Biology subjects

Alvarez, S.

Publications and source records attributed to Alvarez, S..

At least 19 recordsLinked to original sources

bioRxiv2026

3D-bioprinted skeletal muscle with tailored myofibre architecture

Engineered skeletal muscle tissue (eSMT) contractile function strongly depends on the alignment and orientation of its myofibres, as force generation and transmission are intrinsically linked to its microstructural architecture. However, fabricating truly functional eSMT with controlled myofibre architecture remains a major challenge. Here, we present a method that combines extrusion-based 3D bioprinting with custom multi-pillar anchoring systems to guide myofibre orientation. Using this approach, we successfully fabricated functional eSMTs with parallel, fusiform, bipennate, and circular architectures. We then integrated these basic designs into a functional system comprising multiple 3D-bioprinted eSMTs replicating the morphology and myofibre architecture of facial musculature. By recapitulating native muscle architecture, this approach advances the engineering of functional eSMT for multiple biomedical applications and establishes a foundation for next-generation biohybrid robotics.

bioengineering↗

bioRxiv2026

Nasal postbiotic therapy restores NALT architecture and enhances respiratory innate immunity in protein-malnourished mice

BackgroundMalnutrition compromises mucosal immunity, especially in the respiratory tract, increasing susceptibility to pathogens like Streptococcus pneumoniae. This study assessed whether nasal administration of Lacticaseibacillus rhamnosus CRL1505 or its peptidoglycan could promote the recovery of nasopharynx-associated lymphoid tissue (NALT) structure and functionality, thereby enhancing resistance to S. pneumoniae infection in protein-malnourished mice. MethodsMale Swiss albino mice were fed to a protein-free diet to induce malnutrition, followed by nutritional repletion with or without nasal supplementation of CRL1505 or its peptidoglycan. Resistance to S. pneumoniae infection, NALT architecture, immune cell composition in NALT and regional lymph nodes, and nasal cytokine production were evaluated. ResultsProtein deficiency caused marked NALT atrophy, immune cell depletion, and heightened susceptibility to S. pneumoniae. Nutritional repletion alone partially reversed these effects. In contrast, nasal supplementation with CRL1505 or its postbiotic fully restored NALT structure and cellularity, normalized lymphoid and myeloid populations, and reduced pathogen burden. Both treatments increased B and T lymphocytes, immature B cells, dendritic cells, and macrophages. The postbiotic also enhanced MHCII expression and balanced neutrophil-like Gr-1 cells. Notably, immune enhancement was evident even before infection, indicating a mucosal priming effect. Cytokine levels in nasal fluids remained largely unchanged. ConclusionsNasal delivery of L. rhamnosus CRL1505 or its postbiotic effectively reestablished NALT integrity and mucosal immunity in malnourished mice, providing significant protection against respiratory pathogens. These findings support the development of nasal immunobiotic formulations as non-invasive interventions to bolster respiratory defenses in immunocompromised hosts.

immunology↗

bioRxiv2025

Unraveling novel insights into dual-species cariogenic biofilm formation: a comparative analysis on natural vs artificial bioengineered dentin models

Dental caries is the most prevalent biofilm-associated disease affecting billions of people worldwide, including elderly individuals. Conventional biofilm study methods rely on human or animal-derived samples, posing challenges regarding accessibility, cost, and ethical considerations. While in-vitro systems offer a promising alternative, they often fail to replicate the structural characteristics of dentin, which play a crucial role in bacterial adhesion. To bridge this gap, a bioengineered dentin-like construct has recently been developed as a reproducible and accessible model for studying biofilm formation associated with dental aging. Therefore, this study aimed to assess dual-species Streptococcus mutans and Candida albicans biofilm formation on bioengineered dentin substrates and compare it to biofilm formation on natural human aged dentin. For this, S. mutans UA159 and C. albicans (ATCC 90028) were co-cultured on bioengineered and natural dentin slabs, and polymicrobial biofilm formation and EPS production were characterized via high-resolution confocal laser scanning microscopy. Following biofilm formation, image processing was conducted using COMSTAT software to determine biofilm growth parameters. Additionally, fluorescence intensity was quantified via microplate readings, and cell viability was assessed using a Live/Dead viability kit. Overall, results showed similar biofilm formation patterns between the bioengineered and aged dentin, with no significant differences found in biofilm physical properties or viability. These findings suggest that this bioengineered dentin system provides a reliable platform for studying biofilm formation in the context of dental aging, making it a valuable tool for investigating microbial adhesion and cariogenic biofilm development under controlled conditions, potentially facilitating future research in biofilm-related oral diseases. ImportanceDental caries is one of the most common chronic diseases worldwide, which is driven by complex microbial biofilms formed on the tooths surface. However, existing models for studying these biofilms in the laboratory often rely on human or animal tissues, which are difficult to obtain and standardize, and present ethical challenges. In this study, we validate a bioengineered dentin-like model that accurately mimics the microarchitecture of aged human dentin, a key site for root caries in the elderly. By comparing biofilms formed by the clinically significant Streptococcus mutans and Candida albicans on both artificial and natural substrates, we show that the engineered model supports biofilm development under comparable parameters and enables detection of changes in microbial virulence. Overall, this platform provides a reproducible and scalable alternative for studying oral biofilms with potential applications in understanding disease pathogenesis, novel treatment testing, and integration into next-generation organ-on-a-chip systems.

