Biology subjects

Rosales, A.

Publications and source records attributed to Rosales, A..

11 recordsLinked to original sources

bioRxiv2025

Dynamic Stiffening to Improve Vasculogenesis of hiPSC-Derived Endothelial Progenitors

Multiple groups have reported on the impact of hydrogel stiffness on vascular network formation in vitro, with overall findings indicating that less stiff hydrogels better support vasculogenesis. However, the majority of this research utilized hydrogels with static stiffness, even though vasculogenesis occurs in tandem with changes in extracellular matrix material properties. To that end, we hypothesize that dynamic modulation of hydrogel stiffness during the process of vasculogenesis, recapitulating changes observed during embryonic development, would improve vascular network formation. Using our Collagen I/Norbornene-modified hyaluronic acid hydrogel system, we swelled in additional crosslinker and photoinitiator and exposed the hydrogel to UV light, enabling hydrogel stiffening at user-defined time points with no significant effect on cell viability. We observed that in situ stiffening at early time points, prior to the onset of significant cell migration, resulted in more robust vascular network formation relative to unstiffened controls, while stiffening at later time points disrupted existing vascular networks. These trends continued in in vivo experiments in nude mice, with cell-laden hydrogels stiffened at early time points resulting in improved blood flow, while those stiffened at later time points had the opposite effect. We hypothesized that this was due to differential impacts of focal adhesion kinase (FAK) activation following in situ stiffening, as supplementation with a Rho kinase inhibitor, downstream of FAK, partially reversed the effects of in situ stiffening. Taken together, this research demonstrates the benefits of incorporating dynamic cues into hydrogel design to create more physiologically relevant vasculature.

bioengineering↗

bioRxiv2025

Tuning Scaffold Degradation with Non-Natural Peptidomimetics to Control Human Umbilical Vein Endothelial Cell Morphology and Vessel Formation

The in vitro vascularization of 3D tissue constructs, such as hydrogels, remains a paramount challenge in tissue engineering. Extracellular matrix degradation and remodeling are key parts of the vascularization process; however, it is difficult to isolate the effects of degradability in both natural and synthetic matrix models. Naturally-derived matrices typically couple degradability to other material properties, whereas synthetic matrices rely on short peptide sequences to impart degradability, which typically exhibit substrate overlap to many proteases. Here, we present a method to independently and broadly tune 3D hydrogel degradation using crosslinkers with non-natural peptoid (N-substituted glycine) substitutions. Increased peptoid substitutions reduced hydrogel degradability to collagenases without altering hydrogel modulus, swelling ratio, or crosslinker length. Using this approach, human umbilical vein endothelial cells (HUVECs) encapsulated in more degradable hydrogels proliferated more, formed more vessels, exhibited higher metabolic activity, and secreted more extracellular matrix than HUVECs encapsulated in less degradable or non-degradable hydrogels. Interestingly, HUVECs encapsulated in the least degradable hydrogels secreted significantly higher matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) than HUVECs encapsulated in the most degradable hydrogels, suggesting higher MMP secretion to compensate for the reduced matrix degradability. Overall, this work highlights the importance of protease-mediated remodeling on vascularization and suggests that peptoid substitutions are effective for tuning hydrogel degradability for a variety of 3D cell applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/684851v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@c22b3corg.highwire.dtl.DTLVardef@1a4ae14org.highwire.dtl.DTLVardef@a0ad1forg.highwire.dtl.DTLVardef@c5def5_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Regulation of TCR-induced Transcriptional Kinetics by Interleukin 2-inducible T Cell Kinase (ITK) in CD8+ T cells

The initial activation of naive CD8+ T cells induces three major transcription factor pathways, NFAT, NF{kappa}B, and AP-1, predominantly regulated by T-cell receptor (TCR) signaling. Downstream of the TCR, the Tec family tyrosine kinase ITK modulates the transcriptional response in T cells by differentially affecting the kinetics and magnitude of activation of these three transcription factors. How signaling through ITK regulates the duration and/or termination of TCR signaling has not been investigated. To address this, we utilized the "Nr4a3-Tocky" reporter mouse which provides information on the kinetics of TCR signaling over time courses from hours to days. OT-I CD8+ Nr4a3-Tocky T cells were stimulated with peptide ligands of varying affinities, and cells were assayed for a panel of surface and intracellular markers of activation and differentiation at multiple time points post-stimulation. As previously reported, at early time points ITK signaling enhanced the kinetics and magnitude of expression of these markers. At later time points the absence of ITK led to persistent expression of several proteins, indicating an important role for ITK in the termination of TCR-dependent transcriptional responses. Using a dual ITK/ RLK inhibitor (PRN694) at 24h post OVA peptide stimulation it was found that this function of ITK was required in the first 24 hours. These findings reveal a possible key negative regulatory mechanism that is programmed in the first day of activation but impacts CD8+ T cell gene expression patterns days later. One Sentence Summary StatementThe Tec family tyrosine kinase ITK differentially regulates early and late T-cell receptor transcriptional responses.

