Biology subjects

Fernandes, F. F.

Publications and source records attributed to Fernandes, F. F..

14 recordsLinked to original sources

bioRxiv2026

Three-dimensional nano-imaging reveals subtle changes in xylem structure in CAD-deficient sorghum

Lignin plays a central role in the formation and function of secondary cell walls in vascular plants. However, the structural consequences of lignin modification for cell wall properties and cellular function in grasses remain poorly understood. Here, we investigated how cinnamyl alcohol dehydrogenase (CAD) deficiency alters vascular cell architecture in Sorghum bicolor, using the brown midrib-6 (bmr6) mutant as a model system. Biochemical and histochemical analyses confirmed altered lignin chemistry in bmr6, including increased incorporation of hydroxycinnamaldehyde residues and reduced tricin levels. We applied ptychographic X-ray computed tomography (PXCT) to quantify the cell wall geometry, in three dimensions, at nanometer-scale resolution. PXCT enabled measurements of wall thickness distribution and lumen shape along tracheary elements. Analyses revealed no significant differences in wall thickness between wild-type and bmr6 plants. However, three-dimensional morphometric descriptors indicated reduced lumen convexity in bmr6, suggesting localized modifications not detectable by conventional two-dimensional imaging. Water flow numerical simulations through PXCT-derived images indicated reduced vessel permeability and simulated hydraulic conductivity in bmr6, suggesting that subtle geometric changes may influence performance. These findings highlight the value of three-dimensional imaging for resolving cell wall organization and provide new insight into the architectural resilience of grass xylem in response to targeted lignin modification. HighlightThree-dimensional X-ray nano-imaging reveals alterations in the cell wall architecture that affect simulated hydraulic performance under reduced CAD activity in sorghum.

plant biology↗

bioRxiv2026

Correlative Synchrotron X-ray Microscopy Reveals Dose- and Division-Dependent Nanoparticle Redistribution in Macrophages

Understanding the intracellular fate of nanoparticles is essential for designing safer and more effective nanomedicines, yet most studies rely on static observations and lack high-resolution, near-native volumetric information. Here, we establish a synchrotron-based correlative X-ray microscopy framework to investigate how fluorescent silica nanoparticles (SiNPs) redistribute within macrophages as a function of concentration and successive cell-division cycles. SiNPs were internalized by RAW 264.7 macrophages at different concentrations and analyzed using a synchrotron-based correlative X-ray microscopy workflow integrating cryogenic soft X-ray tomography (cryo-SXT), cryogenic structured illumination microscopy (cryo-SIM), and coherent X-ray ptychography, with confocal fluorescence microscopy used to establish population-level uptake tendencies. Cryo-SXT reveals a concentration-dependent redistribution of nanoparticle-containing vesicles from peripheral endosomes toward the perinuclear region, while correlative cryo-SIM confirms strict vesicular confinement, with no evidence of free nanoparticle diffusion into the nucleoplasm. At higher doses, nanoparticles approach the nuclear region via vesicles extending into nuclear-envelope invaginations, rather than by true nuclear entry. Successive cell divisions redistribute the intracellular nanoparticle load and promote stable perinuclear clustering, identifying a long-term sequestration route in macrophages. Coherent X-ray ptychography further reveals nanoscale deformations of the nuclear envelope associated with dense perinuclear vesicles. Together, these results establish synchrotron-based correlative X-ray microscopy as a mechanistic, multiscale platform for unveiling the dynamic intracellular fate of nanoparticles and providing mechanistic insight into their apparent nuclear localization.

cell biology↗

bioRxiv2024

Deuterium Metabolic Imaging Phenotypes Mouse Glioblastoma Heterogeneity Through Glucose Turnover Kinetics

