Biology subjects

Rodrigues, E.

Publications and source records attributed to Rodrigues, E..

11 recordsLinked to original sources

bioRxiv2026

Neocortical astrocyte diversity stems from distinct developmental origins

Key regulators of neural network activity in multiple advanced cognitive processes and essential components of the blood-brain barrier, astrocytes constitute a highly heterogeneous population at the morphological, molecular, and functional levels. However, how this diversity arises during mammalian brain development remains poorly investigated. Here, using a combination of multicolour genetic fate mapping, single-cell transcriptomic analyses, multichannel large-volume imaging and detailed 3D cell morphology reconstructions, we uncover a discrete subpopulation of neocortical astrocytes generated from an early restricted embryonic domain located outside of the dorsal pallium. Besides their separate lineage from pyramidal neurons, these astrocytes exhibit a developmental trajectory that differs from astrocytes produced by dorsal cortical progenitors, including different migratory pathways, spatial distributions and morphology. Overall, our results reveal the diversity of embryonic sources responsible for neocortical astrocyte genesis and provide key insights into the unsuspected complex developmental processes that underlie cortical astrocyte heterogeneity.

neuroscience↗

bioRxiv2026

Mutagenesis study of a Bacteriophytochrome - insights for the development of labels for optical imaging.

Bacteriophytochromes (BphPs) find increasing interest as near-infrared (NIR) labels for imaging. Applications range from whole animal imaging to cell-to super-resolution microscopy. Here we present a comprehensive study of a BphP from Rhizobium etli (ReBphP) allowing mutant-based insights into BphP photophysics. This is complemented by QM/MM-optimized deep-learning structure predictions of ReBphP and variants in their photoswitched states, rationalizing the effects of variants. Pertaining to imaging, based on our study we identify a bright and far red-shifted BphP for imaging in mammalian cells as well as a fluorescent photoswitching BphP. Utilizing the latter, we introduce photoswitching fluorescence background suppression for in vivo whole animal fluorescence imaging achieving higher contrast over background than possible utilizing non-switching labels.

biochemistry↗

bioRxiv2025

Cryptosporidium modifies intestinal microvilli through an exported virulence factor

Cryptosporidium is a common intestinal infection of vertebrates and a significant threat to public health. Within the epithelial layer of the intestine, the parasite invades and replicates. Infected cells are readily detected under the microscope by the presence of elongated microvilli, particularly around the vacuole where the parasite resides. Here, we identify a family of Cryptosporidium virulence factors that are exported into the host cell during infection and localise to the microvilli. We examine the trafficking and function of the most highly expressed family member, MVP1, which appears to control the elongation of microvilli through engagement of host EBP50 and CDC42. Remarkably, this mechanism closely mirrors that of an enteropathogenic Escherichia coli virulence factor, MAP, which is also known to drive host microvilli elongation during infection. This highlights a unique instance where eukaryotic and prokaryotic virulence factors have convergently evolved to modulate host actin structures through a similar mechanism.

microbiology↗

bioRxiv2024

Reversible tuning of membrane sterol levels by cyclodextrin in a dialysis setting

Large unilamellar vesicles (LUVs) are popular membrane models for studying the impact of lipids and bilayer properties on the structure and function of individual membrane proteins. The functional reconstitution of transmembrane in liposomes can be challenging, especially, if the hydrophobic thickness of the protein does not match the thickness of the surrounding lipid bilayer. During the reconstitution procedure Such hydrophobic mismatch causes low yields and protein aggregation, which are exacerbated in sterol-rich membranes featuring low membrane compressibility. Here, we explore new approaches to reversibly tune membrane sterol contents proteoliposomes after their formation. Both cholesterol delivery and extraction are mediated by methyl-{beta}-cyclodextrin in a dialysis setting, which maintains (proteo)liposomes in a confined compartment. This makes it possible to reversibly tune the cholesterol level without losing membrane material simply by placing the dialysis cassette in a new bath containing either empty or cholesterol-loaded methyl-{beta}-cyclodextrin. Cholesterol delivery and removal is monitored with the solvatochromic probe C-Laurdan, which reports on lipid packing. Using Forster-resonance energy transfer, we show that cholesterol delivery to proteoliposomes induces the oligomerization of a membrane property sensor, while the subsequent removal of cholesterol demonstrates the full reversibility. We propose that tuning membrane compressibility by methyl-{beta}-cyclodextrin-meditated cholesterol delivery and removal in a dialysis setup provides a new handle to study its impact on membrane protein structure, function, and dynamics. Statement of significanceGenerating complex, sterol-rich, biomimetic membranes for studying the structure and function of reconstituted membrane proteins is challenging. As an important step towards asymmetric, sterol-rich, complex model membrane systems, we have established a procedure to control the membrane sterol level of liposomes and proteoliposomes using methyl-{beta}-cyclodextrin in a dialysis setup. We demonstrate the feasibility of this approach by C-Laurdan and dehydroergosterol fluorescence spectroscopy and gain control over the membrane sterol content. We explore several parameters that affect the rate of cholesterol delivery and show that the oligomerization of a membrane property sensor, which is on the unfolded protein response sensor protein Ire1, is controlled by the sterol content of the surrounding lipid bilayer.