immunology↗

bioRxiv2025

Myeloid cell IL-15 production in the brain supports Bystander CD8+ T-Cell Neuropathic Immune Responses following Virus infection

The central nervous system (CNS) includes a uniquely regulated immune response that supports homeostasis, response to injury, and response to pathogens. Recent work has shown that virus-associated immune responses in the CNS may contribute to neuronal injury and long-term outcomes such as neurocognitive decline. However, the fundamental mechanisms that regulate acute infiltration of immune cells from vascular compartments into the CNS are not well defined. Using an attenuated Venezuelan equine encephalitis virus TC83 (referred to as TC83) to inoculate using olfactory and intracranial injections, we show that infection in the CNS and olfactory pathways results in rapid infiltration of both CD4+ and CD8+ T-cells as early as 3- and 5-days post-infection. CNS-infiltrating CD8+ T-cells exhibit a bystander, memory phenotype (CD49d+, Tbet+, NKG2D+, Eomes+), are cytotoxic, and are recruited independent of antigen specific responses. We show that infiltration of CD8+ bystander T-cells is supported by microglia and infiltrating macrophage expression of IL-15 and interferon expression in the CNS. These innate antiviral immune signals support activation of bystander CD8+ T-cells in the CNS that contribute to tissue injury independent of virus replication at early time points post-infection. These data support a mechanism by IL-15 stimulates bystander memory CD8+ T-cells to enter the CNS and contribute to injury independent of antigen-specific stimulation. ImportancePrior studies have shown that virus infections in the respiratory and olfactory nerve pathways can result in long term injury in the brain. However, the mechanisms that link virus infection in the olfactory neurons and brain injury are not understood. We show that virus infection of olfactory neurons results in immune stimulation in the brain of resident immune cells to release a cytokine called IL-15 and interferon. This results in infiltration and activation of non-specific T-cells that cause injury of neurons in the brain. This may be an important mechanism by which respiratory viruses and other viruses cause inflammation and injury in the brain.

immunology↗

bioRxiv2025

Spatial and longitudinal tracking of enhancer-AAV vectors that target transgene expression to injured mouse myocardium

Tissue regeneration enhancer elements (TREEs) direct expression of target genes in injured and regenerating tissues. Additionally, TREEs of zebrafish origin were shown to direct the expression of transgenes in border zone regions after cardiac injury when packaged into recombinant AAV vectors and introduced into mice. Future implementation of TREEs into AAV-based vectors as research tools and potential gene therapy modalities will require a more detailed understanding of expression dynamics and potential off-target effects. Here, we applied in vivo bioluminescent imaging to mice systemically injected with AAV vectors containing different combinations of capsids, enhancers, and timing of delivery. Longitudinal tracking of TREE-based expression over time revealed distinct amplitudes and durations of reporter gene expression in the injured heart directed by different TREEs. The liver-de-targeted AAV capsid, AAV.cc84, was able to deliver TREEs either pre- or post-cardiac injury to negate off-target expression in the liver while maintaining transduction in the heart. By screening AAV9-based capsid libraries dosed systemically in mice post-cardiac injury, we discovered a new capsid variant, AAV.IR41, with enhanced transduction at cardiac border zone regions, and with elevated transduction of TREE driven transgenes versus conventional AAV9 vectors. In vivo bioluminescence imaging offers insights into how enhancers and engineered capsids can be implemented to modulate spatiotemporal transgene expression for future targeted therapies.

developmental biology↗

bioRxiv2025

Matrix Stiffness Regulates Mechanotransduction and Vascular Network Formation of hiPSC-Derived Endothelial Progenitors Encapsulated in 3D Hydrogels

The mechanical properties of the extracellular matrix (ECM), particularly stiffness, regulate endothelial progenitor responses during vascular development, yet their behavior in physiologically compliant matrices (<1 kPa) remains underexplored. Using norbornene-modified hyaluronic acid (NorHA) hydrogels with tunable stiffness (190-884 Pa), we investigated how hydrogel stiffness influences cell morphology, endothelial maturation, mechanotransduction, and microvascular network formation in human induced pluripotent stem cell-derived endothelial progenitors (hiPSC-EPs). Our findings reveal a stiffness-dependent tradeoff between mechanotransduction and vascular network formation. At intermediate stiffness (551 Pa), cells exhibited the greatest increase in endothelial marker CD31 expression and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) nuclear translocation, indicating enhanced mechanotransduction and endothelial maturation. However, this did not translate to superior plexus formation. Instead, the most compliant matrix (190 Pa) supported greater vascular connectivity, characterized by longer branches ([~]0.03/volume vs. 0.015 at 551 Pa) and enhanced actin remodeling. 3D cell contraction measurements revealed a 15.6-fold higher basal displacement in compliant hydrogels, suggesting that cell-generated forces and matrix deformability collectively drive vascular morphogenesis. Unlike prior studies focusing on pathological stiffness ranges (>10 kPa), our results emphasize that vascularization is not solely driven by the most mechanotransductive environment but rather by a balance of compliance, contractility, and cell-induced remodeling. These findings underscore the need to design hydrogels that provide sufficient mechanotransduction for endothelial maturation while maintaining compliance to support dynamic vascular morphogenesis. This work provides a mechanically tuned framework for optimizing microenvironments to balance endothelial differentiation and vascular network formation in tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2024