bioengineering↗

bioRxiv2024

Peripheral immune cell response to stimulation stratifies Parkinson's disease progression from prodromal to clinical stages

The motor stage of idiopathic Parkinsons disease (iPD) can be preceded for years by a prodromal stage characterized by non-motor symptoms like REM sleep behavior disorder (RBD). Here, we show that multiple stages of iPD, including the pre-motor prodromal stage, can be stratified according to the inflammatory and immunometabolic responses to stimulation of peripheral blood mononuclear cells ex vivo. We identified increased stimulation-dependent secretion of TNF, IL-1{beta}, and IL-8 in monocytes from RBD patients and showed diminished proinflammatory cytokine secretion in monocytes and T cells in early and moderate stages of PD. Mechanistically, immune activation revealed deficits in CD8+ T-cell mitochondrial health in moderate PD, and relative mitochondrial health in CD8+ T cells was positively correlated with stimulation-dependent T-cell cytokine secretion across the PD spectrum. Dysregulated immunometabolism may drive peripheral inflammation and PD progression, and ex vivo stimulation-based assays have potential to reveal novel biomarkers for patient stratification and progression with immune endophenotypes.

neuroscience↗

bioRxiv2024

Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14{Delta}) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14{Delta} using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14{Delta}-GFP using live cell imaging. We benchmarked out MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was > 15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.

cancer biology↗

bioRxiv2024

Acute inhibition of iron-sulfur cluster biosynthesis disrupts metabolic flexibility in mice

Iron-sulfur clusters (ISCs) are cell-essential cofactors present in [~]60 proteins including subunits of OXPHOS complexes I-III, DNA polymerases, and iron-sensing proteins. Dysfunctions in ISC biosynthesis are associated with anemias, neurodegenerative disorders, and metabolic diseases. To assess consequences of acute ISC inhibition in a whole body setting, we developed a mouse model in which key ISC biosynthetic enzyme NFS1 can be acutely and reversibly suppressed. Contrary to in vitro ISC inhibition and pharmacological OXPHOS suppression, global NFS1 inhibition rapidly enhances lipid utilization and decreases adiposity without affecting caloric intake and physical activity. ISC proteins decrease, including key proteins involved in OXPHOS (SDHB), lipoic acid synthesis (LIAS), and insulin mRNA processing (CDKAL1), causing acute metabolic inflexibility. Age-related metabolic changes decelerate loss of adiposity substantially prolonged survival of mice with NFS1 inhibition. Thus, the observation that ISC metabolism impacts organismal fuel choice will aid in understanding the mechanisms underlying ISC diseases with increased risk for diabetes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/608291v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@863cbaorg.highwire.dtl.DTLVardef@fce90borg.highwire.dtl.DTLVardef@27df04org.highwire.dtl.DTLVardef@1a2a9bf_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Acute ISC inhibition leads to rapid loss of adiposity in mice - Multi-metabolic pathway disruption upon ISC deficiency blocks energy storage - Nfs1 inhibition induces glucose dyshomeostasis due to ISC deficiency in {beta}-cells - Energy distress caused by inhibition of ISC synthesis is attenuated in aged mice

