Biology subjects

D'Adamo, S.

Publications and source records attributed to D'Adamo, S..

6 recordsLinked to original sources

bioRxiv2026

3'tRNAAsp(GTC)-derived fragment links inflammation to post-transcriptional reprogramming in chondrocytes during osteoarthritis.

ObjectiveTransfer RNA-derived fragments (tRFs) belong to an emerging class of small non-coding RNAs that dynamically respond to metabolic stressors and drive different pathological processes, yet their role in osteoarthritis (OA) remains poorly explored. We aimed to define the tRF landscape in OA and investigate the function of 3tRFAsp(GTC) in chondrocyte stress adaptation and translational control. MethodsEx vivo, cartilage specimens from OA patients (n=6) and healthy donors (n=7) were analyzed by small RNA sequencing to define disease-associated tRF signatures. In vitro, primary chondrocytes derived from OA patients were treated with lipopolysaccharide (LPS) to mimic inflammatory environment of OA, used for small RNA sequencing (n=3) and validation analysis (n=6). Functional studies in C28/I2 chondrocytes included antisense oligonucleotide-mediated 3tRFAsp(GTC) inhibition, AGO2-RNA immunoprecipitation (RIP), polysome profiling, stress granule (SG) immunofluorescence, and differential protein analysis. Computational target prediction and pathway enrichment were used to explore tRF-mediated regulatory networks. ResultsBoth OA cartilage and LPS-treated chondrocytes displayed upregulation of 3tRFAsp(GTC) and 5tRFGlu(CTC), indicating a shared inflammatory tRF signature. Predicted targets of upregulated tRFs were enriched in stress-adaptive, proteostasis, and translational control pathways, whereas downregulated tRFs modulated mitochondrial processes. Silencing 3tRFAsp(GTC) inhibited LPS-induced COX2 and MMP13 expression, prevented ER stress, and blocked SG assembly. RIP confirmed selective recruitment of 3tRFAsp(GTC) into AGO2 complexes. Polysome profiling revealed association with 40S ribosomal subunit, mediating translational arrest and influencing selective mRNA expression. Conclusion3tRFAsp(GTC) emerges as a regulator linking inflammation to translational control and SG dynamics in OA. tRFs thus could represent novel therapeutic targets in OA disease.

biochemistry↗

bioRxiv2025

From Light to Lipids: Constraint-Based Metabolic Modeling of Nannochloropsis oceanica Under Light Acclimation Conditions

Oleaginous microalgae, such as Nannochloropsis oceanica, hold strong promise for sustainable lipid bioproduction, but fully realizing this potential requires systems-level insight into their complex metabolism. One of the central challenges in optimizing lipid productivity is the resolution of the highly heterogeneous and incompletely annotated metabolic networks, which respond dynamically to strain-specific traits and cultivation conditions. Here we present iSO1949_N.oceanica, the first genome-scale constraint-based metabolic model (GEM) for this species. Constructed through an orthology-based approach using curated models of related microalgae, the GEM integrates core photosynthetic metabolism and lipid biosynthesis pathways with extensive subcellular localization predictions. To capture environmental dynamics, we introduce two light-acclimation modes derived from continuous cyclostat cultivations, incorporating biomass composition, oxygen exchange, and maintenance rates based on photosynthesis-irradiance curves. Simulations reproduce carbon assimilation under variable light conditions and differentiate acclimated phenotypes. iSO1949_N.oceanica provides a comprehensive framework for exploring photosynthetic metabolism and guiding engineering strategies under photobioreactor-relevant conditions. This resource advances the use of N. oceanica as a chassis for sustainable lipid production and establishes a foundation for systems-level analysis of stramenopile microalgae.

systems biology↗

bioRxiv2025

Design and analysis of synthetic carbon fixation pathways based on novel enzymatic reactions

Biological carbon fixation is currently limited to seven naturally occurring pathways, each with its own limitations and constraints. In recent years, computational analyses of known biochemical reaction networks have identified dozens of theoretical carbon fixation pathways, some of which may have the potential to outperform their natural counterparts. This mix-and-match approach, however, cannot account for those reactions that have not been reported to occur in nature, which heavily limits the possible solution space. Here, we use a bioretrosynthetic approach coupled with expert biochemical knowledge to identify several novel pathways that leverage enzyme promiscuity and the latent biochemical reaction space. We analyze the thermodynamic, stoichiometric, and kinetic parameters of these pathways and compare them to the ubiquitous Calvin-Benson-Bassham cycle and previously proposed synthetic CO2 fixation cycles, highlighting advantages and disadvantages. We identify several promising pathways that could potentially outcompete the Calvin cycle and other previously proposed synthetic CO2 fixation pathways in predicted biomass yield and/or overall pathway activity. In addition, unlike most of the previously proposed efficient mix-and-match pathways, the pathways proposed in this work do not require vitamin B12, which is an advantage for future implementation in plants or microalgae that typically lack B12 biosynthesis. This work highlights the need for enzyme engineering and design in the quest for efficient biological carbon fixation.