Multi-omics analysis of a traditional fermented food reveals a byproduct-associated subpopulation of Neurospora intermedia for waste-to-food upcycling

Fungal solid-state fermentation (SSF) of byproducts has promise for increasing food sustainability and security, but fungal waste-to-food upcycling remains poorly understood at the molecular level. Here we use a multi-omics approach to characterize oncom - a fermented food traditionally produced from byproducts in Java, Indonesia - as a model system for understanding fungal waste conversion. Metagenomic sequencing of two oncom types (red and black) indicated that Neurospora intermedia is the fungus dominating red oncom. Further transcriptomic, metabolomic, and phylogenomic analysis revealed that oncom-derived N. intermedia utilizes pectin and cellulose degradation for substrate conversion and belongs to a distinct byproduct-associated subpopulation that differs from wild strains at the genetic and biochemical level. Finally, we found that N. intermedia grew on a range of industrially relevant byproducts, did not encode for any known mycotoxins, and could be used to create foods that were positively perceived by consumers outside Indonesia. This study uncovers the microbial and genetic basis of a traditional upcycled food, sheds light on human domestication of microbes for sustainability challenges, and establishes the edible N. intermedia as a promising fungus for byproduct upcycling in SSF and beyond.

microbiology↗

bioRxiv2024

Repurposing CRISPR-Cas13 systems for robust mRNA trans-splicing

Type VI CRISPR enzymes have been developed as programmable RNA-guided Cas proteins for eukaryotic RNA editing. Notably, Cas13 has been utilized for site-targeted single base edits, demethylation, RNA cleavage or knockdown and alternative splicing. However, the ability to edit large stretches of mRNA transcripts remains a significant challenge. Here, we demonstrate that CRISPR-Cas13 systems can be repurposed to assist trans-splicing of exogenous RNA fragments into an endogenous pre-mRNA transcript, a method termed CRISPR Assisted mRNA Fragment Trans-splicing (CRAFT). Using split reporter-based assays, we evaluate orthogonal Cas13 systems, optimize guide RNA length and screen for optimal trans-splicing site(s) across a range of intronic targets. We achieve markedly improved editing of large 5 and 3 segments in different endogenous mRNAs across various mammalian cell types compared to other spliceosome-mediated trans-splicing methods. CRAFT can serve as a versatile platform for attachment of protein tags, studying the impact of multiple mutations/single nucleotide polymorphisms, modification of untranslated regions (UTRs) or replacing large segments of mRNA transcripts.

bioengineering↗

bioRxiv2023

Gene Expression Profiling Unveils the Temporal Dynamics of CIGB-300-Regulated Transcriptome in AML Cells

Protein kinase CK2 activity is implicated in the pathogenesis of various hematological malignancies like Acute Myeloid Leukemia (AML) that remains challenging concerning treatment. Consequently, here we used Illumina HT-12 microarray gene RNA expression profiling to study the molecular events that might support the anti-leukemic effect of CIGB-300 peptide which targets both CK2 substrates and the CK2 catalytic subunits on HL-60 and OCI-AML3 cell lines. As a result, 185 and 812 genes appeared significantly modulated in HL-60 cells at 30 min and 3 h of incubation with CIGB-300 for p< 0.01 and FC>[boxv]1.5[boxv], respectively; while 222 and 332 genes appeared modulated in OCI-AML3 cells. Importantly, functional enrichment analysis evidenced that genes and transcription factors related to apoptosis, cell cycle, leukocyte differentiation, signaling by cytokines/interleukins, and NF-kB, TNF signaling pathways were significantly represented in AML cells transcriptomic profiles. The influence of CIGB-300 on these biological processes and pathways is dependent on the cellular background, in first place, and treatment duration. Of note, the impact of the peptide on NF-kB signaling was corroborated by the quantification of selected NF-kB target genes, as well as the measurement of p50 binding activity and soluble TNF- induction. Quantification of CSF1/M-CSF and CDKN1A/P21 by PCR supports peptide effects on differentiation and cell cycle. Overall, here we explore for the first time the temporal dynamics of the gene expression profile regulated by CIGB-300 and provide fresh molecular clues concerning the antineoplastic effect of CIGB-300 in two relevant AML backgrounds.

