here, using a combination of approaches, including confocal microscopy with deconvolution, in situ analysis, and biochemical cell fractionation, we show that a substantial fraction of NS1 (up to 30%) translocates to the nucleus during infection. We identified a conserved, structurally exposed bipartite nuclear localization signal (NLS) within NS1. Pharmacological inhibition with ivermectin and site-directed mutagenesis of the NLS in recombinant confirmed that nuclear import of NS1 is an active process, dependent on the classical importin /{beta} pathway. Notably, both dimeric and multimeric forms of NS1 were detected in the nucleus in association with nuclear lamin. Introduction of the NLS mutations into DENV2 infectious clones resulted in a non-viable virus. Production of virus progeny and completion of the replicative cycle by the mutant genomes could be rescued by trans-complementation with wild-type NS1, but not with an NLS-mutated NS1, indicating that an NS1 nuclear phase is required for a productive infection. Transcriptomic analysis by RNA-seq further revealed that NS1 functions depend on its subcellular location. Nuclear NS1 induced the overexpression of genes associated with DNA-binding transcription factors, whereas NLS-mutated NS1, retained in the cytoplasm, failed to induce these genes and instead triggered pro-inflammatory and metabolic responses. Together, these findings reveal a previously unrecognized nuclear phase of NS1 that is required for an efficient viral life cycle, redefining NS1 as a modulator of the host transcriptional environment. These findings also suggest new avenues for antiviral and vaccine development. Authors summaryDengue virus NS1 is a glycoprotein of approximately 45-50 kDa that rapidly dimerizes after proteolytic maturation. Dimeric NS1 is located in the lumen of the endoplasmic reticulum where it acts as a scaffold component of the viral replication complexes. In addition, NS1 is secreted from infected cells as a tetramer or hexamer and circulates in the serum of infected individuals during the acute phase of dengue disease. Circulating NS1 is widely used as a diagnostic marker and has also been associated with dengue pathogenesis through several mechanisms. Here, we expand the current understanding of DENV NS1 by identifying a previously unrecognized and essential nuclear location of this protein. We show that NS1 contains a conserved nuclear localization signal that mediates import into the nucleus via the classical import pathway. Using wild-type and NLS-mutated infectious clones, we demonstrated that nuclear localization of NS1 is required for completion of the DENV replicative cycle. Transcriptomic analysis further revealed that nuclear NS1 promotes the expression of host genes involved in nucleic acid metabolism, whereas retention of NS1 in the cytoplasm triggers an antiviral and inflammatory response. Together, these findings identify the nucleus as an important site of dengue virus-host interactions and redefine NS1 as a regulator of the host transcriptional environment during infection.

microbiology↗

bioRxiv2025

The dengue virus NS1 protein alters Aedes aegypti midgut permeability and favors virus dissemination

The dengue virus is an arbovirus of public health importance transmitted by Aedes aegypti mosquitoes. The NS1 protein is a multifunctional glycoprotein, highly conserved among Orthoflaviviruses, that is secreted from infected cells, circulate in the sera of dengue patients at high concentrations and has been involved with pathogenesis by several mechanisms, including vascular leakage and immune evasion. However, the role of NS1 within the mosquito vector is not fully understood. In this study, we observed that feeding female Aedes aegypti mosquitoes with blood meals supplemented with recombinant NS1 resulted in increased midgut permeability, as evaluated by the diffusion of the non-toxic dye Brilliant Blue FCF into the hemocoel. Histological and transmission electron microscopy analysis revealed epithelial damage and disruption of the midgut cellular junctions. Immunofluorescence assays further demonstrated the delocalization of septate junction proteins essential for epithelial integrity. In addition, metalloproteinase expression in the midgut was also reduced. All these effects were abolished when NS1 was heat-inactivated indicating that they were NS1-dependent. Significantly, virus blood meals containing NS1 resulted in enhanced dissemination of DENV to secondary organs and earlier virus presence in the mosquito salivary glands, in comparison with meals treated with specific NS1 antibodies. Our findings reveal a novel function for NS1 in mosquitoes, and expand the understanding of NS1 functions beyond its human pathophysiological role. In addition, highlight NS1 as a strategic intervention point to reduce transmission efficiency in mosquitoes. ImportanceDengue is the most important virus disease transmitted by mosquitoes to humans. Thus, a deeper understanding of the virus-vector interaction is required for the development of control measures and the mosquito transmission capacity. In this work, we present evidence indicating that the NS1 protein plays a role in the establishment and dissemination of the dengue virus infection in Aedes aegypti mosquitoes. Ingested NS1 was found to disrupt the septate junctions and to inhibit metalloproteinase expression of the midgut, allowing the virus to escape the digestive tract. In addition, NS1 was found to promote virus dissemination into the hemocoel, carcass and salivary glands. These findings uncover new functions for NS1 in the mosquito and highpoint ways to interfere with vector competence.

