Biology and Life Sciences

Abstract: Rare actinobacteria are promising sources of specialized metabolites, yet genome mining still prioritizes genomes encoding many biosynthetic gene clusters (BGCs) rather than clusters that are distant from characterized chemistry. Here, we describe the isolation, for the first time, of a novel Jiangella strain reported from a marine, animal-associated plastisphere, and place its biosynthetic repertoire in the context of the whole genus. Digital DNA-DNA hybridization (57.8%) and OrthoANIu (94.65%) values against its closest relative, J. alba DSM 45237ᵀ, support its assignment as a candidate novel species. antiSMASH predicted twelve BGCs, within the range observed across the genus (7-13), of which only one matched a characterized pathway, and the Biosynthetic Novelty Index of the genome was 865.3, the fourth highest among the twelve genomes analyzed. Across these genomes, 124 BGCs grouped into 70 gene cluster families, 74% of them strain-specific. Two families without any characterized representative, a lasso-peptide family and a RiPP recognition element-containing family, were instead conserved across a subclade, down to nearly identical precursor peptides. No antimicrobial activity was detected under the conditions tested. Genus-wide comparison therefore identifies conserved yet uncharacterized families as rational priorities that cluster counts alone would miss.

Abstract: Gonadal toxicity from cancer treatments highlights the need for efficient spermatogonial stem cell (SSC) isolation and expansion protocols for fertility preservation. This study aimed to isolate and culture rat SSCs and evaluate the cytoprotective and antioxidant effects of mangiferin (M) and sodium selenite (S), individually and in combination (M+S). Rat testicular tissue was isolated through a two-step enzymatic digestion protocol, and baseline passage 1 (P1) cells were characterized using RT-qPCR and immunocytochemistry. Cultured cells were allocated into four experimental groups: control, 100 μg/mL mangiferin alone, 2 ng/mL sodium selenite alone, and combined co-treatment. Supplementation with sodium selenite alone reduced viability to 62.00%, whereas mangiferin alone demonstrated high biocompatibility (81.00% viability). The combined M+S group partially mitigated selenite-induced toxicity, restoring viability to 75.50%. Notably, co-supplementation exerted a potent combined cryoprotective action, elevating post-thaw cellular viability to 92.67% compared to 71.33% in the untreated control. Molecular and immunocytochemical profiling confirmed the enrichment of a functional undifferentiated germline population expressing stemness markers (Gfra1, Zbtb16, Vimentin) without somatic lineage markers (CD18, CD31, AE1/AE3). In conclusion, the co-supplementation of mangiferin and sodium selenite effectively counteracts in vitro hyperoxic and cryogenic oxidative insults, establishing a robust framework for germ cell cryobanking and male fertility restoration.

Abstract: Background/Objectives: Among endocrine malignancies, thyroid cancer is the most prevalent and encompasses a broad clinical spectrum, ranging from indolent differentiated tumors to highly aggressive poorly differentiated and anaplastic thyroid carcinomas. Histopathology and molecular testing incompletely capture the spatial organization of tumor, stromal, and immune compartments that drives invasion, dedifferentiation, and treatment resistance. Spatial transcriptomics preserves tissue architecture while profiling gene expression, mapping molecular programs onto histologic context. We systematically reviewed its platforms, applications, and biological insights in thyroid cancer. Methods: PubMed/MEDLINE, Scopus, and Web of Science Core Collection were searched for English-language studies combining spatial-transcriptomic and thyroid-cancer concepts, following PRISMA 2020 guidance. Primary studies that applied a spatially resolved transcriptomic method to thyroid cancer tissue and reported spatial gene-expression findings were included; data were extracted on subtype, platform, complementary methods, and principal findings. Results: Of 104 records (47 after de-duplication), 21 studies met the inclusion criteria. Most used capture-based whole-transcriptome profiling, particularly 10x Visium, while GeoMx digital spatial profiling supported FFPE-compatible biomarker studies. Recurrent findings included POSTN+ and SERPINE1+ cancer-associated fibroblast programs at invasive fronts, APOE-linked immunosuppressive states, organized immune-evasion niches, and progressive tumor-microenvironment remodeling accompanying dedifferentiation toward ATC. Spatial transcriptomics also clarified invasion, lymph node and distant metastasis, and cell–cell communication. Conclusions: Spatial transcriptomics is opening new avenues for dissecting thyroid cancer progression, but current evidence is limited by small cohorts, platform concentration, and variable validation. Larger, standardized, clinically annotated studies are needed to define reproducible spatial biomarkers.