physiology↗

bioRxiv2023

Netrin1 patterns the dorsal spinal cord through modulation of Bmp signaling

We have identified an unexpected role for netrin1 as a suppressor of bone morphogenetic protein (Bmp) signaling in the developing dorsal spinal cord. Using a combination of gain- and loss-of-function approaches in chicken, embryonic stem cell (ESC), and mouse models, we have observed that manipulating the level of netrin1 specifically alters the patterning of the Bmp-dependent dorsal interneurons (dIs), dI1-dI3. Altered netrin1 levels also change Bmp signaling activity, as measured by bioinformatics, and monitoring phosophoSmad1/5/8 activation, the canonical intermediate of Bmp signaling, and Id levels, a known Bmp target. Together, these studies support the hypothesis that netrin1 acts from the intermediate spinal cord to regionally confine Bmp signaling to the dorsal spinal cord. Thus, netrin1 has reiterative activities shaping dorsal spinal circuits, first by regulating cell fate decisions and then acting as a guidance cue to direct axon extension.

developmental biology↗

bioRxiv2023

Microfabrication-based engineering of biomimetic dentin-like constructs to simulate dental aging

1.Human dentin is a highly organized dental tissue displaying a complex microarchitecture consisting of micrometer-sized tubules encased in a mineralized type-I collagen matrix. As such, it serves as an important substrate for the adhesion of microbial colonizers and oral biofilm formation in the context of dental caries disease, including root caries in the elderly. Despite this issue, there remains a current lack of effective biomimetic in-vitro dentin models that facilitate the study of oral microbial adhesion by considering the surface architecture at the micro- and nanoscales. Therefore, the aim of this study was to develop a novel in-vitro microfabricated biomimetic dentin surface that simulates the complex surface microarchitecture of exposed dentin. For this, a combination of soft lithography microfabrication and biomaterial science approaches were employed to construct a micropitted PDMS substrate functionalized with mineralized type-I collagen. These dentin analogues were subsequently glycated with methylglyoxal (MGO) to simulate dentin matrix aging in-vitro and analyzed utilizing an interdisciplinary array of techniques including atomic force microscopy (AFM), elemental analysis, and electron microscopy. AFM force-mapping demonstrated that the nanomechanical properties of the biomimetic constructs were within the expected biological parameters, and that mineralization was mostly predominated by hydroxyapatite deposition. Finally, dual-species biofilms of Streptococcus mutans and Candida albicans were grown and characterized on the biofunctionalized PDMS microchips, demonstrating biofilm specific morphologic characteristics and confirming the suitability of this model for the study of early biofilm formation under controlled conditions. Overall, we expect that this novel biomimetic dentin model could serve as an in-vitro platform to study oral biofilm formation or dentin-biomaterial bonding in the laboratory without the need for animal or human tooth samples in the future.

bioengineering↗

bioRxiv2023

Nitrogen starvation leads to TOR kinase-mediated downregulation of fatty acid synthesis in the algae Chlorella sorokiniana and Chlamydomonas reinhardtii

The accumulation of triacylglycerol (TAG) as a storage compound in eukaryotic algae has been the subject of extensive studies over the last 50 years. The model industrial alga Chlorella sorokiniana accumulates TAG and other storage compounds under nitrogen (N)-limited growth. Previously we used transcriptomics to explore the regulation of TAG synthesis in C. sorokiniana. Surprisingly, our analysis showed that the expression of several key genes encoding enzymes involved in plastidic fatty acid synthesis are significantly repressed. Metabolic labeling with radiolabeled acetate showed that de novo fatty acid synthesis is indeed downregulated under N-limitation. Likewise, inhibition of the Target of Rapamycin kinase (TOR), a key regulator of metabolism and growth, decreased fatty acid synthesis. We compared the changes in proteins and phosphoprotein abundance using a proteomics and phosphoproteomics approach in C. sorokiniana cells under N-limitation or TOR inhibition and found extensive overlap between the N-limited and TOR-inhibited conditions. We also identified changes in the phosphorylation levels of TOR complex proteins, TOR-kinase and RAPTOR, under N-limitation, indicating that TOR signaling is altered. Our results indicate that under N-limitation there is significant metabolic remodeling, including fatty acid synthesis, mediated by TOR signaling. We find that TOR-mediated metabolic remodeling of fatty acid synthesis under N-limitation is conserved in the chlorophyte algae Chlorella sorokiniana and Chlamydomonas reinhardtii.

plant biology↗

bioRxiv2023

Bimodality in Ras signaling originates from processivity of the Ras activator SOS without classic kinetic bistability