Glioblastomas are aggressive brain tumors with dismal prognosis. One of the main bottlenecks for developing more effective therapies for glioblastoma stems from their histologic and molecular heterogeneity, leading to distinct tumor microenvironments and disease phenotypes. Effectively characterizing these features would improve the clinical management of glioblastoma. Glucose flux rates through glycolysis and mitochondrial oxidation have been recently shown to quantitatively depict glioblastoma proliferation in mouse models (GL261 and CT2A tumors) using dynamic glucose-enhanced (DGE) deuterium spectroscopy. However, the spatial features of tumor microenvironment phenotypes remain hitherto unresolved. Here, we develop a DGE Deuterium Metabolic Imaging (DMI) approach for profiling tumor microenvironments through glucose conversion kinetics. Using a multimodal combination of tumor mouse models, novel strategies for spectroscopic imaging and noise attenuation, and histopathological correlations, we show that tumor lactate turnover mirrors phenotype differences between GL261 and CT2A mouse glioblastoma, whereas recycling of the peritumoral glutamate-glutamine pool is a potential marker of invasion capacity in pooled cohorts, linked to secondary brain lesions. These findings were validated by histopathological characterization of each tumor, including cell density and proliferation, peritumoral invasion and distant migration, and immune cell infiltration. Our study bodes well for precision neuro-oncology, highlighting the importance of mapping glucose flux rates to better understand the metabolic heterogeneity of glioblastoma and its links to disease phenotypes.

cancer biology↗

bioRxiv2024

Evidence for a push-pull interaction between superior colliculi in monocular dynamic vision mode

Visual perception can operate in two distinct vision modes - static and dynamic - that have been associated with different neural activity regimes in the superior colliculus (SC). The static vision mode (low flashing frequencies) is associated with strong SC activation modulated by cortical gain and inhibitory intertectal effects, while the dynamic vision mode (high flashing frequencies) evokes the continuity illusion, with associated suppression of SC neural activity. However, the pathway-wide mechanisms underpinning the dynamic vision mode remain poorly understood, especially in terms of corticotectal and tectotectal feedback. Here, we harness rat functional MRI combined with brain lesions to investigate whole-pathway interactions in the dynamic vision mode. In the SC, we find contralateral suppression of activity opposing positive ipsilateral neural activation upon monocular visual stimulation in the dynamic vision mode. A cortical amplification effect was confirmed for both static and dynamic vision modes through cortical lesions, while further lesioning ipsilateral SC led to a boost in the contralateral negative signals, suggesting an active push-pull interaction between ipsilateral and contralateral SCs during the dynamic vision mode regime. This push-pull interaction is specific to the dynamic vision mode; in the static vision mode, both SCs show similar response polarities. These results highlight hitherto unreported frequency-dependent modulations in the tectotectal pathway and further challenge the contemporary notion that intertectal connections solely serve as reciprocal inhibitory mechanisms for avoiding visual blur during saccade occurrence. One Sentence SummaryOpposing signals between superior colliculi in the dynamic vision mode suggest an active push-pull interaction within the tectotectal commissural pathway.

neuroscience↗

bioRxiv2022

Paracoccin impacts on Paracoccidioides brasiliensis virulence and susceptibility to antifungal drugs by modifying the expression of genes related to remodeling of the yeasts cell wall

The study of the paracoccin lectin (PCN) has provided knowledge about its role in the biology of Paracoccidioides brasiliensis and in the pathogenesis of paracoccidioidomycosis (PCM). In this context, PCN has proved to be a promising immunomodulatory agent for the exploration of vaccine target molecules and/or for diagnostic or therapeutic purposes. Previous investigations allowed establishing PCN as a factor of fungal virulence. However, the effect PCN exerts on the yeasts resistance to antifungal pharmacological agents used to treat human PCM are not known. Therefore, this work characterizes the role of PCN functional duality on virulence and susceptibility of P. brasiliensis to antifungal drugs. We show that the PCN overexpression increases the virulence of P. brasiliensis yeasts in an alternative model of infection, induces high susceptibility in vitro and in vivo of P. brasiliensis yeasts to antifungal therapy, and impact reducing relative mRNA expression of genes encoding proteins related to cell wall degradation. Conversely, PCN silencing minimized the yeasts virulence in Galleria mellonella, correlates with the lowest susceptibility to treatment with antifungal agent in vivo and impact differently from the PCN overexpression on the relative expression of markers related to P. brasiliensis yeasts cell wall remodelling. Our study demonstrates the impact of endogenous PCN on the P. brasiliensis yeasts virulence vs. susceptibility to antifungal drugs, the fungal biology, and the relationship of the yeasts-host cells.