Abstract: Public burn transcriptomic datasets remain difficult to compare because of differences in tissue source, study design, platform, annotation and statistical analysis. We developed BurnOmicsDB (https://peneapple.github.io/2026-BurnOmicsDB/), a curated and gene-centred web resource integrating six human burn-related Gene Expression Omnibus series comprising 1,013 public expression profiles. Dataset-specific quality control and differential-expression workflows were applied to 22 predefined contrasts across skin, wound margin, eschar, scar and blood, generating 448,735 standardized gene–contrast records. Gene identifiers and aliases were harmonized to support retrieval by official symbols, historical aliases, NCBI Gene IDs, Ensembl IDs and HGNC identifiers. Cross-tissue analysis showed that 564 of 1,444 genes with clear significant directions in both skin and blood exhibited opposite directions, highlighting the context dependence of burn-associated transcriptional responses. BurnOmicsDB enables code-free exploration and cross-study comparison of burn transcriptomic evidence for research and clinical reference, but is not intended for diagnosis or treatment decisions.

Abstract: Nowadays Cannabis sativa L. is primarily valued for the cannabinoids produced and accumulated in its glandular trichomes. Consequently, it is of special relevance to address studies related to the biochemical, molecular, and morphological panorama of trichomes. This review highlights some of the most recent publications on the morphological, phenological, biochemical and molecular characterization of trichomes and mechanisms underlying their development. Particular emphasis is placed on the transcription factors related to trichome cell identity, drawing on studies conducted in model organisms and in Cannabis sativa L. Finally, we discuss emerging approaches for studying trichomes and highlight biotechnological perspectives that may facilitate future research and crop improvement.

Abstract: Honey biosynthesis is a biochemical transformation process in which nectar-derived phenolic compounds undergo selective modification during collection, enzymatic processing and maturation within the hive. This study investigated the influence of geographical origin (GeO), botanical origin (BO) and drying treatment (DT) on the phenolic composition and antioxidant activity of nectariferous flowers and their corresponding honeys from Yucatán, Mexico. A 5 × 8 × 2 multifactorial design was applied to floral biomass, whereas 4² and 3 × 6 multifactorial designs were used for monofloral and multifloral honeys, respectively. Total phenolic content (TPC), antioxidant activity (DPPH) and individual phenolic compounds were determined by the Folin–Ciocalteu assay, DPPH radical scavenging assay and UPLC-DADFreeze-dried flowers showed higher total phenolic content, antioxidant capacity, and flavonoid concentrations than oven-dried flowers. Among the floral samples, Gymnopodium floribundum from Peto showed the highest total phenolic content (1,754.69 ± 128.67 mg GAE/100 g DM) and antioxidant capacity (96.82 ± 1.09% DPPH inhibition), whereas Bursera simaruba from the same locality had the highest catechin concentration (998.72 ± 1.45 mg/100 g DM). In contrast, honey displayed a more selective phenolic profile, characterized by lower flavonoid abundance and the predominance of protocatechuic acid (84.76 ± 0.09 mg/100 g DM). These findings indicate that honey does not proportionally preserve the floral phenolic profile but instead emerges from a selective biochemical reorganization in which individual metabolites are differentially retained, transformed, enriched, or depleted during nectar conversion, providing new insight into the metabolomic transition from nectar to honey.

