Abstract: Cardiac fibrosis is a central determinant of heart failure progression and arises from pathological remodeling characterized by fibroblast activation, myofibroblast differentiation, and excessive extracellular matrix deposition. In contrast, physiological remodeling permits adaptive cardiac growth without net fibrosis. Pregnancy represents an underexplored physiological model of reversible cardiac remodeling. In response to hemodynamic load, the maternal heart undergoes hypertrophic growth that resolves postpartum, constituting a natural paradigm of fibrosis-resistant cardiac adaptation. Pregnancy and lactation are accompanied by profound endocrine and immune reprogramming of maternal tissues. We propose that this hormonal milieu orchestrates coordinated crosstalk among endothelial cells, fibroblasts, and immune cell populations to suppress profibrotic pathways and preserve extracellular matrix homeostasis. Candidate regulators include estrogen, progesterone, prolactin family peptides, relaxin, oxytocin, and components of the renin–angiotensin–aldosterone system. During the postpartum and lactational period, prolactin and oxytocin may further promote reverse remodeling. These hormones likely act by modulating local cytokine and growth factor networks that otherwise drive fibroblast activation. By focusing on non-myocyte cardiac cells and extracellular matrix dynamics, this review positions pregnancy as a translational model to uncover endogenous anti-fibrotic mechanisms and identify novel therapeutic strategies for cardiac fibrosis.

Abstract: Vascular endothelial cells (ECs) coordinate with osteogenic processes to establish the specialized vasculature of bone tissue, where endothelial cells and bone cells interact, and bone cells regulate EC proliferation and differentiation. However, it remains unclear how ECs and bone cells are coordinated during early bone formation and whether these interactions differ between endochondral ossification (e.g., femur) and intramembranous ossification (e.g., skull). To address this question, we analyzed endothelial and osteogenic marker expression in the femur and skull between postnatal days 3 and 39. We identified distinct expression patterns of endothelial markers (Endomucin, VE-cadherin and CD31) and osteogenic markers (Osterix, Cbfa1 and BGLP) during osteogenesis in these tissues. In the femurs, endothelial marker expression alternated with the expression of osteogenic markers, suggesting potential reciprocal regulation. In contrast, in the skull, endothelial and osteogenic markers exhibited similar temporal expression patterns without alternation. We also analyzed the expression of VEGF and its receptor FLK1. In the femur, VEGF expression paralleled osteogenic marker expression, whereas in the skull VEGF expression differed from both osteogenic and endothelial marker patterns. Together, these results demonstrate that the coordination of endothelial and osteogenic marker expression, as well as VEGF signaling, differs between endochondral and intramembranous ossification, suggesting distinct modes of interaction between endothelial and bone cells during the formation of long and flat bones.

Abstract: Epigenomic regulation, particularly DNA methylation, plays a critical role in gene expression control and has emerged as an important source of biomarkers for disease diagnosis, risk prediction, and longitudinal health monitoring. As high-throughput sequencing technologies have expanded, epigenomic research has rapidly grown, producing a large and complex body of biomedical literature. This review presents an AI-driven literature-level analysis aimed at uncovering structural patterns and research trends related to epigenomic biomarker discovery. Using a large corpus of full-text articles collected from PubMed and PubMed Central, we applied text mining techniques including keyword frequency analysis, document-level co-occurrence analysis, topic clustering, contextual concordance analysis, and temporal trend analysis. Rather than evaluating individual experiments, this approach examines the broader research landscape to identify recurring conceptual structures and methodological patterns. The analysis reveals that epigenomic biomarker research is organized into several interconnected domains, including disease-focused epigenomics, chromatin regulation studies, transcriptomic integration research, and cancer-related epigenomic investigations. The rapid growth of publications since 2010 further reflects the increasing importance of high-throughput epigenomic profiling and biomarker-driven research. These findings demonstrate that AI-driven literature mining provides a scalable framework for uncovering epigenomic biomarker knowledge and translating it toward AI-enabled health monitoring systems. Such approaches may support biomarker prioritization, early disease detection, and data-driven health monitoring within precision health environments.