Ras is a small GTPase that is central to important functional decisions in diverse cell types. An important aspect of Ras signaling is its ability to exhibit bimodal, or switch-like activity. We describe the total reconstitution of a receptor-mediated Ras activation-deactivation reaction catalyzed by SOS and p120-RasGAP on supported lipid membrane microarrays. The results reveal a bimodal Ras activation response, which is not a result of classic kinetic bistability, but is rather driven by the distinct processivity of the Ras activator, SOS. Furthermore, the bimodal response is controlled by the condensation state of the scaffold protein, LAT, to which SOS is recruited. Processivity-driven bimodality leads to stochastic bursts of Ras activation even under strongly deactivating conditions. This behavior contrasts classic kinetic bistability and is distinctly more resistant to pharmacological inhibition.

biophysics↗

bioRxiv2023

In vivo functional phenotypes from a computational epistatic model of evolution

Computational models of evolution are valuable for understanding the dynamics of sequence variation, to infer phylogenetic relationships or potential evolutionary pathways and for biomedical and industrial applications. Despite these benefits, few have validated their propensities to generate outputs with in vivo functionality, which would enhance their value as accurate and interpretable evolutionary algorithms. We demonstrate the power of epistasis inferred from natural protein families to evolve sequence variants in an algorithm we developed called Sequence Evolution with Epistatic Contributions. Utilizing the Hamiltonian of the joint probability of sequences in the family as fitness metric, we sampled and experimentally tested for in vivo {beta}-lactamase activity in E. coli TEM-1 variants. These evolved proteins can have dozens of mutations dispersed across the structure while preserving sites essential for both catalysis and interactions. Remarkably, these variants retain family-like functionality while being more active than their WT predecessor. We found that depending on the inference method used to generate the epistatic constraints, different parameters simulate diverse selection strengths. Under weaker selection, local Hamiltonian fluctuations reliably predict relative changes to variant fitness, recapitulating neutral evolution. SEEC has the potential to explore the dynamics of neofunctionalization, characterize viral fitness landscapes and facilitate vaccine development.

evolutionary biology↗

bioRxiv2023

Multiplex single-cell chemical genomics reveals the kinase dependence of the response to targeted therapy

Chemical genetic screens are a powerful tool for exploring how cancer cells response to drugs is shaped by their mutations, yet they lack a molecular view of the contribution of individual genes to the response to exposure. Here, we present sci-Plex-Gene-by-Environment (sci-Plex-GxE), a platform for combined single-cell genetic and chemical screening at scale. We highlight the advantages of large-scale, unbiased screening by defining the contribution of each of 522 human kinases to the response of glioblastoma to different drugs designed to abrogate signaling from the receptor tyrosine kinase pathway. In total, we probed 14,121 gene-by-environment combinations across 1,052,205 single-cell transcriptomes. We identify an expression signature characteristic of compensatory adaptive signaling regulated in a MEK/MAPK-dependent manner. Further analyses aimed at preventing adaptation revealed promising combination therapies, including dual MEK and CDC7/CDK9 or NF-kB inhibitors, as potent means of preventing transcriptional adaptation of glioblastoma to targeted therapy.

genomics↗

bioRxiv2022

Extracellular Vesicles derived from Apis mellifera Royal Jelly promote wound healing by modulating inflammation and cellular responses

Apis mellifera Royal Jelly (RJ) is a well-known remedy in traditional medicine around the world and its versatile effects range from antibacterial to anti-inflammatory properties and pro-regenerative properties. Several active compounds have been identified, however, the mechanisms of action still remain widely unknown. As a glandular product, RJ has been shown to contain a substantial number of extracellular vesicles (EVs) and in this study, we aimed to investigate the extent of involvement of RJEVs in wound healing associated effects. Molecular analysis of RJEVs verified the presence of important conserved exosomal markers such as CD63 and syntenin, as well as cargo molecules MRJP1, defensin-1 and jellein-3. RJEV internalization analysis demonstrated the involvement of membrane fusion as well as macropinocytosis or clathrin-dependent endocytosis into mammalian cells. Furthermore, RJEVs have demonstrated to modulate MSCs differentiation and secretome, as well as decrease LPS-induced inflammation in RAW 264.7 macrophages by blocking the MAPK pathway. In vivo studies confirmed anti-bacterial effects of RJEVs, and demonstrated an acceleration of wound healing in a splinted mouse model. Summarizing, this study suggests that RJEVs of potentially exosomal origin play a crucial role in the known effects of RJ by modulating the inflammatory phase and cellular response in wound healing.

bioengineering↗

bioRxiv2022

Variation in morpho-physiological and metabolic responses to low nitrogen stress across the sorghum association panel