Abstract: Non-specific lipid transfer proteins (nsLTPs) are plant-specific proteins involved in lipid transport, cuticle formation, and responses to abiotic and biotic stresses. Here, we functionally characterized AtLTP12, a member of the nsLTP1 subfamily in Arabidopsis thaliana, using a T-DNA insertion knockout mutant. The atltp12 mutant exhibited reduced seed germination rates, increased leaf water loss, and enhanced sensitivity to salt, drought, osmotic, and oxidative stress. Additionally, the mutant plants also showed compromised basal resistance, while their response to inducing systemic acquired resistance remained unaffected. In vitro assays confirmed lipid-binding activity of recombinant AtLTP12, with a preference for phospholipids. Subcellular localization analysis indicated its targeting to the extracellular space. These results demonstrate that AtLTP12 positively regulates multi-stress tolerance through lipid homeostasis and ROS regulation and contributes to basal immune responses. AtLTP12, therefore, represents a potential target for improving stress-resilience in plants.

Abstract: Predicting CRISPR-Cas9 guide RNA efficiency and off-target activity is a precondition for precise genome editing. Computational models have progressively incorporated chromatin accessibility and epigenetic descriptors into their feature sets, yet synthesizing findings from independently published studies—especially when those studies contradict one another—remains an unresolved methodological gap. Large Language Models (LLMs) have been proposed as a route to automate cross-study synthesis, but their utility depends on a constraint that receives less attention than model architecture: how much of the source text actually reaches the model at inference time. Cloud-based models process 48,000-token corpora without hardware limitations, but at the cost of data leaving the local environment and with limited reproducibility across API versions. Local RAG systems avoid the cloud dependency while fragmenting the input, discarding the global context needed to link biological arguments that are distributed across separate papers. We benchmark these strategies using a corpus of four CRISPR-Cas9 efficiency prediction studies and apply the Reduced Interaction Sampling (RIS) engine—a local sparse attention method—to retain the full sequence within the memory envelope of a laboratory server. Preserving that context surfaces three undocumented contradictions. The static epigenetic markers used in DeepCRISPR (CTCF, DNase I) show near-zero Spearman correlations with off-target cleavage (ρ ≤ 0.07), while nucleosome positioning scores from the Block Decomposition Method reach ρ = 0.388–0.423. The sequence-only Apindel model was published in June 2022 without incorporating nucleosome descriptors reported in the concurrent literature. The benchmark review by Konstantakos et al. attributed 10–20% of rank correlation to epigenetics—a figure that reflects the weak feature subset evaluated, not a ceiling on chromatin influence. These discrepancies are invisible when papers are read individually or retrieved as chunks; they become traceable only when the full corpus is processed as a single context window. An independent empirical analysis of 2,000 CRISPR-Cas9 off-target cleavage events confirms the pattern: static epigenetic markers yield |ρ| ≤ 0.11, whereas computed NuPoP Affinity descriptors reach r = −0.622 (p < 10⁻²¹⁰). On a 30-question crossstudy synthesis benchmark (5 independent seeds), Baseline accuracy is 53.33%, RAG 60.00%, and RIS (30 seeds, 3% density) 70.00% (p < 0.0001, t-test vs. RAG, σ = 0.00% for all configurations).

Abstract: Recombinant AAV (rAAV) production in HEK293 cells has advanced through separate optimizations of individual intervention points: raising ATP levels, inhibiting the proteasome, suppressing apoptosis, or increasing Rep52/40 stoichiometry. However, when these results are considered together, each intervention produces counterintuitive trade-offs. This paper argues that these trade-offs are not anomalies but the expected behavior of a coupled system in which intracellular ATP serves as a shared control variable for three manufacturing outputs: particle yield, capsid integrity, and producer-cell longevity. I describe four mechanistic couplings that link ATP to these outputs. First, ATP directly fuels the Rep52/40 packaging motor, making the encapsidation rate sensitive to the cytosolic energy supply. Second, physiological millimolar ATP suppresses 26S proteasome activity in a biphasic, concentration-dependent manner, sparing nascent capsid precursors and Rep proteins (productive arm) while simultaneously stabilizing p53 and sensitizing cells to apoptosis (destructive arm). Third, proteasome suppression also spares the MRN replication-restriction complex, predicting a non-monotonic, window-shaped yield response to increasing ATP. Fourth, the apoptosis node receives at least three mechanistically distinct death inputs, none of which is fully addressed by single-branch interventions. From this coupling structure, the model makes five falsifiable commitments, each specifying experimental results that would challenge it. The framework re-derives recent trade-off findings in the field, including contradictory results from pan-caspase and HIF1α inhibition, as predictable outcomes of a constrained system rather than isolated anomalies. It further predicts that coordinated improvement in yield requires joint control of both the ATP set-point and the apoptosis node.