microbiology↗

bioRxiv2022

Extensive topographic remapping and functional sharpening in the adult rat visual pathway upon first visual experience

Understanding the dynamics of stability/plasticity balances during adulthood is pivotal for learning, disease, and recovery from injury. However, the brain-wide topography of sensory remapping remains unknown. Here, using a first-of-its-kind setup for delivering patterned visual stimuli in a rodent Magnetic Resonance Imaging (MRI) scanner, coupled with biologically-inspired computational models, we noninvasively mapped brain-wide properties - receptive fields (RFs) and spatial frequency (SF) tuning curves - that were insofar only available from invasive electrophysiology or optical imaging. We then tracked the RF dynamics in the chronic Visual Deprivation Model (VDM) of plasticity, and found that light exposure progressively promoted a large-scale topographic remapping in adult rats. Upon light exposure, the initially unspecialized visual pathway progressively evidenced sharpened RFs (smaller and more spatially selective) and enhanced spatial frequency tuning curves. Our findings reveal that visual experience following VDM reshapes both structure and function of the visual system and shifts the stability/plasticity balance in adults.

neuroscience↗

bioRxiv2022

StandardRat: A multi-center consensus protocol to enhance functional connectivity specificity in the rat brain

Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows comparison with invasive or terminal procedures. To date, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. We introduce StandardRat, a consensus rat functional MRI acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired in rats from 46 centers. We developed a reproducible pipeline for the analysis of rat data acquired with diverse protocols and determined experimental and processing parameters associated with a more robust functional connectivity detection. We show that the standardized protocol enhances biologically plausible functional connectivity patterns, relative to pre-existing acquisitions. The protocol and processing pipeline described here are openly shared with the neuroimaging community to promote interoperability and cooperation towards tackling the most important challenges in neuroscience.

neuroscience↗

bioRxiv2022

Brain-wide sensory aberrations in a Parkinson's Disease mouse model revealed by functional MRI

Parkinsons disease (PD) is associated with irreparable damage to dopaminergic neurons in brain areas involved in movement. Frequently, a very early symptom of PD involves an impaired sense of smell, while other studies suggest a broader impairment of other sensory modalities. Still, the associated brain-wide mechanisms underlying this phenomenon remain unclear. Here, we harness in vivo functional Magnetic Resonance Imaging (fMRI) at 9.4T to investigate how the olfactory and visual systems are affected in an -Synuclein (Syn) mouse model of PD. We find significant aberrations in fMRI responses along both sensory pathways including decreases in activation extent and in activation amplitude. Deficits in the olfactory system were larger than in the visual pathway, but nevertheless both sensory systems were clearly impacted, suggesting a more overarching mechanism may be involved. To further ensure that the aberrations observed in the fMRI signals were not driven solely by putative vascular deficiency, we quantified c-FOS expression in the olfactory system and found statistically significant decreases in the PD group when compared to age-matched controls. Hence, our findings indicate a neural origin for the observed fMRI aberrations. Our findings thus demonstrate a more global deficiency in sensory systems in the (Syn) mouse model. One Sentence SummaryParkinsons disease (PD) is associated with an impaired sense of smell and more generally sensory deficits, but the associated brain-wide mechanisms remain unclear; here, we investigate aberrations in BOLD-fMRI responses along sensory pathways in an SYN PD mouse model.