Abstract: Regulation of all-trans retinoic acid (ATRA) signalling is crucial to early embryonic de-velopment. We show here that in embryonic stem (ES) cell-derived gastruloids, which mimic normal development in response to the Wnt/beta-catenin agonist CHIR9901, expression of retinoic acid receptor (RAR) gamma was spatially restricted to primitive cells that co-expressed ES cell and early progenitor cell markers, i.e., Nanog, Sox2, and Oct4. In contrast, RAR alpha expression was ubiquitous. mRNAs for the key enzymes involved in ATRA synthesis (Aldh1a2) and degradation (Cyp26a1) were not seen in cells that ex-pressed RAR gamma. Treatment of ES cell-derived gastruloids with physiologically relevant (10nM) levels of ATRA or with a highly selective RAR gamma agonist blocked normal developmental processes, preventing symmetry-breaking and axial elongation. This was not seen following treatments with an RAR alpha agonist, where there was a tendency for enhanced axial elongation. Brachyury (TBXT) immuno-positive cells localised in the posterior end of elongated gastruloids in control- and RAR alpha agonist-treated cultures, with Sox2 immuno-positive cells seen more widely, whilst both TBXT and Sox2 immu-no-positive cells were randomly distributed throughout ATRA- and RAR gamma agonist-treated gastruloids. Concurrent treatment of gastruloids with 10nM ATRA and 100nM of an RAR gamma antagonist partially abrogated the ATRA-mediated block to axial elongation. Conversely, 10nM RAR gamma antagonist treatments were associated with the formation of multi-axis gastruloid elongations, with comparatively little effect seen after treatments with an RAR alpha antagonist. These findings reveal that RAR gamma plays a crucial role in the development of embryonic tissues.

Abstract: Breast cancer is classified into distinct molecular subtypes, including Luminal A, Lu-minal B, HER2-enriched, Basal-like, and Claudin-low. While traditional studies mostly use 2D cell cultures, 3D models better mimic in vivo tumour conditions. In this study, we generated and characterized 3D multicellular tumour spheroids (MCTS) from breast cancer cell lines representing different molecular subtypes. Morphologically, spheroids were either compact (MCF-7/AZ, T47D, BT474, MDA-IBC-3, BT-20, SUM149PT) or loosely adhered (MDA-MB-468, SK-BR-3, MDA-MB-231), while retain-ing key parental subtype biomarkers. Cell viability decreased with increasing spheroid size, but apoptotic cCasp3 staining was restricted to basal-like spheroids. Compact spheroids expressed E- and/or P-cadherin, indicating epithelial or epithelial–mesenchymal transition (EMT) hybrid traits, while loose spheroids showed vimentin expression linked to a mesenchymal phenotype. Overall, EMT status, rather than mo-lecular subtype, primarily determined spheroid morphology.

Abstract: Background: Etiologies of neural tube defects (NTDs) are multifactorial. Genetic, epigenetic and environmental factors may contribute to their reported variation in prevalence across the globe. Ethiopia has among the highest reported NTD prevalence globally, making investigation of genetic determinants in this high-risk population particularly important for advancing understanding of NTD etiology. Genes involved in folate metabolism, such as the reduced folate carrier 1 (RFC1), have been investigated for the potential associations with NTDs, but findings throughout the literature remain inconsistent and inconclusive. Objective: The aim of this study was to determine an association of RFC-1 polymorphism at rs1131596 and rs1051266 loci (functional variants previously implicated in folate transport efficiency and NTD susceptibility) among mothers with the occurrence of NTDs in their offspring in Ethiopia. Methods: A case-control study involving 250 mothers (187 controls and 63 cases) of children with or without NTDs was conducted in Addis Ababa, Ethiopia between April, 2022, to September, 2024. A total of 250 maternal whole blood samples were systematically collected and subjected to genetic analysis at loci rs1131596 and rs1051266 by PCR (polymerase chain reaction) and Sanger sequencing. Results: Detection of heterozygous (TC) and homozygous (CC) genotypes for SNP rs1131596 (-43T>C) in the RFC1 gene was 27.2%, with heterozygous (TC) comprising 10.4% and homozygous (CC) 16.8 %. In contrast, for the rs1051266 (80A>G), the prevalence of the AG polymorphism was 28% while the GG polymorphism was 16.4%, resulting in a cumulative prevalence of 44.4%. The presence of maternal RFC-1 polymorphism at these two locations did not show significant association (p =0.601 & p = 0.225 respectively) with increased risk for NTD births. Conclusion: This study did not reveal significant association between maternal RFC-1 gene polymorphisms and NTD-affected births. Comprehensive whole-genome sequencing of affected off springs is essential to identify specific mutations or polymorphisms that may individually or collaboratively affect the risk of NTDs in the Ethiopian context.