Abstract: This study aims to investigate osmoprotectant rhizobacteria producing glycine betaine, marked with the Green Fluorescent Protein (GFP) gene, in the rhizosphere of mung bean plants under drought stress conditions. The transformation of the GFP gene was performed on isolates Bl-19 and Am-7. Seeds were soaked for 30 minutes in bacterial suspensions containing either Bl-19-gfp or Am-7-gfp at a density of 106 cells mL-1, in a volume of 500 mL. The seeds were then planted under drought stress at 60% field ca-pacity and sterile media. The results showed that inoculation of Bl-19-gfp isolates with an initial density of 10⁶ CFU led to a decrease in bacterial density to 1.4 x 10⁵ CFU 1 week after inoculation. Similarly, inoculation of Am-7-gfp isolates with an initial density of 10⁶ CFU caused a decrease to 1.7 x 10⁵ CFU after 1 week of inoculation. Subsequently, the densities of both isolates remained stable from week 2 to week 4. The dry weight of mung bean plants at soil moisture conditions of 60% field capacity using isolates Bl-19 and Am-7 increased by 40.13% and 52.44%, respectively. Based on the results, both isolate increased dry weight below 60% of field capacity in sterile media.

Abstract: Gallic acid (GA) is a value-added phenolic compound widely applied in the food, pharmaceutical, and cosmetic industries. Traditionally obtained through chemical hydrolysis of tannins, its production faces limitations related to environmental impact, substrate dependency, and process sustainability. In recent years, microbial platforms have emerged as promising bioproduction alternatives, driven by advances in metabolic engineering and synthetic biology. This review discusses the transition from conventional enzymatic tannin hydrolysis to the engineering of microbial cell factories to enable de novo GA biosynthesis via shikimate pathway reprogramming. This work summarizes reported strategies, including tannase-mediated bioconversion, exploration of native GA-producing microorganisms, and rational pathway reconstruction in model chassis such as Escherichia coli, Corynebacterium glutamicum, and Pseudomonas putida. Emphasis is given to recent approaches involving carbon flux redirection, deletion of competing catabolic routes, enzyme engineering, dynamic regulatory circuits, and tolerance improvement. Current challenges related to metabolic bottlenecks, product toxicity, and industrial scalability are critically analyzed. By integrating enzymatic, metabolic, and systems-level strategies, this review highlights the progress achieved in microbial GA production and outlines perspectives for the development of robust and sustainable biotechnological platforms.

Abstract: Chinese hamster ovary (CHO) cells are the principal platform for manufacturing recombinant biopharmaceuticals, but sustained production can exceed the protein-folding capacity of the endoplasmic reticulum (ER). Binding immunoglobulin protein (BiP/GRP78), encoded by Hspa5, is a major ER chaperone that supports folding and modulates unfolded protein response (UPR) signaling, yet its overexpression has been reported as beneficial or inhibitory depending on the product. Here, we stably overexpressed the Chinese hamster Hspa5 coding sequence in a CHO line producing a dulaglutide analogue comprising a modified GLP-1 peptide fused to human IgG4 Fc and compared BiP-overexpressing (DUL-BiP) cells with empty-vector (DUL-Neo) controls in fed-batch culture. At constant temperature, BiP overexpression did not alter viable cell density or viability, but DUL-BiP cells maintained higher late-stage cell-specific productivity, prolonged productive secretion by approximately two days, and reached a 40% higher final titer; under a temperature shift, maximal cell density decreased. Control cells induced endogenous BiP only at late culture stages, whereas DUL-BiP cells maintained an elevated BiP level from the start. Intracellular GLP-1–Fc did not accumulate progressively, and culture-associated changes in cleaved ATF6, eIF2α phosphorylation, and CHOP persisted in both lines. Constitutive host-derived BiP expression therefore enlarges the secretory reserve, sustaining late-stage secretion rather than increasing growth; the benefit depends on product-specific folding requirements.

