Biology subjects

Romo, D.

Publications and source records attributed to Romo, D..

11 recordsLinked to original sources

bioRxiv2026

From nest to hatchling: nidobiome assembly and host selection in oviparous vertebrates without parental care

Early microbial assembly is fundamental for host development and fitness, yet in oviparous vertebrates lacking parental care, the mechanisms by which eggs and neonates acquire their microbiome remain poorly understood. The nidobiome concept predicts that maternal and environmental sources, and host selection, collectively shape early microbial communities. To test this, we conducted a longitudinal study on microbial assembly in the yellow-spotted Amazon river turtle (Podocnemis unifilis). We sampled maternal mucosa, freshly laid outer eggshells, and nest sand before incubation, along with hatchery sand, outer and inner eggshells, and hatchling cloaca after incubation. Using 16S and ITS amplicon sequencing to quantify microbial diversity, we observed that freshly laid outer eggshells shared bacterial diversity with maternal mucosa, whereas nest sand harboured a distinct community, providing evidence of early vertical transmission. After incubation, eggshell microbial communities became more even without a change in richness, while nest sand communities exhibited the opposite pattern, reflecting divergent host-environment assembly dynamics. SourceTracker analyses confirmed contributions from both maternal and environmental sources, yet inner eggshells and hatchling cloaca assembled distinct communities compared with outer eggshells, indicating that host selection is largely deterministic. Interkingdom interactions favoured beneficial microorganisms, suggesting active host filtering throughout development. Even in the presence of fusariosis, a widespread threat in turtle nests, host selection enriched protective taxa, linking microbiome composition to hatching success. These findings demonstrate that maternal seeding, priority effects, and deterministic host selection govern early-life microbiome assembly in oviparous reptiles without parental care, with direct implications for microbe-informed conservation strategies. ImportanceUnderstanding early-life microbial assembly is critical for uncovering the ecological and evolutionary drivers of host-microbiome interactions. This study reveals how microbial communities develop in oviparous vertebrates lacking parental care, highlighting key mechanisms such as vertical transmission, host selection, and environmental modulation that shape early-life microbiomes. Our results demonstrate that maternal microbiota seeding and priority effects play fundamental roles in structuring egg-associated microbial communities, directly influencing host fitness and survival. By identifying deterministic processes that enrich beneficial microorganisms during early development, this research underscores the pivotal role of microbiomes in host resilience and adaptation. These insights carry urgent conservation implications, offering microbe-informed strategies to mitigate emerging threats, such as diseases in vulnerable species like the yellow-spotted Amazon river turtle, and emphasising the broader importance of microbiome stewardship for ecosystem health.

ecology↗

bioRxiv2025

Ophiobolin A selectively alters mitochondria, metabolism and redox biology in breast cancer and mammary epithelial cells which have undergone epithelial to mesenchymal transition

Breast cancer progression is facilitated by the epithelial to mesenchymal transition (EMT), generating cancer cells with enhanced metastatic capacity and resistance to chemotherapeutics. EMT is known to impart changes in metabolic pathways and mitochondrial function. Here, we show a natural product possesses EMT-specific cytotoxic activity via alterations in metabolic and mitochondrial functions. The fungus-derived sesterterpenoid, ophiobolin A (OpA), possesses nanomolar cytotoxic activity and a high therapeutic index, although its target and mechanism of action remain unknown. Here, we utilized a model of mammary epithelial cells and breast cancer cell lines with and without EMT features to characterize the mechanism of selectivity towards EMT(+) cells by OpA and to identify novel targets for the treatment of EMT-enriched breast cancer. Proteins interacting with OpA in EMT(+) cells were identified through proteomic studies. We utilized trans-mitochondrial cybrids to determine that mitochondria mediate sensitivity to OpA. Furthermore, we report effects on glycolysis, oxidative metabolism, and disruption of metabolite abundance in the TCA cycle. Alterations to mitochondrial iron concentrations and glutathione production, mediated partly by OpA engagement with the metabolic proteins CISD3 and SLC25A40, were also detected in EMT(+) cells. Antioxidant mechanisms are activated by OpA in EMT(+) cells via the NRF2-ARE pathway, verified by decreased cytotoxicity in EMT(+) cells pretreated with the NRF2 activator CDDO. Collectively, we conclude that OpA selectivity toward EMT is mediated by the mitochondria, and at sub-cytotoxic levels, generates a metabolic shift leading to cell death countered by antioxidant mechanisms.