neuroscience↗

bioRxiv2022

Resonant waves drive long-range correlations in fMRI signals

Spontaneous fluctuations in functional Magnetic Resonance Imaging (fMRI) signals correlate across distant brain areas, shaping functionally relevant intrinsic networks. However, the generative mechanism of fMRI signal correlations - and in particular their link with locally-detected ultra-slow oscillations - remain unclear. To investigate this link, we record ultrafast ultrahigh field fMRI signals (9.4 Tesla, temporal resolution = 38 milliseconds) from rat brains across three anesthesia conditions. Power at frequencies extending up to 0.3 Hz is detected consistently across rat brains, and is modulated by anesthesia level. Principal component analysis reveals a repertoire of modes, in which transient oscillations organize with fixed phase relationships across distinct cortical and subcortical structures. Oscillatory modes are found to vary between conditions, resonating at faster frequencies under medetomidine sedation and reducing both in number, frequency, and duration with the addition of isoflurane. Peaking in power within clear anatomical boundaries, these oscillatory modes point to an emergent systemic property, questioning current assumptions regarding the local origin of oscillations detected in fMRI and providing novel insights into the organizing principles underpinning spontaneous long-range functional connectivity. One Sentence SummaryOscillations in fMRI signals organize in stationary wave patterns driving condition-specific long-range correlations across brain structures

neuroscience↗

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↗

bioRxiv2020

Morphological aspects of immature stages of Migonemyia migonei (Diptera: Psychodidae, Phlebotominae) an important vector of Leishmaniosis in South America by scanning electron microscopy

We describe the immature stages of Migonemyia migonei, which is the vector of Leishmania (Viannia) braziliensis, the aetiological agent of cutaneous leishmaniasis in South America, and a putative vector of Leishmania infantum chagasi. Scanning Electron Microscopy (SEM) was used to refine the description of the structures of eggs, all instar larvae and pupa. The eggs have polygonal cells on the egg exochorion, and differences between larval and pupal chaetotaxy are highlighted. Different sensillary subtypes were observed in the larval stages, among the types trichoidea, basiconica, coelonica and campanoformia. These results contribute to the taxonomy of Mg. migonei and may contribute to future studies on the phylogeny of this important species vector.

zoology↗

bioRxiv2019

Galectin-3 inhibits Paracoccidioides brasiliensis growth and impacts paracoccidioidomycosis through multiple mechanisms

The thermodimorphic pathogenic fungi Paracoccidioides brasiliensis and Paracoccioidioides lutzii are the etiologic causes of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Galectin-3 (Gal-3), an animal {beta}-galactoside-binding protein, modulates important roles during microbial infections, such as triggering a Th2-polarized immune response in PCM. Herein, we demonstrate that Gal-3 also plays other important roles in P. brasiliensis infection. We verified Gal-3 levels are upregulated in human and mice infections and establish that Gal-3 inhibits P. brasiliensis growth by inhibiting budding. Furthermore, Gal-3 affects disruption and internalization of extracellular vesicles (EV) from P. brasiliensis by macrophages. Our results suggest important roles for Gal-3 in P. brasiliensis infection, indicating that increased Gal-3 production during P. brasiliensis infection may account for affecting the fungal growth and EV stability, promoting a benefic course of experimental PCM. IMPORTANCEParacoccidiodomycosis (PCM) is the most prevalent systemic mycosis in Latin America. Although the immune mechanisms to control PCM are still not fully understood, several events of the host innate and adaptive immunity are crucial to determine the progress of the infection. Mammalian {beta}-galactoside-binding protein Galectin-3 (Gal-3) plays significant roles during microbial infections, and has been studied for its immunomodulatory roles but it can also have direct antimicrobial effects. We asked whether this protein plays a role in P. brasiliensis. We report herein that Gal-3 indeed has direct effects on fungal pathogen, inhibiting fungal growth and reducing extracellular vesicles stability. Our results suggest a direct role for Gal-3 in P. brasiliensis infection, with beneficial effects for the mammalian host.

microbiology↗

bioRxiv2019

Paracoccin overexpression in Paracoccidioides brasiliensis reveals the influence of chitin hydrolysis on fungal virulence and host immune response

