Biology subjects

Galindo, A.

Publications and source records attributed to Galindo, A..

6 recordsLinked to original sources

bioRxiv2026

Hyaluronic Acid-Alginate Hydrazone Crosslinked Hydrogels Support the Generation and Maturation of V2a Interneurons

Injury to the central nervous system (CNS) causes inflammation, cell death, and glial scar formation that inhibits tissue repair. Injectable hydrogels modified with extracellular matrix (ECM)-derived peptides can provide biochemical cues to promote neural tissue repair and serve as a vehicle to deliver therapeutics across the blood-spinal/blood-brain barrier in a minimally invasive manner. We developed an injectable hydrazone crosslinked hyaluronic acid-alginate (HA-Alg) hydrogel for neural tissue repair. We fabricated hydrogels with a range of polymer concentrations and evaluated their physicochemical properties to identify formulations that mimic the stiffness and viscoelastic properties of the CNS tissue environment. Hyaluronic acid was further modified with ECM-derived, cell-adhesive peptides (RGD and IKVAV) to enhance neuronal adhesion and viability. To evaluate the therapeutic potential of our hydrogel platform, we embedded mouse embryonic stem cell aggregates and differentiated them toward mature V2a interneurons. These interneurons are critical for relaying motor signals and represent a promising therapeutic cell population for treating spinal cord injuries. We demonstrated successful enrichment for V2a interneurons in HA-Alg hydrogels containing ECM-derived peptides. Interestingly, both our newly described HA-Alg and established crosslinked HA-HA hydrogels containing IKVAV peptides demonstrated significantly increased neurite length in interneuron enriched cultures compared to hydrogels without peptides. This study demonstrates that the addition of ECM-derived peptides is essential to support the neuronal adhesion and viability required for functional CNS tissue repair. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/729352v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@666b1aorg.highwire.dtl.DTLVardef@17d4borg.highwire.dtl.DTLVardef@1c8ece2org.highwire.dtl.DTLVardef@37fdd5_HPS_FORMAT_FIGEXP M_FIG C_FIG Statement of SignificanceDevelopment of injectable, peptide-modified hyaluronic acid alginate hydrogels to support neuronal maturation for central nervous system tissue repair.

bioengineering↗

bioRxiv2025

ATG8i Autophagy activation is mediated by cytosolic Ca2+ under osmotic stress in Arabidopsis thaliana

Autophagy is a highly conserved catabolic process in eukaryotic cells that enables the degradation and recycling of damaged or unnecessary cytoplasmic components. It plays essential roles in both development and responses to environmental stress. In this study, we investigated the regulation of autophagy in response to osmotic stress, focusing on the dynamics of the RFP-tagged ATG8i protein and the potential involvement of cytosolic calcium ion (Ca{superscript 2}) in this process. Our findings indicate that both osmotic stress and Ca{superscript 2} signaling modulate the accumulation of RFP-ATG8i-labeled autophagosomes in a plant organ-specific manner. Furthermore, the observed colocalization of RFP-ATG8i with the endoplasmic reticulum (ER) marker HDEL suggests a significant role for ATG8i in ER-phagy, highlighting its potential contribution to ER turnover under stress conditions.

plant biology↗

bioRxiv2025

Isolation and primary culture of Octopus vulgaris cells: Assessment of their proliferative capacity

The octopus cell culture system represents a useful tool in various fields, such as biological studies and sustainable octopus production. In this paper, we describe the isolation procedure and culture protocols, metabolic activity, and the proliferative capacity assessment of the common octopus (Octopus vulgaris) cells, at three different life stages: embryo, paralarvae and adult. Isolation of cells was carried out with minimum essential medium (MEM) or Leibovitz L-15 medium, using 0.4 % type I collagenase for 2 (paralarvae and embryo) or 3 h (adult) at 25 {degrees}C. Isolated cells showed significant metabolic activity, demonstrating their viability for in vitro studies. The culture of isolated cells in MEM or L-15 supplemented with different sera (4 % fetal bovine serum (FBS), 10 % FBS and 5 % haemolymph (HEMO)) was viable for 168 h, although the survival of different cell types was variable. A detachment protocol using trypsin was also successfully developed. Finally, isolated muscle cells from both paralarval and adult specimens were marked with the mitotic indicator PHH3. Results from flow cytometry analysis showed that all developmental stages examined contained cells in mitotic phase, with embryos XII-XIII showing the highest proportion. This study provides novel guidelines for the in vitro culture of O. vulgaris cells, which are useful for the future development of cellular aquaculture, understood as the production of lab-grown meat for this species. Moreover, the results reported in this communication could contribute to reduce the use of animals for scientific purposes as well as providing a new option for human consumption of cephalopods.

cell biology↗

bioRxiv2022

Tethering by Uso1 is dispensable: The Uso1 monomeric globular head domain interacts with SNAREs to maintain viability.

Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Paradoxically, genetic studies placed Uso1 acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S substitutions in the GHD permitting growth of otherwise inviable rab1-deficient Aspergillus nidulans. Remarkably, the double mutant suppresses the complete absence of RAB1. Full-length Uso1 and CTR{Delta} proteins are dimeric and the GHD lacking the CC/CTR is monomeric irrespective of whether they carry or not E6K/G540S. Microscopy showed recurrence of Uso1 on puncta (60 sec half-life) colocalizing with RAB1 and less so with early Golgi markers Sed5 and GeaA/Gea1/Gea2. Localization of Uso1 but not of Uso1E6K/G540S to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. By S-tag-coprecipitation we demonstrate that Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Bos1 and Bet1 bind the Uso1 GHD directly, but Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. AlphaFold2 predicts that G540S actually increases binding of GHD to the Bos1 Habc domain. In contrast, E6K seemingly increases membrane targeting of an N-terminal amphipathic -helix, explaining phenotypic additivity. Overexpression of E6K/G540S and wild-type GHD complemented uso1{Delta}. Thus, a GHD monomer provides the essential Uso1 functions, demonstrating that long-range tethering activity is dispensable. Therefore, when enhanced by E6K/G540S, Uso1 binding to Bos1/Bet1 required to regulate SNAREs bypasses both the contribution of RAB1 to Uso1 recruitment and the reported role of RAB1 in SNARE complex formation (Lupashin and Waters, 1997), suggesting that the latter is consequence of the former.

cell biology↗

bioRxiv2022

Development of a novel high-throughput screen for the identification of new inhibitors of protein S-acylation

Protein S-acylation is a reversible post-translational modification that modulates the localisation and function of many cellular proteins. S-acylation is mediated by a family of zinc finger DHHC domain-containing proteins encoded by 23 distinct ZDHHC genes in the human genome. These enzymes catalyse S-acylation in a two-step process involving "auto-acylation" of the cysteine residue in the catalytic DHHC motif followed by transfer of the acyl chain to a substrate cysteine. S-acylation is essential for many fundamental physiological processes, and there is growing interest in zDHHC enzymes as novel drug targets for a range of disorders. However, there is currently a lack of chemical modulators of S-acylation either for use as tool compounds or for potential development for therapeutic purposes. In this study, we developed and implemented a novel FRET-based high throughput assay for the discovery of compounds that interfere with auto-acylation of zDHHC2, an enzyme that is implicated in neuronal S-acylation pathways. A screen of >350,000 compounds identified two related tetrazole containing compounds (TTZ-1 and -2) that inhibited both zDHHC2 auto-acylation and substrate S-acylation in cell-free systems. Furthermore, these compounds were also active in HEK293T cells, where they inhibited substrate S-acylation mediated by different zDHHC enzymes, with some apparent isoform selectivity. Resynthesis of the hit compounds confirmed their activity, providing sufficient quantities of material for further investigations. The assays developed herein provide novel strategies to screen for zDHHC inhibitors, and the identified compounds add to the chemical toolbox for interrogating the cellular activities of S-acylation and zDHHC enzymes.

cell biology↗

bioRxiv2020

A cryo-EM structure of metazoan TRAPPIII, the multisubunit complex that activates the GTPase Rab1

The TRAPP complexes are highly conserved nucleotide exchange factors, with TRAPPIII activating Rab1 and TRAPPII acting primarily on Rab11. The two complexes share a core of small subunits that affect nucleotide exchange, but are distinguished by additional large subunits that are essential for activity in vivo, and are mutated in a range of human disorders. Crystal structures of the core subunits have revealed the mechanism of Rab activation, but how and why the large subunits associate with the core remains unclear. We report here a cryo-EM structure of the entire TRAPPIII complex from Drosophila. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact bound Rab1, indicating how the arms could alter the Rab specificity of the core. A lower resolution structure of TRAPPII shows a similar architecture, and suggests that the TRAPP complexes evolved from a single ur-TRAPP.

cell biology↗