cancer biology↗

bioRxiv2025

PANoptosis, a combination of inflammatory cell death mechanisms, induced 1 by Ophiobolin A in breast cancer cell lines

An unmet challenge in managing breast cancer is treatment failure due to resistance to apoptosis-inducing chemotherapies. Thus, it is important to identify novel non-apoptotic therapeutic agents. Several non-apoptotic programmed cell death pathways utilize specific cellular signaling events to trigger lytic and pro-inflammatory cell death. PANoptosis, which encompasses pyroptosis, apoptosis and necroptosis, is of paramount importance in the regulation of cell death and immune responses. Our study illustrates that ophiobolin A (OpA) is an anti-cancer agent that triggers lytic cell death in breast cancer cells, including triple-negative breast cancer (TNBC), via a mechanism dependent on RIPK1. This study reveals that OpA induces typical pyroptosis-like characteristics, including cellular swelling, plasma membrane rupture, GSDMD cleavage and release of cytokines in breast cancer cells. The involvement of caspase 3, RIPK1, and GSDMD suggests that PANoptosis is activated upon OpA treatment in breast cancer. The induction of pro-inflammatory cell death suggests potential applications for OpA in cancer treatment.

cancer biology↗

bioRxiv2024

Combined HDAC and eIF4A inhibition: A novel epigenetic therapy for pancreatic adenocarcinoma

Pancreatic ductal adenocarcinoma-(PDAC) needs innovative approaches due to its 12% 5-year survival despite current therapies. We show marked sensitivity of pancreatic cancer cells to the combination of a novel eIF4A inhibitor, des-methyl pateamine A (DMPatA), and a histone deacetylase inhibitor, romidepsin, inducing epigenetic reprogramming as an innovative therapeutic strategy. Exploring the mechanistic activity of this combination showed that with a short duration of romidepsin at low doses, robust acetylation persisted up to 48h with the combination, while histone acetylation rapidly faded with monotherapy. This represents an unexpected mechanism of action against PDAC cells that triggers transcriptional overload, metabolic stress, and augmented DNA damage. Structurally different class I HDAC inhibitors exhibit the same hyperacetylation patterns when co-administered with DMPatA, suggesting a class effect. We show efficacy of this combination regimen against tumor growth in a MIA PaCa-2 xenograft model of PDAC with persistent hyperacetylation confirmed in tumor samples. STATEMENT OF SIGNIFICANCEPancreatic ductal adenocarcinoma, a significant clinical challenge, could benefit from the latent potential of epigenetic therapies like HDAC inhibitors-(HDIs), typically limited to hematological malignancies. Our study shows that a synergistic low dose combination of HDIs with an eIF4A-inhibitor in pancreatic cancer models results in marked pre-clinical efficacy, offering a promising new treatment strategy.

cancer biology↗

bioRxiv2023

Pateamine A mediates RNA sequence-selective translation repression by anchoring eIF4A and DDX3 to GNG motifs

Small-molecule compounds that elicit mRNA-selective translation repression have attracted interest due to their potential for expansion of druggable space. However, only limited examples have been reported to date. Here, we show that pateamine A (PatA) represses translation in an mRNA-selective manner by clamping eIF4A, a DEAD-box RNA-binding protein, on GNG motifs. Through a systematic comparison of multiple eIF4A inhibitors by ribosome profiling, we found that PatA has unique mRNA selectivity in translation repression. Unbiased Bind-n-Seq revealed that PatA-targeted eIF4A exhibits a sequence preference for GNG motifs in an ATP-independent manner. This unusual RNA binding sterically hinders scanning by 40S ribosomes. In silico simulation, combination of classical molecular dynamics simulation and quantum chemical calculation, and the subsequent development of an inactive PatA derivative revealed that the positive charge of the tertiary amine on the trienyl arm induces G selectivity. Moreover, we identified DDX3, another DEAD-box protein, as an alternative target of PatA, showing the same effect as on eIF4A. Our results provide an example of the sequence-selective anchoring of RNA-binding proteins and mRNA-selective inhibition of protein synthesis by small-molecule compounds.

molecular biology↗

bioRxiv2023

Spatial organization and function of RNA molecules within phase-separatedcondensates are controlled by Dnd1