Abstract: Animal studies have shown that early life exposure to general anesthetics may impair brain development. However, the implications of this phenomenon in human patients remain unclear. In this study, we use an induced pluripotent stem cell (iPSC)-derived human brain microphysiological system (bMPS) to investigate the effects of early sevoflurane (SEV) exposure on human brain development. Human iPSCs were cultured and differentiated into neural progenitor cells (NPCs) and then into bMPS. At week 8, bMPSs were exposed to 2.4% SEV for 4h. Four weeks after exposure, immunofluorescence, Western blotting, and quantitative real time polymerase chain reaction (qPCR) were conducted to evaluate the alteration of nerve cells in bMPS. After SEV exposure, number of apoptotic cells increases and the level of neural differentiation marker decreases. The ratio of mature neurons over NPCs and mature oligodendrocytes over oligodendrocyte progenitor cells (OPCs) are reduced which leads to reduction of myelination. SEV also impedes the development of astrocytes and synaptogenesis, especially the formation of excitatory synapses. Meanwhile, SEV increases the expression of molecules in mammalian target of rapamycin (mTOR) signal pathway. In conclusion, early SEV exposure substantially disrupts the development of human brain tissue. The mTOR signal pathway is involved in this alteration.

Abstract: Background: The transformative potential of liquid biopsy in precision oncology is currently limited by a critical structural challenge: nomenclature inconsistency. Historically, the term "TME" was employed to denote Tumor Microemboli—multicellular clusters of circulating tumor cells (CTCs) that drive high-efficiency metastasis. In contemporary cancer biology, however, "TME" has transitioned into the universal shorthand for the Tumor Microenvironment, representing the complex systemic ecosystem of malignant and non-malignant components. Objective: This dual usage has led to conceptual ambiguity, where the same term describes both a physical cellular aggregate and a biological landscape, hindering the standard reporting of clinical results. This review seeks to resolve this inconsistency by proposing a rigorous taxonomic framework to decouple these entities and highlight their clinical utility in therapeutic decision-making. Proposed Taxonomy: We advocate for the adoption of Circulating Tumor Microenvironment (cTME) as the inclusive term for the systemic environment, encompassing non-cellular factors such as ctDNA, extracellular vesicles, and biophysical attributes. Conversely, physical cellular clusters should be strictly classified as Circulating Tumor Emboli (CTE). Crucially, we define Circulating Tumor-Associated Cells (C-TACs) as the functional cellular subset within the cTME, encompassing single CTCs, CTE, and supporting non-malignant cells like CTECs and CAFs. Clinical Applications: Establishing this distinction allows for the seamless integration of molecular profiling (NGS) and functional assays. We highlight evidence that C-TACs serve as the primary substrate for Chemo-Response Profiling (CRP), demonstrating high concordance with clinical outcomes. Furthermore, identifying these functional units, particularly perioperative CTE, acts as a critical predictor for the efficacy of adjuvant chemotherapy in early-stage malignancies. Conclusion: Adopting this unified taxonomy is essential for advancing precision oncology. By recognizing the cTME as the superordinate ecosystem and C-TACs as its functional executors, clinicians can more accurately interpret multi-modal liquid biopsy data, transforming these technologies into actionable platforms for personalized real-time cancer management.