biophysics↗

bioRxiv2024

Birth order specified recruitment of motor circuits during spontaneous neural activity in zebrafish embryo

Modular organization of spinal neural circuits control dynamic regulation of locomotion. However, it is unknown when or how the distinct microcircuits emerge during development. We carried out high-resolution calcium imaging of neural activity driving the first motor behavior in one day old zebrafish embryo. During this period, at least two waves of neurogenesis occur to generate primary and secondary motoneurons. We found that embryos first display a single highly synchronized rhythmic neuronal circuit containing interneurons and motoneurons. Later, two distinct interneuron-motoneuron circuits emerge with one containing early-born motoneurons displaying low-frequency activity and the other containing later-born motoneurons with high-frequency activity. The results indicate a mode of birth order determined microcircuits where neurons that are born together are recruited together. Nicotine affected neuronal activity frequency, revealing a functional role for cholinergic signaling in the emergence of patterned spinal circuits. Indeed, we found aberrant arrhythmic synchronized activity in mutants for cholineacetyltransferase-a where acetylcholine is no longer synthesized. Overall, we reveal the sequential recruitment of birth order specified microcircuits during the emergence of the earliest motor behavior and highlight a conserved role for cholinergic signaling in regulating rhythmic neural activity in the embryonic spinal cord.

neuroscience↗

bioRxiv2023

A new method for the reproducible development of aptamers (Neomers)

The development of aptamers has been almost exclusively performed based on the SELEX method since their inception. While this method represents a powerful means of harnessing the in vitro evolution of sequences that bind to a given target, there are significant constraints in the design. The most significant constraint has been the reliance on counter selection on off-targets to drive specificity. Counter selection has not been as effective at driving aptamer specificity as the presence of immune tolerance, the capacity of the immune system to remove antibodies that bind to host targets, is for antibody development. This deficiency has constrained the commercial efficacy of aptamers to date. These limitations have been addressed with our design of a novel platform for aptamer identification. This new design is based on what we refer to as a Neomer library with sixteen random nucleotides interspersed with fixed sequences. The fixed sequences are designed to minimize the potential for hybridization, such that secondary structure is driven by the random nucleotides. The use of sixteen random nucleotides reduces the possible library sequences to 4.29 x 109. This enables the application of the same sequences to either the same target or different targets while maintaining a high level of structural diversity. In effect, this introduces the capacity for reproducibility in aptamer selection and in silico approach to replicating immune tolerance We provide here an overview of the new method and a description of the performance of aptamers selected for interleukin 6 developed using this approach.

biochemistry↗

bioRxiv2021

Carbohydrate sulfation as a mechanism for fine-tuning Siglec ligands

The immunomodulatory family of Siglecs recognize sialic acid-containing glycans as self, which is exploited in cancer for immune-evasion. The biochemical nature of Siglec ligands remains incompletely understood with emerging evidence suggesting the importance of carbohydrate sulfation. Here, we investigate how specific sulfate modifications affect Siglec ligands by overexpressing eight carbohydrate sulfotransferases (CHSTs) in five cell lines. Overexpression of three CHSTs (CHST1, CHST2, or CHST4) significantly enhances the binding of numerous Siglecs. Unexpectedly, two other CHSTs (Gal3ST2 and Gal3ST3) diminish Siglec binding, suggesting a new mode to modulate Siglec ligands via sulfation. Results are cell type dependent, indicating that the context in which sulfated glycans are presented is important. Moreover, pharmacological blockade of N- and O-glycan maturation reveals a cell type-specific pattern of importance for either class of glycan. Production of a highly homogenous CD33 (Siglec-3) fragment enabled a mass spectrometry-based binding assay to determine 10-fold and 3-fold enhanced affinity for Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4GlcNAc and Neu5Ac2-3Gal{beta}1-4(6-O- sulfo)GlcNAc, respectively, over Neu5Ac2-3Gal{beta}1-4GlcNAc. CD33 showed significant additivity in affinity (36-fold) for the disulfated ligand, Neu5Ac2-3(6-O-sulfo)Gal{beta}1-4(6-O-sulfo)GlcNAc. Moreover, overexpression of both CHST1 and CHST2 in cells greatly enhanced the binding of several Siglecs, including CD33. Finally, we reveal that CHST1 is upregulated in numerous cancers, correlating with poorer survival rates and sodium chlorate sensitivity for the binding of Siglecs to cancer cell lines. These results provide new insights into carbohydrate sulfation as a modification that is a general mechanism for tuning Siglec ligands on cells, including in cancer.