microbiology↗

bioRxiv2024

A sustainable and bee-pollinated coffee to start your day

The integration of managed pollinators into agricultural systems represents a practical strategy for boosting crop production, optimising sustainable farming practices, and improving rural livelihoods. However, the use of managed bees to supplement the contribution of wild pollinators still faces challenges, such as over-reliance on pesticides in intensively managed agroecosystems. Here, we placed managed bee colonies on conventional farms to assess their impact on arabica coffee yield, quality and market value, while also to evaluate colony health in coffee fields treated with field rates of thiamethoxam-based products. We show that supplementing farms with managed bees increased farmer income by enhancing coffee yield and beverage quality, which is rewarded by specialty coffee markets. Moreover, our field-based study reveals that, even when exposed to field-realistic levels of the neonicotinoid thiamethoxam, managed bee colonies exhibited no significant adverse effects on their health within coffee fields. Our results underline that pollination is a key input on intensively managed coffee farms, and that agricultural production and environmental conservation interact synergically to maximize profitability, which is fundamental to encourage farmers to be good stewards of their croplands by adopting nature-positive practices.

ecology↗

bioRxiv2024

Non-invasive MRI of Blood-Cerebrospinal Fluid-Barrier Function: a Functional Biomarker of Early Alzheimer's Disease Pathology

INTRODUCTIONChoroid plexus (CP) dysfunction is thought to contribute to toxic protein build-up in neurodegenerative disorders, including Alzheimers disease (AD). However, the dynamics of this process remain unknown, mainly due to the paucity of in-vivo methods capable of assessing CP function. METHODSHere, we harness recent developments in Arterial Spin Labelling MRI to measure water delivery across the blood cerebrospinal fluid barrier (BCSFB) as a proxy for CP function, as well as cerebral blood flow (CBF), at different stages of AD progression in the widely used triple transgenic mouse model (3Tg), which recapitulates aspects of disease pathology. RESULTSTotal BCSFB-mediated water delivery is significantly higher in 3Tg mice (>50%) from 8 weeks (preclinical stage), while tissue parameters such as CBF and T1 are not different between groups at all ages. DISCUSSIONOur work shows changes in BCSFB function in the early stages of AD, providing a novel biomarker of pathology.

neuroscience↗

bioRxiv2024

Mosaic sarbecovirus vaccination elicits cross-reactive responses in pre-immunized animals

Immunization with mosaic-8b [60-mer nanoparticles presenting 8 SARS-like betacoronavirus (sarbecovirus) receptor-binding domains (RBDs)] elicits more broadly cross-reactive antibodies than homotypic SARS-CoV-2 RBD-only nanoparticles and protects against sarbecoviruses. To investigate original antigenic sin (OAS) effects on mosaic-8b efficacy, we evaluated effects of prior COVID-19 vaccinations in non-human primates and mice on anti-sarbecovirus responses elicited by mosaic-8b, admix-8b (8 homotypics), or homotypic SARS-CoV-2 immunizations, finding greatest cross-reactivity for mosaic-8b. As demonstrated by molecular fate-mapping in which antibodies from specific cohorts of B cells are differentially detected, B cells primed by WA1 spike mRNA-LNP dominated antibody responses after RBD-nanoparticle boosting. While mosaic-8b- and homotypic-nanoparticles boosted cross-reactive antibodies, de novo antibodies were predominantly induced by mosaic-8b, and these were specific for variant RBDs with increased identity to RBDs on mosaic-8b. These results inform OAS mechanisms and support using mosaic-8b to protect COVID-19 vaccinated/infected humans against as-yet-unknown SARS-CoV-2 variants and animal sarbecoviruses with human spillover potential.

immunology↗

bioRxiv2022

Optimisation and Pre-clinical Demonstration of Temporal Diffusion Ratio for Imaging Restricted Diffusion