biochemistry↗

bioRxiv2025

Modular in vivo engineering of the reductive methylaspartate cycles for synthetic CO2 fixation

Biological carbon fixation is currently limited to seven naturally occurring pathways. Synthetic carbon fixation pathways have the potential to surpass aerobic natural pathways in efficiency, but none have been realized in living cells. Here, we present the reductive methylaspartate cycles (rMASP), a novel family of 4 energy-efficient aerobic CO2 fixation pathway variants. These cycles have the potential to outperform the yields and/or rates of the Calvin cycle and previously proposed synthetic CO2 fixation cycles. To realize these designs, we adopted a modular engineering approach in Escherichia coli. Several pathway modules up to a cascade of 11 enzymes were realized via engineering and evolution. We demonstrate crotonyl-CoA carboxylase dependent growth for both elevated and ambient CO2 conditions, and show in vitro activity of the other CO2-fixing 2-oxoglutarate carboxylase, which requires further activity optimization for mesophilic temperatures. This work demonstrates important steps toward realizing efficient synthetic carbon fixation pathways in living organisms.

synthetic biology↗

bioRxiv2025

A recursive pathway for isoleucine biosynthesis arises from enzyme promiscuity

Enzyme promiscuity can be the starting point for the evolution of new enzymatic activities and pathways. Previously Cotton et al. (2020) identified underground isoleucine biosynthesis routes that can replace the canonical route in Escherichia coli, after they deleted the enzymes that catalyze the formation of its precursor 2-ketobutyrate. Using this strain and short-term evolution we identify a new pathway for isoleucine biosynthesis based on the promiscuous activity of the native enzyme acetohydroxyacid synthase II. We demonstrate that this enzyme catalyzes the previously unreported condensation of glyoxylate with pyruvate to generate 2-ketobutyrate in vivo. The gene encoding this enzyme, ilvG, is inactivated by a frameshift mutation in the laboratory model strain E. coli K-12 MG1655. Its evolutionary reactivation we report here points to a potential natural role in isoleucine biosynthesis in E. coli. Isoleucine biosynthesis proceeds with a further condensation step of 2-ketobutyrate with pyruvate, again catalyzed by AHAS, giving the proposed pathway the unusual property of recursivity. The discovered enzyme activity uses glyoxylate and pyruvate as direct central metabolic precursors for isoleucine biosynthesis instead of its canonical indirect biosynthesis via the amino acid threonine. Unlike previously discovered underground isoleucine routes by Cotton et al., this route is more likely to play a role in natural isoleucine biosynthesis in E. coli due to the use of ubiquitous metabolites and its activity in aerobic conditions. The discovered route further expands the known metabolic space for isoleucine biosynthesis in E. coli and potentially other organisms, and could find applications in biotechnological isoleucine production.

microbiology↗

bioRxiv2025

5'tRNA-derived fragments modulate β-cell homeostasis and islet macrophage activation in type 2 diabetes

During obesity and type 2 diabetes, pancreatic {beta}-cells face chronic environmental stress, while islet-resident macrophages (iMACs) undergo metabolic reprogramming that exacerbates {beta}-cell dysfunction. Stress-induced cleavage of transfer RNAs (tRNAs) generates tRNA-derived fragments (tRFs), whose role in this context is not fully understood. We identify elevated levels of 5tRFGlu(CTC) and 5tRFGly(GCC) in {beta}-cells and iMACs from db/db mice and in islets from type 2 diabetic patients. Notably, 5tRFGlu(CTC) is also induced under prediabetic conditions and inversely correlates with insulin secretion. Lipotoxic stress triggers their production via Angiogenin-mediated cleavage. Blocking 5tRFGlu(CTC) in islets protects against {beta}-cell apoptosis and restores insulin secretion under palmitate stress. Using a {beta}-cell/macrophage co-culture system, we show that {beta}-cell contact shapes a unique macrophage phenotype (iMAC-like) that shifts upon palmitate exposure--recapitulating in vivo observations. Inhibiting 5tRFGlu(CTC) in iMAC-like cells prevents this activation switch, reduces {beta}-cell stress, and improves insulin secretion. Mechanistically, 5tRFGlu(CTC) interacts with RNA-binding proteins to regulate transcriptional and post-transcriptional pathways linked to immune activation, extracellular matrex remodeling, neurogenesis, and oxidative stress. Our study identifies 5tRFs as key mediators of islet microenvironment remodeling in diabetes, offering new insights into intercellular stress signaling in metabolic disease.

cell biology↗