immunology↗

bioRxiv2020

A Versatile Soluble Siglec Scaffold for Sensitive and Quantitative Detection of Glycan Ligands

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are immunomodulatory receptors that are regulated by their glycan ligands. A growing connection between Siglecs and human disease motivates improved methods to detect Siglec ligands. Here, we describe a new versatile set of Siglec-Fc proteins for glycan ligand detection. Enhanced sensitivity and selectivity are enabled through multimerization and avoiding Fc receptors, respectively. Using these Siglec-Fc proteins, Siglec ligands were systemically profiled on healthy and cancerous cells and tissues, revealing many unique patterns. Additional features enable the production of small, homogenous Siglec fragments and development of a quantitative ligand-binding mass spectrometry assay. Using this assay, the ligand specificities of several Siglecs were clarified. For CD33, we demonstrate that it recognizes both 2-3 and 2-6 sialosides in solution and on cells, which has implications for its link to Alzheimers disease susceptibility. These soluble Siglecs reveal the abundance of their glycan ligands on host cells as self-associated molecular patterns.

biochemistry↗

bioRxiv2020

Modulate Siglec-7 Signaling via in situ Created High-affinity cis-Ligands

Sialic acid-binding immunoglobulin-like lectins, also known as Siglecs, have recently been designated as glyco-immune checkpoints. Through their interactions with sialylated glycan epitopes overexpressed on tumor cells, inhibitory Siglecs on innate and adaptive immune cells modulate signaling cascades to restrain anti-tumor immune responses. However, the mechanisms underlying these processes are just starting to be elucidated. We discover that when human natural killer (NK) cells attack tumor cells, glycan remodeling occurs on the target cells at the immunological synapse. This remodeling occurs through both transfer of sialylated glycans from NK cells to target tumor cells and accelerated de novo synthesis of sialosides on the tumor cell themselves. The functionalization of NK cells with a high-affinity ligand of Siglec-7 leads to multifaceted consequences in modulating Siglec-7-regulated NK-activation. At high levels, the added Siglec-7 ligand suppresses NK-cytotoxicity through the recruitment of Siglec-7, whereas at low levels the same ligand triggers the release of Siglec-7 from the cell surface into the culture medium, preventing Siglec-7-mediated inhibition of NK cytotoxicity. These results suggest that glycan engineering of NK cells may provide a means to boost NK effector functions.

biochemistry↗

bioRxiv2020

Genetically Encoded, Multivalent Liquid Glycan Array (LiGA)

The Central Dogma of Biology does not allow for the study of glycans using DNA sequencing. We report a "Liquid Glycan Array" (LiGA) platform comprising a library of DNA barcoded M13 virions that display 30-1500 copies of glycans per phage. A LiGA is synthesized by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins such as CD22 on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identifies the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo; measurements that cannot be performed with canonical glass slide-based glycan arrays. DedicationThe paper is dedicated to Laura L. Kiessling on the occasion of her 60th birthday.

biochemistry↗

bioRxiv2018

Changes in thalamocortical connectivity as a potential mechanism of cross-modal plasticity in congenitally blind individuals

Cross-modal plasticity in blind individuals has been reported over the past decades showing that non-visual information is carried and processed by visual brain structures. However, the structural underpinnings of cross-modal plasticity in congenitally blind individuals remain unclear despite multiple efforts. We mapped thalamocortical connectivity and assessed cortical thickness and integrity of white matter of ten congenitally blind individuals and ten sighted controls. We hypothesized an aberrant thalamocortical pattern of connectivity taking place in the absence of visual stimuli from birth as a potential mechanism of cross-modal plasticity. In addition to the increased cortical thickness of the primary visual cortex and reduced integrity of visual white matter bundles, we observed structural connectivity changes between the thalamus and occipital and temporal cortices. Specifically, the thalamic territory dedicated to connections with the occipital cortex was smaller and displayed weaker connectivity in congenitally blind individuals. In contrast, those connecting with the temporal cortex showed greater volume and increased connectivity compared to sighted controls. The abnormal pattern of thalamocortical connectivity included the lateral and medial geniculate nuclei and the pulvinar nucleus. For the first time in humans, a remapping of structural thalamocortical connections involving both unimodal and multimodal thalamic nuclei has been demonstrated, shedding light on the possible mechanisms of cross-modal plasticity in humans. The present findings may help understand the functional adaptations commonly observed in congenitally blind individuals.

neuroscience↗