Abstract: Bioactive peptides are an important source of therapeutic molecules and molecular scaffolds involved in defense, signaling, and immune regulation. Despite the extraordinary diversity of Coleoptera, the structural landscape of beetle-derived bioactive peptides remains largely unexplored, limiting our understanding of their evolutionary diversity and biotechnological potential. Here, we performed a large-scale structural survey of predicted toxin-like peptide scaffolds across publicly available Coleoptera transcriptomes by integrating transcriptome mining, peptide maturation prediction, physicochemical characterization, AlphaFold 3 structural modeling, structural similarity analyses, and interpretable machine learning. We identified 291 candidate peptides, of which 155 contained canonical signal peptides and 273 produced mature peptides within the expected size range of known bioactive peptides. Structural analyses revealed that, despite extensive sequence diversity, many candidates converged toward a comparatively restricted repertoire of compact cysteine-rich architectures, suggesting that structural conservation exceeds primary sequence conservation during peptide diversification. Comparative structural analyses further identified recurrent protein architectures shared across multiple beetle lineages, while machine learning prioritization integrated structural and biochemical descriptors to identify high-confidence candidates for future functional characterization. These analyses establish the first structural atlas of predicted toxin-like peptides across Coleoptera and demonstrate that structure-guided transcriptome mining provides a powerful framework for uncovering evolutionarily conserved bioactive peptide scaffolds that would remain largely undetected using sequence-based approaches alone. Beyond expanding our understanding of peptide evolution in beetles, this resource is a foundation for future structural, functional, and biotechnological exploration of bioactive peptides in underexplored animal groups.

Abstract: Natural products remain a major source of structurally diverse and biologically active small molecules, yet traditional activity-guided discovery is labor-intensive and prone to rediscovery, while untargeted genome mining often lacks efficient prioritization criteria for biosynthetic gene clusters (BGCs). Self-resistance-gene guided discovery has emerged as a powerful strategy to address this limitation. In producing organisms, toxic metabolites are typically accompanied by genetically encoded self-protection mechanisms, such as resistant target homologs, duplicated housekeeping genes, detoxification enzymes, repair systems, or transporters. When co-localized with BGCs, these determinants serve as functional markers for predicting bioactivity and, in some cases, molecular targets prior to compound isolation. Over the past decade, this concept has evolved into a target-directed genome mining framework supported by tools and databases including ARTS, FunARTS, antiSMASH, and MIBiG. This review summarizes the biological basis, workflow, representative advances, and limitations of this strategy. Self-resistance genes can thus be viewed as functional beacons for accelerating bioactive natural product discovery.

Abstract: Maternal psychological stress during pregnancy is linked to adverse birth outcomes, but the biochemical pathways involved remain poorly defined. We previously reported that the human placenta buffers the fetus from several effects of maternal perceived stress in a cohort of term pregnancies. Here, we extend that cohort with a broader steroid panel and, for the first time, a targeted bile acid metabolomic panel across four maternal–fetal compartments: maternal plasma, placental tissue, cord blood, and maternal hair. Nulliparous women were recruited during the third trimester at UCSF Mission Bay Hospital and stratified into Low- and High-Perceived Stress Scale (PSS) groups using the 10-item PSS. Steroid and bile acid metabolites were quantified by UPLC–MS/MS and compared between groups using nonparametric methods. Most steroid and bile acid metabolites did not differ significantly between High-PSS and Low-PSS groups, consistent with a broadly preserved placental metabolic barrier. However, three metabolites were selectively reduced in the High-PSS group: placental estrone (E1), cord blood glycochenodeoxycholic acid (GCDCA), and maternal plasma ursodeoxycholic acid (UDCA) (all p < 0.05), while estradiol and estriol concentrations were unaffected. Maternal hair showed poor con-cordance with plasma and placental compartments, suggesting limited value as a non-invasive proxy for these analytes in this setting. These findings reinforce the concept that placental buffering of maternal stress is broadly selective rather than absolute and identify E1, GCDCA, and UDCA as candidate stress-sensitive metabolites for validation in larger, longitudinal cohorts.