Germ granules, condensates of phase-separated RNA and protein, are organelles essential for germline development in different organisms The patterning of the granules and its relevance for germ cell fate are not fully understood. Combining three-dimensional in vivo structural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that localization of RNA molecules to the periphery of the granules, where ribosomes are localized depends on translational activity at this location. In addition, we find that the vertebrate-specific Dead end (Dnd1) protein is essential for nanos3 RNA localization at the condensates periphery. Accordingly, in the absence of Dnd1, or when translation is inhibited, nanos3 RNA translocates into the granule interior, away from the ribosomes, a process that is correlated with loss of germ cell fate. These findings highlight the relevance of sub-granule compartmentalization for posttranscriptional control, and its importance for preserving germ cell totipotency.

cell biology↗

bioRxiv2022

Patterning of phase-separated condensates by Dnd1 controls cell fate

Germ granules, condensates of phase-separated RNA and protein, are essential for germline development, but how these molecules are organized within the granules and whether such an organization is relevant for germ cell fate is unclear. Combining three-dimensional in vivo structural and functional analyses, we study the dynamic spatial organization of molecules within zebrafish germ granules. We find that the vertebrate-specific Dead end protein is essential for positioning nanos3 RNA at the condensates periphery, where ribosomes are located. Without Dead end, or when translation is inhibited, nanos3 RNA translocates into granule interiors, far from the ribosomes location. These findings reveal the molecular mechanisms controlling the spatial organization of RNA within the phase-separated organelle and the importance of subgranule RNA localization for preserving germ cell totipotency.

cell biology↗

bioRxiv2022

Roles of the second-shell amino acid R266 in other members of the MLE subgroup of the enolase superfamily

Catalytic promiscuity is the coincidental ability to catalyze non-biological reactions in the same active site as the native biological reaction. Several lines of evidence show that catalytic promiscuity plays a role in the evolution of new enzyme functions. Thus, studying catalytic promiscuity can help identify structural features that predispose an enzyme to evolve new functions. This study identifies such a pre-adaptive residue in an N-succinylamino acid racemase/o-succinylbenzoate synthase (NSAR/OSBS) enzymes from the NSAR/OSBS subfamily. Previously, we identified a point mutation, R266Q, in the catalytically promiscuous Amycolatopsis sp. T-1-60 NSAR/OSBS that has a deleterious effect on NSAR activity with a lesser effect on OSBS activity (Truong et al., in preparation). We demonstrated that R266 was a pre-adaptive feature that enabled the emergence and evolution of NSAR activity in AmyNSAR/OSBS. We examined the role of the residue R266 in the evolution of NSAR activity by examining the effects of the single substitution R266Q in other members of the NSAR/OSBS subfamily including Enterococcus faecalis NSAR/OSBS, Roseiflexus castenholzii NSAR/OSBS, Lysinibacillus varians NSAR/OSBS, and Listeria innocua NSAR/OSBS, which have been previously characterized to carry out both OSBS and NSAR activities efficiently. RcNSAR/OSBS, LvNSAR/OSBS, EfNSAR/OSBS, and LiNSAR/OSBS are 49, 48, 32, and 28% identical, respectively, to AmyNSAR/OSBS. We found that while the R266Q mutation decreases NSAR activity more than OSBS activity, as expected, in most NSAR/OSBS members, the differential effects of the R266Q substitution on NSAR and OSBS activities are not as striking as observed in AmyNSAR/OSBS. In some homologs, the R266Q mutation has very deleterious effects on both OSBS and NSAR activities. Furthermore, the mutation unexpectedly decreases OSBS activity more than NSAR activity in LiNSAR/OSBS. Thus, the effects of R266Q on NSAR and OSBS activities depend on differences in sequence context between members of the NSAR/OSBS subfamily, demonstrating the complex role of epistasis in the evolution of NSAR activity in the NSAR/OSBS subfamily.

biochemistry↗

bioRxiv2020

Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2) selectively binds an ascaroside (ascr#3)