Abstract: This study presents a theoretical mechanobiological model explaining the multilaminated architecture of retroperitoneal fasciae. Classical peritoneal fusion theories cannot account for either these organized laminae or the 10‑week delay between early visceral fixation and definitive fascial formation. We propose that early localized tension at 10–12 gestational weeks forms the inner renal fascial layer, whereas a systemic tension field emerging around 20 weeks—driven by axial skeletal ossification, pelvic expansion, and exponential volumetric growth—induces orthogonal Poisson‑effect compression, poroelastic fluid exudation, and LOX‑mediated cross‑linking to generate the laminated outer layer. To illustrate this framework, we examined a pure clinical cohort of adult renal vacancy (n=3) from 5,509 CT scans. Despite lifelong absence of the kidney, a continuous outer fascial layer persisted, indicating that its formation is tension‑driven rather than organ‑dependent. This natural subtraction phenomenon resolves the long‑standing discrepancy between classical dissection and modern imaging and supports a systemic mechanobiological origin for retroperitoneal fascial lamination.

Abstract: Plerixafor is a clinically approved CXCR4 antagonist that mobilizes hematopoietic stem cells by disrupting CXCL12/CXCR4 retention signaling. However, its biochemical effects on melanocytes and pigmentation remain unexplored. We investigated how plerixafor modulates CXCR4 signaling in melanocytes and evaluated its potential as a pro-melanogenic agent using in vitro and in vivo approaches. Human PIG1 melanocytes were treated with plerixafor (1–10 nM) with or without hydroquinone, followed by qPCR for MITF and tyrosinase expression, flow cytometry for CXCR4/CXCR7 and integrin pro-filing, Transwell migration assays, β-arrestin siRNA knockdown, Western blotting, sub-cellular fractionation, and ChIP-qPCR for β-catenin binding to MITF regulatory regions. A murine HQ-induced depigmentation model was used to test topical plerixafor (0.1–10 mM) on pigmentation, hair follicles, melanogenic gene expression, and systemic safety markers. Plerixafor significantly increased MITF and tyrosinase mRNA and enhanced melanocyte migration, while counteracting HQ-induced suppression of melanogenic genes. Plerixafor reduced cell-surface CXCR4 (consistent with β-arrestin–mediated recep-tor internalization) without altering CXCR7, c-KIT, or N-cadherin. β-Arrestin knockdown abolished plerixafor-induced ERK phosphorylation and melanogenic responses, con-firming β-arrestin dependence. Plerixafor promoted β-catenin nuclear translocation and direct β-catenin occupancy at MITF promoter/enhancer TCF/LEF motifs (5- to 8-fold en-richment, p< 0.05). In vivo, topical plerixafor restored HQ-induced depigmentation, in-creased hair follicle number and melanin content, and upregulated cutaneous MITF and tyrosinase without hepatic, renal, or inflammatory toxicity. Plerixafor functions as a bi-ased CXCR4 ligand in melanocytes, engaging a β-arrestin–β-catenin–MITF signaling axis to drive melanogenesis and repigmentation. These findings identify CXCR4 biased an-tagonism as a tractable pharmacologic strategy for therapeutic repigmentation in pig-mentary disorders.