Abstract: The Mass Action Law (MAL) Median Effect equation, fa/fu = (D/Dm)^m, leads to the Unified General Dynamics Theory and algorithm, which provides interdisciplinary and cross-disciplinary common linkage parameters for computerized, digital, efficient, cost-effective, Econo-green scientific R&D and data science informatics. The MAL- general theory for drug and entity dynamics actions with common parameters (Dm and m); and dose-effect interaction in combinations with a unified combination index (CI), which allows digital simulation of synergism (CI < 1), additive effect (CI =1), and antagonism (CI > 1). The MAL-based input-output sequence, pattern transition, and combinatorics of enzyme reactions led to the discovery of the Second Degree (Squared) Pascal triangle. Surprisingly, life sciences dynamics can be linked to Riemann’s zeta hypothesis, ζ(s) at s = 2, for the critical line of prime distribution and the critical strip (0 – 1), and Euler’s Prime Product Function and its properties. The universe has two domains: Life and Non-Life, which manifest the dimensionless relativity ratio with basic codes. For life is a/b = a/(1-a) = (1-b)/b (Floating Ratio), and for Non-Life is a/b = (a + b)/a = 1 + b/a, or (Golden Ratio). Life and Non-Life can be linked by the two fractional finite and continuous distribution functions of “1”. Life is finite, discrete, and binary, cyclable with the Median-based equilibrium, symmetry, harmony, homeostasis, and optimal conditions; Non-Life is open, fractal, and extendable to infinity, as in mathematics, physics, and AI. Despite numerous mathematical equations having been derived and established since antiquity, the relevance of fundamental principles in biomedical science constitutes only a small fraction of them. Human existence constitutes a tiny fraction of the universe. However, humans evolve and decide the units and methods of measurement in all branches of science. This paper presents a unified MAL median-based top-down R&D, distinct from traditional, statistics-based, bottom-up R&D. The MAL input-output dynamics have yielded deterministic digital informatics through computer simulations and automation, with efficient, cost-effective features that are not accessible in traditional, specific-aimed, consensus-based regulated R&D.

Abstract: The intestinal barrier (IB) is a critical interface that maintains internal homeostasis, and its functional compromise—termed intestinal hyperpermeability—permits luminal antigens such as lipopolysaccharide to enter the circulation, triggering metabolic endotoxemia and systemic low-grade inflammation that underpin both intestinal and extra-intestinal diseases. Exercise has emerged as a promising non‑pharmacological strategy to modulate IB function. This review systematically examines the molecular pathways through which exercise exerts its effects, focusing on: (i) the regulation of tight junction proteins, including zonula occludens-1, occludin, and claudins; (ii) the suppression of TLR4/NF-κB-driven inflammation and activation of Nrf2-mediated antioxidant defenses; and (iii) the beneficial remodeling of gut microbiota composition and short-chain fatty acid production. We also explore the peripheral–gut crosstalk mediated by myokines (irisin, IL-6, BDNF) and autonomic signals, and compare the differential impacts of exercise modalities—moderate‑intensity continuous training, resistance training, and high‑intensity interval training—on barrier function. A J‑shaped dose–response relationship is proposed, wherein moderate exercise confers protection while excessive endurance exercise may precipitate transient hyperpermeability. Finally, we discuss the therapeutic potential of exercise in intestinal disorders, identify current challenges including the translational gap and lack of large‑scale trials, and outline future directions in precision exercise medicine and multi‑omics integration to support IB‑targeted exercise prescriptions.