During their infective stages, hookworms release excretory-secretory (E-S) products, including small molecules and proteins, to help evade and suppress the hosts immune system. Small molecules found in E-S products of mammalian hookworms include nematode derived metabolites like ascarosides, which are composed of the sugar ascarylose linked to a fatty acid side chain. Ascarosides play vital roles in signaling, development, reproduction, and survival. The most abundant proteins found in hookworm E-S products are members of the protein family known as Ancylostoma secreted protein (ASP). ASP belongs to the SCP/TAPS (sperm-coating protein / Tpx / antigen 5 / pathogenesis related-1 / Sc7) superfamily of proteins, members of which have previously been shown to bind to eicosanoids and fatty acids. These molecules are structurally similar to the fatty acid moieties of ascarosides. The objective of this study was to determine if the hookworm ASP; N. americanus Ancylostoma secreted protein 2 (Na-ASP-2) binds to the ascarosides or their fatty acid moieties. We describe investigations of our hypothesis that there is a functional relationship between the major secreted proteins and signaling small molecules found in hookworm E-S products. To accomplish this, several ascarosides and their fatty acid moieties were synthesized and tested for in vitro binding to Na-ASP-2 using a ligand competition assay and microscale thermophoresis. Our results reveal that the fatty acid moieties of the ascarosides, bind specifically to the palmitic acid binding cavity of Na-ASP-2. Additionally, ascr#3, an ascaroside that is present in mammalian hookworm E-S products binds to the palmitic acid binding cavity of Na-ASP-2, whereas oscr#10 which is not found in hookworm E-S products does not bind. Future studies are required to determine the structural basis of ascaroside binding by Na-ASP-2 and to understand the physiological significance of these observations.

biochemistry↗

bioRxiv2020

Epithelial-mesenchymal transition sensitizes breast cancer cells to cell death via the fungus-derived sesterterpenoid ophiobolin A

The epithelial-mesenchymal transition (EMT) imparts properties of cancer stem-like cells, including resistance to frequently used chemotherapy, necessitating the identification of molecules that induce cell death specifically in stem-like cells with EMT properties. Herein, we demonstrate that breast cancer cells enriched for EMT features are more sensitive to cytotoxicity induced by ophiobolin A (OpA), a sesterterpenoid natural product. Using a model of experimentally induced EMT in human mammary epithelial (HMLE) cells, we show that EMT is both necessary and sufficient for OpA sensitivity. Moreover, prolonged, sub-cytotoxic exposure to OpA is sufficient to reduce migration, sphere formation, and resistance to doxorubicin. OpA is well-tolerated in mice and treatment with OpA alone reduces tumor burden. These data identify a driver of EMT-driven cytotoxicity with significant potential for use either in combination with standard chemotherapy or for tumors enriched for EMT features.

cancer biology↗

bioRxiv2018

Occidiofungin, an actin binding antifungal with in vivo efficacy in a vulvovaginal candidiasis infection

Current antifungal treatment options are plagued with rapidly increasing occurrence of resistance, high degree of toxicity and a limited spectrum of activity. The need to develop a novel antifungal with a unique target, wider spectrum of activity, and reduced toxicity to the host, is urgent. We have identified and characterized one such compound named occidiofungin that is produced by the soil bacterium Burkholderia contaminans MS14. This study identifies the primary cellular target of the antifungal, which was determined to be actin. Actin binding metabolites are generally characterized by their ability to inhibit polymerization or depolymerization of actin filaments, which presumably accounts for their severe toxicity. Occidiofungin, instead, has a subtler effect on actin dynamics that triggers apoptotic cell death. We were able to demonstrate the effectiveness of the antifungal in treating a vulvovaginal yeast infection in a murine model. This discovery puts occidiofungin in a unique class of actin-binding antifungal compounds with minimal reported toxicity to the host. The results of this study are important for the development of a novel class of antifungals that could fill the existing gap in treatment options for fungal infections.\n\nAuthor summaryWidespread resistance to antifungal compounds currently in use has been alarming. Identification and development of a new class of antifungals with a novel cellular target is desperately needed. This study describes the assays carried out to determine the molecular target and evaluate efficacy of one such novel antifungal compound called occidiofungin. Occidiofungin modified with a functional alkyne group enabled affinity purification assays and localization studies in yeast. These studies led to the identification of the actin binding property of occidiofungin. Actin-binding by secondary metabolites often exhibit severe host toxicity, but this does not appear to be the case for occidiofungin. We have previously been able to administer occidiofungin to mice at concentrations in the range of 5 mg/kg without any serious complications. We were able to demonstrate the effectiveness of the antifungal in treating a vaginal fungal infection in a murine model. The results outlined in this manuscript establish that occidiofungin is an efficacious compound with a novel molecular target, putting it in a completely new class of antifungals.

microbiology↗