Abstract: Basement membrane (BM) is an essential part of epithelial cell architecture. BM is a specialized extracellular matrix. BM assembles on the basal side outside of plasma membrane. It carries out important biological functions such as maintaining tissue shape, enabling cellular migration, and facilitating communication between cells. Defects in BM assembly or composition can cause a range of diseases. However, the regulatory mechanisms governing polarized deposition of BM are not well understood. To study this process, I use the follicular epithelium of the Drosophila melanogaster ovary as a model system. In this study, I analyzed the role of SNARE proteins in intracellular trafficking, secretion and polarized deposition of the BM. SNAREs are crucial for membrane fusion. I performed a genetic screen targeting all SNARE proteins in Drosophila. Individual knockdowns of multiple SNARE family members resulted in BM mislocalization. Three distinct mislocalization phenotypes were observed: Intracellular accumulation of BM, apical deposition of BM, or a combination of apical deposition and intracellular accumulation of BM. Taken together, these observations suggest that multiple SNARE family members contribute to the intracellular trafficking, secretion, and polarized deposition of BM proteins.

Abstract: Mammalian cells contain numerous membrane-bound organelles, among which endosomes serve as the initial destination for endocytosed materials. Drugs and pathogens are internalized by cells and transported to endosomes or phagosomes, and are subsequently delivered to lysosomes for degradation. Therefore, internalized drugs must escape from endosomes into the cytosol before lysosomal degradation occurs. However, endosomal escape is often inefficient in artificial drug delivery systems (DDSs). In contrast, many pathogens such as bacteria are phagocytosed and subsequently escape into the cytosol where they proliferate successfully. Studies on bacterial phagosomal escape have revealed molecular mechanisms by which host cells detect damage to organelle membrane. These host cellular machineries for sensing membrane damage can also detect membrane damage caused by artificial drugs. In this review, we summarize current knowledge of the cellular machinery involved in sensing membrane damage, including galectins, ESCRT complexes, sphingomyelin, stress granules, phosphatidylinositol 4-phosphate (PI4P) at membrane contact sites, and annexins. Although the aim of this review is to identify the molecules involved in endosomal membrane damage, many of these molecules were initially discovered through studies of bacterial infection and damage to the plasma membrane or lysosomes. Research on membrane damage not only advances our understanding of cellular responses to organelle damage, but also provides insights into the toxicity induced by inorganic materials and contributes to the rational design of more effective DDSs.

Abstract: Syntaxins are SNARE proteins that play essential roles in membrane fusion. In this review, I focus on Drosophila Syntaxins to provide a valuable insight in understanding the conserved principle of SNARE mediated membrane fusion in intracellular trafficking. This review aims to provide a comprehensive overview of how individual Drosophila Syntaxins contribute to distinct intracellular pathways, including secretory/Golgi trafficking, endocytic/endosomal sorting, and autophagy/endolysosomal fusion pathway. My studies reveal that each Drosophila Syntaxin executes a unique and essential function within intracellular transport networks.

Abstract: Muscle stem cells (MuSC) are the cellular source for generation and regeneration of skeletal muscle. To ensure correct muscle growth, MuSC self-renewal and differentiation need to be tightly regulated. Several signaling systems have been implicated in the control of MuSCs, among them Bone Morphogenetic Proteins (BMPs) and Notch, both of which promote MuSC proliferation and suppress differentiation. To better understand the mechanisms of function and the target genes regulated by BMP signaling in myogenesis, we investigated the transcriptional responses of adult mouse MuSCs to BMP6/4 using RNA-sequencing. BMP6/4-stimulation of freshly isolated MuSCs for one hour rapidly increased the expression of classical BMP target genes like Id1 and strongly induced expression of genes of the Notch pathway (Hes1, Hey1, Lfng, Snai1). In parallel, using Cleavage Under Targets and Tagmentation (CUT&Tag), we generated whole-genome binding profiles for the BMP pathway effectors pSMAD1/5/9 and SMAD4 and detected binding in promoters and potential regulatory elements of BMP targets and Notch pathway genes (Hes1, Hey1, Lfng, Snai1) indicating that BMP signaling directly influences Notch and that crosstalk between the two pathways regulates myogenesis.