Abstract: Hematopoietic malignancies are commonly associated with oncogenic chromosomal translocations involving histone methylases. Also, the phosphoinositide 3-kinases (PI3Ks) mediate growth factor signaling and are implicated in cancer as drivers of cellular transformation and as targets for anticancer agents. While histone methylation and growth factor signals interact at gene promoters, the interaction mechanisms are not well understood. Pan-specific and paralog-selective class I PI3K inhibitors were tested for the ability to inhibit the proliferation of the leukemia cells lines THP-1 and U937. Further, the transcriptional effects in U937 cells were studied with RNA-seq. Differential cytotoxicity was observed with taselisib being the most cytotoxic compound followed by idelalisib and wortmannin. Alpelisib and seralisib had lower levels of cytotoxicity in both cell lines. Morphological studies showed cell shrinkage and nuclear fragmentation in wortmannin and taselisib treated U937 cells. The transcriptional effects of idelalisib, taselisib, and wortmannin were studied in U937 cells. Consistent with a myeloid lineage, PI3Kδ was most abundant PI3K followed by PI3Kγ at 2-fold lower, and PI3Kβ at 4-fold lower. PI3Kα and class III PI3Ks were detected at much lower levels. Histone lysine methyltransferases and related proteins also displayed marked differences in expression levels. Taselisib caused the most changes in gene expression patterns with the dominant pathways being RNA processing, cell cycle and inflammatory pathways. The results identify genes in the PI3K/AKT pathway as well and histone modification enzymes that correlate with treatment with specific PI3K inhibitors and that could provide insights into the mechanisms of blockage of cell proliferation by the inhibitors.

Abstract: Cellular identity determination and lineage tracing is a pivotal technique in modern biological research. Conceptually simple yet efficient cell labeling techniques offering broader applicability are warranted despite many methods requiring intricate construction procedures do exist. One such technique is ADAR (adenosine deaminase acting on RNA)-mediated RNA sensing, a live cell labeling technique that is based on the expression and abundance of cell-type-specific RNAs. Here, we utilized the optimized version, CellREADR (Cell access through RNA sensing by Endogenous ADAR)1, to establish a feasible tracing system for mouse spermatogonial stem cells (mSSCs) which are refractory to CRISPR-based reporter gene knock-in. We identified several previously unreported features of CellREADR, including RNA interference induced by double-strand RNA formation that is obligatorily generated during the normal operation of the CellREADR system. More importantly, we established its application for long-term labelling of mSSCs and monitoring mSSCs differentiation induced by retinoic acid (RA) treatment in vitro. This work offers a practical solution for dynamic monitoring of mSSCs self-renewal and differentiation and supports that CellREADR can be developed into more versatile and efficient tools in stem cell research.

Abstract: Supraphysiological levels of oxygen are often used as therapy for acute respiratory distress and other severe pulmonary morbidities but can cause excessive generation of reactive O₂ and nitrogen species resulting in oxidative and inflammatory injury. Aurothioglucose (ATG), a FDA approved, gold-containing pharmaceutical, potently and irreversibly inhibits TrxR1 and in adult and neonatal mice, ATG treatment preserves reduced glutathione levels, and attenuates hyperoxic lung injury. Adult C3H mice were treated with saline or ATG and exposed to room air or >95% O₂. All mice had succumbed or were euthanized at 200h of hyperoxia exposure and lung tissues were collected. Metabolomic analyses were conducted and comparisons were performed between room air and >95% O₂ exposure, saline and ATG treatment, and male and female sex. Profound differences in survival between sexes with and without ATG treatment with ATG-treated females surviving longer than all other hyperoxia-exposed groups. Comparisons between groups identified metabolites in the glutathione pathway as significantly different. Metabolomic analysis revealed keratin sulfate (KS) biosynthesis and glycosphingolipid (GSL) biosynthesis as the primary pathways in the saline O₂ vs ATG O₂ comparison. Carnitine shuttle was identified as the primary pathway between sexes both with ATG and O₂. The current data suggests that the improved survival of hyperoxia-exposed ATG-treated female C3H mice is likely driven by enhanced glutathione synthesis, energy production, and metabolism resulting in decreased lung injury through modulation of KS and GSL levels. These findings may provide direction for further research to improve outcomes after hyperoxia exposure.