cancer biology↗

bioRxiv2022

Crosslinker Structure Modulates Bulk Mechanical Properties and Dictates hMSC Behavior on Hyaluronic Acid Hydrogels

Synthetic hydrogels are attractive platforms due in part to their highly tunable mechanics, which impact cell behavior and secretory profile. These mechanics are often controlled by altering the number of crosslinks or the total polymer concentration in the gel, leading to structure-property relationships that inherently couple network connectivity to the overall modulus. In contrast, the native extracellular matrix (ECM) contains structured biopolymers that enable stiff gels even at low polymer content, facilitating 3D cell culture and permeability of soluble factors. To mimic the hierarchical order of natural ECM, this work describes a synthetic hydrogel system in which mechanics are tuned using the structure of sequence-defined peptoid crosslinkers, while fixing network connectivity. Peptoid crosslinkers with different secondary structures are investigated: 1) a helical, molecularly stiff peptoid, 2) a non-helical, less stiff peptoid, and 3) an unstructured, relatively flexible peptoid. Bulk hydrogel storage modulus increases when crosslinkers of higher chain stiffness are used. In-vitro studies assess the viability, proliferation, cell morphology, and immunomodulatory activity of human mesenchymal stem cells (hMSCs) on each hydrogel substrate. Matrix mechanics regulate the morphology of hMSCs on the developed substrates, and all of the hydrogels studied upregulate IDO production over culture on TCP. Softer substrates further this upregulation to a plateau. Overall, this system offers a biomimetic strategy for decoupling hydrogel storage modulus from network connectivity, enabling systematic study of biomaterial properties on hMSC behavior and enhancement of cellular functionality for therapeutic applications. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/502671v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@11c5785org.highwire.dtl.DTLVardef@1b60763org.highwire.dtl.DTLVardef@19f2acdorg.highwire.dtl.DTLVardef@b31021_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

Quantification of iPSC-derived vascular networks in novel phototunable angiogenic hydrogels

Vascularization of engineered scaffolds remains a critical obstacle hindering the translation of tissue engineering from the bench to the clinic. Previously, we demonstrated the robust micro-vascularization of collagen hydrogels with induced pluripotent stem cell (iPSC)-derived endothelial progenitors; however, physically cross-linked collagen hydrogels compact rapidly and exhibit limited strength. To address these challenges, we synthesized an interpenetrating polymer network (IPN) hydrogel comprised of collagen and norbornene-modified hyaluronic acid (NorHA). This dual-network hydrogel combines the natural cues presented by collagens binding sites and extracellular matrix (ECM)-mimicking fibrous architecture with the in situ modularity and chemical cross-linking of NorHA. We modulated the stiffness and degradability of this novel IPN hydrogel by varying the concentration and sequence, respectively, of the NorHA peptide cross-linker. Rheological characterization of the photo-mediated gelation process revealed that the stiffness of the IPN hydrogel increased with cross-linker concentration and was decoupled from the bulk NorHA content. Conversely, the swelling of the IPN hydrogel decreased linearly with increasing cross-linker concentration. Collagen microarchitecture remained relatively unchanged across cross-linking conditions, although the mere addition of NorHA delayed collagen fibrillogenesis. Upon iPSC-derived endothelial progenitor encapsulation, robust, lumenized microvascular networks developed in IPN hydrogels over two weeks. Subsequent computational analysis showed that an initial rise in stiffness increased the number of branch points and vessels, but vascular growth was suppressed in high stiffness IPN hydrogels. These results suggest that an IPN hydrogel consisting of collagen and NorHA is highly tunable, compaction resistant, and capable of stimulating angiogenesis. STATEMENT OF SIGNIFICANCEWe have synthesized the first tunable collagen and norbornene functionalized hyaluronic acid (NorHA) interpenetrating polymer network hydrogel. This unique biomaterial allows for control over hydrogel stiffness, independent of the total polymer concentration, by varying the concentration of a peptide cross-linker and was specifically designed to produce a biomimetic vasculogenic microenvironment. Using the system, we performed a detailed study of the vasculogenesis of induced pluripotent stem cell-derived (iPSC) endothelial progenitors, a poorly studied cell source with considerable therapeutic potential. Our results show that vascular growth can be tuned by altering the stiffness and degradability of the scaffolds independently. Finally, we improved upon our open-source computational pipeline programmed in ImageJ and MATLAB to further quantify vascular topologies in three dimensions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/259630v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1d3ee04org.highwire.dtl.DTLVardef@af739aorg.highwire.dtl.DTLVardef@cec470org.highwire.dtl.DTLVardef@c2ae18_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