Access to biologically available nitrogen is a key constraint on plant growth in both natural and agricultural settings. Variation in tolerance to nitrogen deficit stress and productivity in nitrogen limited conditions exists both within and between plant species. Here we quantified variation in the metabolic, physiological, and morphological responses of a sorghum association panel assembled to represent global genetic diversity to long term, moderate, nitrogen deficit stress and the relationship of these responses to grain yield under both conditions. Grain yield exhibits substantial genotype by environment interaction while many other morphological and physiological traits exhibited consistent responses to nitrogen stress across the population. Large scale nontargeted metabolic profiling for a subset of lines in both conditions identified a range of metabolic responses to long term nitrogen deficit stress as well as several metabolites associated with variation in the degree of yield plasticity specific sorghum genotypes exhibited in response to nitrogen deficit stress.

plant biology↗

bioRxiv2022

Functional and structural deficiencies of Gemin5 variants associated with neurological disease

Dysfunction of RNA-binding proteins are often linked to a wide range of human disease in general, and particularly with neurological conditions. Gemin5 is a member of the survival of motor neuron (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 protein have been reported but the functional consequences of these variants remain elusive. Here we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)-like dimerization module and the non-canonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization while the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, abolishing the cap-dependent and selective translation control of Gemin5, and establishing a functional difference with the wild type protein. Our study provides a molecular basis of disease associated with malfunction of Gemin5 protein.

molecular biology↗

bioRxiv2022

Functional Requirements for a Samd14-Capping Protein Complex in Stress Erythropoiesis

Acute anemia induces rapid expansion of erythroid precursors and accelerated differentiation to replenish erythrocytes. Paracrine signals - involving cooperation between SCF/c-Kit signaling and other signaling inputs - are required for the increased erythroid precursor activity in anemia. Our prior work revealed that the Sterile Alpha Motif (SAM) Domain 14 (Samd14) gene increases the regenerative capacity of the erythroid system and promotes stress-dependent c-Kit signaling. However, the mechanism underlying Samd14s role in stress erythropoiesis is unknown. We identified a protein- protein interaction between Samd14 and the - and {beta} heterodimers of the F-actin capping protein (CP) complex. Knockdown of the CP {beta} subunit increased erythroid maturation in ex vivo cultures and decreased colony forming potential of stress erythroid precursors. In a genetic complementation assay for Samd14 activity, our results revealed that the Samd14-CP interaction is a determinant of erythroid precursor cell levels and function. Samd14-CP promotes SCF/c-kit signaling in CD71med spleen erythroid precursors. Given the roles of c-Kit signaling in hematopoiesis and Samd14 in c-Kit pathway activation, this mechanism may have pathological implications in acute/chronic anemia.

cell biology↗

bioRxiv2021

A two-component protein condensate of EGFR and Grb2 regulates Ras activation at the membrane

We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the [~]200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and the dimerization of Grb2, which provides the crosslinking element for condensation with EGFR. The Grb2 Y160 residue plays a structurally critical role in dimer formation, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that EGFR condensation controls the ability of SOS to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.

biophysics↗

bioRxiv2021

Natural variation in root exudation of GABA and DIMBOA impacts the maize root endosphere and rhizosphere microbiomes

Root exudates are important for shaping root-associated microbiomes. However, studies on a wider range of metabolites in exudates are required for a comprehensive understanding about their influence on microbial communities. We identified maize inbred lines that differ in exudate concentrations of DIMBOA and GABA using a semi-hydroponic system. These lines were grown in the field to determine the changes in microbial diversity and gene expression due to varying concentrations of DIMBOA and GABA in exudates using 16S rRNA amplicon sequencing and metatranscriptomics. Results showed individual and interaction effects of DIMBOA and GABA on the rhizosphere and root endosphere {beta}-diversity, most strongly at the V10 growth stage. The main bacterial families affected by both compounds were Ktedonobacteraceae and Xanthomonadaceae. Higher concentrations of DIMBOA in exudates affected the rhizosphere metatranscriptome, enriching for KEGG pathways associated with plant disease. This study validated the use of natural variation within plant species as a powerful approach for understanding the role of root exudates on microbiome selection. We also showed that a semi-hydroponic system can be used to identify maize genotypes that differ in GABA and DIMBOA exudate concentrations under field conditions. The impact of GABA exudation on root-associated microbiomes was shown for the first time.

microbiology↗