Abstract: The present study examined the effect of Enterococcus durans cell free supernatant (CFS) on interleukin (IL) 8, 10 and 1β gene expressions in the intestinal cell line HT-29 treated with Staphylococcus aureus CFS. HT-29 cells were incubated with E. durans CFS or S. aureus CFS, or S. aureus CFS plus E. durans CFS. All concentrations of E. durans CFS did not show cytotoxicity, while the highest treatment (44.9 μg/mL) with S. aureus CFS induced significant cell death. S. aureus CFS did not modify IL-1β gene expression, while E. durans CFS alone or in combination with S. aureus CFS reduced it. Treatment with S. aureus CFS induced greater expression of the IL-8 gene compared to S. aureus CFS plus E. durans CFS. S. aureus CFS alone or in combination with E. durans CFS increased the expression of the IL-10 gene, while E. durans CFS alone did not modify it. These results suggest a potential protective role of the E. durans secretome in mitigating the inflammatory environment in intestinal cells. This treatment could be useful to protect against possible contact with dangerous soluble microbial products present in food.

Abstract: Chronic alcohol consumption increases the risk of osteoporosis and fracture by disrupting bone remodeling, in part by enhancing osteoclastogenesis. However, the cellular mechanisms underlying this process remain incompletely defined. We analyzed scRNA-seq data from osteoclasts differentiated in vitro from bone marrow mononuclear cells obtained from macaques following 12 months of chronic ethanol or isocaloric control solution consumption. Module scoring, trajectory inference with generalized additive modeling (tradeSeq), and CellChat-based analyses of intercellular communication were applied to uncover ethanol-induced changes in metabolic reprogramming, lineage progression, and signaling network dynamics. Module scoring indicated metabolic reprogramming toward oxidative phosphorylation, with reduced glycolytic, migratory, and phagocytic activities. Pseudotime analysis revealed accelerated osteoclast lineage commitment, broader intermediate differentiation states, and stabilization of mature osteoclasts. CellChat analysis showed globally amplified intercellular signaling, with mature osteoclasts functioning as dominant communication hubs sustained by autocrine feedback. Together, chronic alcohol consumption rewired osteoclastogenesis through early fate priming, metabolic adaptation, and hierarchical remodeling of intercellular communication, promoting enhanced osteoclastogenesis. These findings provide mechanistic insight into alcohol-induced bone pathology and highlight potential targets for therapeutic intervention.

Abstract: Tooth development or odontogenesis is a complex morphogenetic process that requires tightly regulated interactions between the oral epithelium and mesenchyme of neural crest origin. In this narrative review we compile existing knowledge regarding gene regulatory networks and epigenetic factors throughout tooth development from initiation to eruption. Signaling between epithelium and mesenchyme is mediated by four conserved pathways—Wnt/β-catenin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Sonic hedgehog (Shh)—which operate iteratively and interact through extensive crosstalk at each developmental stage. Transcription factors such as PAX9, MSX1, PITX2 and LEF1 interpret these signals to control cell fate decisions and differentiation. Epigenetic modifications, including DNA methylation, histone modifications, and microRNA-mediated regulation, provide additional layers of control that fine-tune gene expression programs. Unlike existing reviews that address these regulatory mechanisms separately, here we integrate signaling pathways, transcription factor networks, epigenetic regulation, human genetic disorders, dental stem cell biology, and recent single-cell transcriptomic insights into a unified framework. We discuss opportunities to apply developmental biology knowledge towards regenerative dentistry goals, including iPSC-derived dental models and spatially resolved multi-omics approaches, while acknowledging the considerable gap between preclinical findings and clinical application.