Temporal Diffusion Ratio (TDR) is a recently proposed dMRI technique (DellAcqua, 2019) which provides contrast between areas with restricted diffusion and areas either without restricted diffusion or with length scales too small for characterisation. Hence, it has a potential for mapping pore sizes, in particular large axon diameters or other cellular structures. TDR employs the signal from two dMRI acquisitions obtained with the same, large, b-value but with different diffusion times and gradient settings. TDR is advantageous as it employs standard acquisition sequences, does not make any assumptions on the underlying tissue structure and does not require any model fitting, avoiding issues related to model degeneracy. This work for the first time optimises the TDR diffusion sequences in simulation for a range of different tissues and scanner constraints. We extend the original work (which considers substrates containing cylinders) by additionally considering the TDR signal obtained from spherical structures, representing cell soma in tissue. Our results show that contrasting an acquisition with short gradient duration and short diffusion time with an acquisition with long gradient duration and long diffusion time improves the TDR contrast for a wide range of pore configurations. Additionally, in the presence of Rician noise, computing TDR from a subset (50% or fewer) of the acquired diffusion gradients rather than the entire shell as proposed originally further improves the contrast. In the last part of the work the results are demonstrated experimentally on rat spinal cord. In line with simulations, the experimental data shows that optimised TDR improves the contrast compared to non-optimised TDR. Furthermore, we find a strong correlation between TDR and histology measurements of axon diameter. In conclusion, we find that TDR has great potential and is a very promising alternative (or potentially complement) to model-based approaches for mapping pore sizes and restricted diffusion in general. HighlightsO_LITemporal Diffusion Ratio (TDR) 2-seq approach maps areas with restricted diffusion C_LIO_LIOptimised gradient waveform pair is: long {delta} + low G and short {delta} + high G C_LIO_LIIf data is noisy calculating TDR using HARDI acquisition subsets increases accuracy C_LIO_LIWe demonstrate TDR for the first time pre-clinically in rat spinal cord C_LIO_LIPre-clinical TDR values are strongly correlated with axon diameter C_LI

bioengineering↗

bioRxiv2022

Deep learning-based system for real-time behavior recognition and closed-loop control of behavioral mazes using depth sensing

Robust quantification of animal behavior is fundamental in experimental neuroscience research. Systems providing automated behavioral assessment are an important alternative to manual measurements avoiding problems such as human bias, low reproducibility and high cost. Integrating these tools with closed-loop control systems creates conditions to correlate environment and behavioral expressions effectively, and ultimately explain the neural foundations of behavior. We present an integrated solution for automated behavioral analysis of rodents using deep learning networks on video streams acquired from a depth-sensing camera. The use of depth sensors has notable advantages: tracking/classification performance is improved and independent of animals coat color, and videos can be recorded in dark conditions without affecting animals natural behavior. Convolutional and recurrent layers were combined in deep network architectures, and both spatial and temporal representations were successfully learned for a 4- classes behavior classification task (standstill, walking, rearing and grooming). Integration with Arduino microcontrollers creates an easy-to-use control platform providing low-latency feedback signals based on the deep learning automatic classification of animal behavior. The complete system, combining depth-sensor camera, computer, and Arduino microcontroller, allows simple mapping of input-output control signals using the animals current behavior and position. For example, a feeder can be controlled not by pressing a lever but by the animal behavior itself. An integrated graphical user interface completes a user-friendly and cost-effective solution for animal tracking and behavior classification. This open-software/open-hardware platform can boost the development of customized protocols for automated behavioral research, and support ever more sophisticated, reliable and reproducible behavioral neuroscience experiments.

animal behavior and cognition↗

bioRxiv2021

Soma and Neurite Density MRI (SANDI) of the in-vivo mouse brain

Diffusion MRI (dMRI) provides unique insights into the neural tissue milieu by probing interaction of diffusing molecules and tissue microstructure. Most dMRI techniques focus on white matter tissues (WM) due to the relatively simpler modelling of diffusion in the more organized tracts; however, interest is growing in gray matter characterisations. The Soma and Neurite Density MRI (SANDI) methodology harnesses a model incorporating water diffusion in spherical objects (assumed to be associated with cell bodies) and in impermeable "sticks" (representing neurites), which potentially enables the characterisation of cellular and neurite densities. Recognising the importance of rodents in animal models of development, aging, plasticity, and disease, we here sought to develop SANDI for preclinical imaging and provide a validation of the methodology by comparing its metrics with the Allen mouse brain atlas. SANDI was implemented on a 9.4T scanner equipped with a cryogenic coil, and experiments were carried out on N=6 mice. Pixelwise, ROI-based, and atlas comparisons were performed, and results were also compared to more standard Diffusion Kurtosis MRI (DKI) metrics. We further investigated effects of different pre-processing pipelines, specifically the comparison of magnitude and real-valued data, as well as different acceleration factors. Our findings reveal excellent reproducibility of the SANDI parameters, including the sphere and stick fraction as well as sphere size. More strikingly, we find a very good rank correlation between SANDI-driven soma fraction and Allen brain atlas contrast (which represents the cellular density in the mouse brain). Although some DKI parameters (FA, MD) correlated with some SANDI parameters in some ROIs, they did not correlate nearly as well as SANDI parameters with the Allen atlas, suggesting a much more specific nature of the SANDI parameters. We conclude that SANDI is a viable preclinical MRI technique that can greatly contribute to research on brain tissue microstructure.

neuroscience↗