Paracoccidioides brasiliensis and P. lutzii, etiological agents of paracoccidioidomycosis (PCM), develop as mycelia at 25-30 {degrees}C and as yeast at 35-37 {degrees}C. Only a few Paracoccidioides spp. proteins are well characterized. Thus, we studied paracoccin (PCN) from P. brasiliensis, its role in the fungus biology, and its relationship with the host innate immune cells. Cloning and heterologous expression analysis revealed its lectin, enzymatic, and immunomodulatory properties. Recently, we employed a system based on Agrobacterium tumefaciens-mediated transformation to manipulate P. brasiliensis yeast genes to obtain clones knocked-down for PCN, which after all, are unable to transit from yeast to mycelium forms, causing a mild pulmonary disease. Herein, we generate P. brasiliensis overexpressing PCN (ov-PCN). To date, it was not explored the overexpressing of endogenous components in Paracoccidioides spp. Therefore, we investigate the role of PCN in fungal biology and pathogenesis. Augmented levels of PCN mRNA and protein, and N-acetylglucosaminidase activity confirmed PCN overexpression in ov-PCN of P. brasiliensis yeasts. Interestingly, PCN overexpression did not affect the yeasts growth or viability and favored cell separation. The ov-PCN clones transitioned faster to the mycelium form than the wt-PCN yeasts. Concerning infection, while most of mice infected with the wt-yeasts (90%) survive at least until the 70th day, all mice infected with ov-PCN yeasts were already died at the 35th day post-infection. In vitro assays showed that ov-PCN were more susceptible to phagocytosis by macrophages. Finally, it was verified that the chitin particles isolated from the ov-PCN cells were smaller than those obtained from the wt-PCN yeasts. Macrophages stimulated with the chitin isolated from ov-PCN produce IL-10, whereas the particles with a wider size range harvested from wt-PCN yeasts induced TNF- and IL-1{beta} secretion. The anti-inflammatory microenvironment from macrophage stimulation with small chitin particles hampers the development of a protective immune response against the fungus. We postulated that the high grade of chitin cleavage, as the results of augmented PCN expression, favors pathogenesis following P. brasiliensis infection. Thus, PCN is a relevant virulence fungal factor.\n\nAUTHOR SUMMARYParacoccidioides spp. are pathogenic fungi that cause paracoccidioidomycosis (PCM) in humans, the main deep mycosis of Latin America. Recently, by knocking down the paracoccin gene, our group showed that this lectin is necessary for the morphological transition from yeast to hyphae, and that this decrease results in low P. brasiliensis virulence. Here, after overexpress PCN, we revealed the importance of the yeast chitin hydrolysis to the host response. Infection of mice with ov-PCN yeasts causes severe lung disease compared to moderate disease caused by wt-PCN yeasts. The release of smaller chitin particles was as a result of an accelerated chitin hydrolysis provided by ov-PCN yeasts. Interestingly, these smallest chitin particles are able to modulate host response by increasing IL-10 in the meantime that decrease TNF- secretion, thus hampering Th1 immune response that is crucial in the fight against this fungi. These findings represent a significant advance in the knowledge about the role of PCN chitinase in P. brasiliensis.

microbiology↗

bioRxiv2017

Toxoplasma gondii microneme proteins 1 and 4 bind to Toll-like receptors 2 and 4 N-glycans triggering innate immune response

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex, secreted on the parasite surface and function to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages to produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-{gamma} in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.\n\nAUTHOR SUMMARYToxoplasmosis is caused by the protozoan Toxoplasma gondii, belonging to the Apicomplexa phylum. This phylum comprises important parasites able to infect a broad diversity of animals, including humans. A particularity of T. gondii is its ability to invade virtually any nucleated cell of all warm-blooded animals through an active process, which depends on the secretion of adhesin proteins. These proteins are discharged by specialized organelles localized in the parasite apical region, and termed micronemes and rhoptries. We show in this study that two microneme proteins from T. gondii utilize their adhesion activity to stimulate innate immunity. These microneme proteins, denoted MIC1 and MIC4, recognize specific sugars on receptors expressed on the surface of mammalian immune cells. This binding activates these innate immune cells to secrete cytokines, which promotes efficient host defense mechanisms against the parasite and regulate their pathogenesis. This activity promotes a chronic infection by controlling parasite replication during acute infection.

immunology↗