Abstract: Developmental processes are usually described through dynamical systems and gradient-driven cellular rearrangements, yet their topological constraints are not well characterized. We introduce a mathematical approach linking morphogenesis with the Gömböc, a convex body whose equilibrium structure is minimal under topological constraints. We model developmental dynamics as gradient flows defined on a configuration space of tissue states where a morphogenetic potential integrates mechanical, chemical and adhesive cellular interactions. To explore how varying landscape parameters affect the stability of critical configurations and developmental trajectories, we simulated morphogenetic systems governed by gradient flows with Morse-type potentials. We found that systems approaching minimal critical-point structures display large basins of attraction and convergent trajectories despite diverse initial states. Developmental systems may operate near Gömböc-like dynamical regimes in which the topological properties of the configuration space constrain the number of accessible states, while attractors and gradient dynamics may induce a causal order. Our framework generates testable predictions. Developmental trajectories should concentrate into a small number of preferred channels, with transverse dispersion showing an exponential decay over time. In exponential morphogen gradients, migration time is expected to scale approximately linearly with the initial distance from the source. Saddle-like transitional configurations should appear as intermediate states in morphogenetic landscapes, detectable as brief phases of reduced migration speed and increased directional fluctuations. Overall, a quantitative framework is provided for analyzing developmental robustness, identifying transition bottlenecks in morphogenetic landscapes and predicting how physical or biochemical parameters could reshape developmental trajectories in synthetic and regenerative contexts.

Abstract: Haploid embryos constitute a valuable model for genetic and epigenetic studies; however, their developmental competence is reduced compared with diploid counterparts. This study evaluated whether supplementation of the culture medium with specific small molecules could improve developmental competence and outgrowth establishment of parthenogenetic haploid embryos. The effects of TGF-β inhibition (A83-01), WNT pathway modulation (CHIR99021 and IWR-1), and activin A (AA) supplementation were assessed from the morula stage onward under serum-free conditions. A83-01 treatment did not improve blastocyst formation or morphology and was associated with reduced total cell numbers relative to IVF controls. CHIR99021 supplementation increased the number of SOX2-positive cells compared with IWR-1 and vehicle-treated embryos, suggesting partial support of pluripotency; however, overall developmental progression remained inferior to diploid controls. In contrast, activin A significantly increased the proportion of haploid morulae developing into blastocyst and improved hatching rates. Nevertheless, AA supplementation did not restore CDX2-positive cell numbers or total cell counts to diploid levels. Furthermore, neither CHIR99021 nor AA affect DNA fragmentation levels, although a tendency toward increased TUNEL-positive cells was observed. Activin A treatment also failed to improve embryonic outgrowth formation. Collectively, these findings demonstrate that although activin A enhances blastocyst yield and hatching in bovine haploid embryos, modulation of TGF-β or WNT signaling alone is insufficient to restore diploid-like proliferative developmental competence.

Abstract: Here, we review the history, advancements, and broad utility of the NTR/prodrug system, and suggest future strategies for developing versatile ablation models. As a chemogenetic tool, the nitroreductase (NTR)/prodrug system enables precise spatiotemporal control over cell ablation. The technology leverages bacterial nitroreductase enzymes (e.g., nfsB) to convert inert prodrugs into cytotoxic agents, thereby allowing researchers to induce targeted cell death. Following its landmark application in zebrafish with metronidazole (MTZ) in 2007, the system's utility has expanded to other essential model organisms, including Drosophila, Nematostella, Xenopus, medaka, and rodents, facilitating detailed studies of tissue damage and regeneration.This review highlights how the NTR system has been deployed to model a spectrum of human diseases, including Parkinson's disease, retinal degeneration, demyelinating disorders, and kidney disease. These models provide valuable platforms to study pathogenesis in vivo. Furthermore, the precise and controllable nature of NTR ablation makes it an ideal tool for high-throughput chemical and genetic screens aimed at discovering pro-regenerative and protective compounds.The development of NTR2.0, an enzyme variant with over 100-fold greater activity, along with more potent prodrugs such as ronidazole (RNZ), has dramatically broadened experimental possibilities. These improvements permit chronic ablation and long-term disease modeling at well-tolerated drug concentrations. Here we present some key considerations including transgenic design for optimal cell-type specificity, calibrating expression levels for desired ablation kinetics, and suitable controls to allow interpretation. These best practices will allow the researcher to develop a precise, reproducible, and versatile platform for either modeling human disease or dissecting regenerative mechanisms.