The use of early subcultures to reduce culturomics handling time.

Brief guide to Drosophila maintenance and husbandry.

Continuous evolving humanoid for advanced cellular models.

Aureobasidium pullulans is used as a bio-protectant of crops and wood and can stimulate plant growth. This fungus forms different cell types including blastoconidia, swollen cells, and hyphae. The role of these cells in bio-protectant performance of A. pullulans is not yet described. Here, dynamics in formation and differentiation of blastoconidia, swollen cells, and hyphae were quantitatively assessed in liquid static cultures of four isolates. During 16h of culturing, blastoconidia were formed by cell division of swollen cells, hyphae, and existing blastoconidia, while blastoconidia differentiated into swollen cells and swollen cells differentiated into hyphae. As a result, blastoconidia were no longer the most dominant cell type after 10h of culturing, although they represented more than 75 % of the cells at the moment of inoculation. A mathematical model was developed based on Ordinary Differential Equations to describe the time-dependent cellular dynamics of the neotype strain A. pullulans CBS 584.75 under fixed culture conditions. The quantitative data of its 16h static liquid cultures was used as input with the incubation time as input variable. The model predicted that blastoconidia would again be the most prominent cell type from 18h of incubation onwards with relative abundances of the blastoconidia, swollen cells and hyphae of 57 %, 32 %, and 11 %, respectively, after 72h of growth. The quantitative assessment of cell division and differentiation and its mathematical model can be used in future studies to optimize performance of A. pullulans as a bio-protectant and to stimulate plant growth.

Patient-derived malignant effusion organoids guide tailored therapy and dissect third-generation EGFR-tyrosine kinase inhibitor resistance mechanisms in lung cancer.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease lacking effective therapies. Mesenchymal stem cells (MSCs) show therapeutic potential; however, their efficacy is often limited. Aggregates of human umbilical cord-derived MSCs (hUC-MSCs) were generated using AggreWell™ 400 plates. The paracrine and mechanotransduction profiles of three-dimensional (3D)-MSCs were assessed by reverse transcription quantitative PCR and western blot. MASH was induced in C57BL/6J mice via a 32-week high-fat, high-fructose, and high-cholesterol (HFFC) diet, followed by tail vein injection of PBS, two-dimensional (2D)-MSCs, or 3D-MSCs. Therapeutic efficacy was evaluated via histological staining, immunohistochemistry, and serum biochemistry. RNA sequencing was performed to elucidate underlying molecular mechanisms. Under 3D culture conditions, hUC-MSCs formed relatively uniform aggregates with preserved MSC phenotypic characteristics after recovery. 3D aggregate culture enhanced the paracrine, anti-inflammatory, mechanosensitive phenotype, pro-survival, and matrix-remodeling properties of hUC-MSCs, evidenced by increased expression of hepatocyte growth factor, tumor necrosis factor-stimulated gene 6, cyclooxygenase-2, prostaglandin E synthase, B-cell lymphoma-2, and mechanotransduction-related genes, accompanied by elevated matrix metalloproteinase-2 (MMP2) and MMP9 expression in vitro. In vivo fluorescence imaging showed no significant difference in early hepatic retention between intravenously infused single-cell hUC-MSCs and 3D aggregates. In the MASH model, 3D-MSCs more effectively reduced lipid accumulation, collagen deposition, α-smooth muscle actin, hepatomegaly, and serum total cholesterol levels compared with 2D-MSCs. Moreover, 3D-MSCs enhanced the expression of anti-inflammatory interleukin-10, while suppressing inducible nitric oxide synthase. 3D-MSC therapy induced broader transcriptional remodeling than conventional MSCs, with enrichment in extracellular matrix organization, focal adhesion, and the phosphatidylinositol 3-kinase-protein kinase B signaling pathway. Consistently, western blot analysis showed that 3D-MSCs more effectively reduced hepatic p-PI3K and p-AKT levels, indicating stronger inhibition of PI3K/AKT pathway activation. 3D aggregate culture enhances the therapeutic efficacy of hUC-MSCs against MASH, supporting its translational potential for MSC-based liver therapy.

Engineering aspartate metabolism improves β-alanine-based 3-hydroxypropionic acid production in Saccharomyces cerevisiae.

Evaluation of dimethyl sulfoxide-free formulations for cryopreservation of human T cells.

Strawberry ( Fragaria × ananassa ) is an economically important fruit crop worldwide. Although substantial progress has been achieved through conventional breeding, improvement efforts are constrained by long breeding cycles, complex octoploid genetics, extensive heterozygosity, and strong genotype × environment interactions, all of which limit the speed and precision of trait development. Moreover, introgression of desirable traits through conventional hybridization often leads to linkage drag and unpredictable phenotypic outcomes, particularly for disease resistance and fruit quality traits. In contrast, genetic transformation and genome editing enable precise modification of target genes without disrupting elite genetic backgrounds, providing a powerful approach for the timely and targeted introduction of desirable traits. However, the successful application of these technologies relies on robust and reproducible in vitro regeneration platforms, which are essential for recovering edited plants, overcoming genotype-dependent barriers, and accelerating precision breeding in strawberries. In this study, an efficient in vitro regeneration system was developed to support genome-editing applications in strawberries. Optimal regeneration conditions were established for the cultivar Festival using in vitro leaf explants by evaluating thidiazuron (TDZ) and 6-benzylaminopurine (BAP), applied alone or in combination with low concentrations of naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid, under both light and dark culture conditions. Dark incubation markedly enhanced leaf regeneration efficiency, significantly increasing both regeneration frequency and the number of regenerated shoots compared with light conditions. The genotype dependency of regeneration was further assessed by validating the selected conditions across four additional cultivars (Albion, Chandler, Monterey, and Rocco). Notably, this study represents the first report of successful in vitro regeneration of the strawberry cultivars Monterey and Rocco. Finally, all regenerated shoots were successfully rooted and acclimatized using an optimized regeneration, rooting, and acclimatization protocol.

Optimization of biotechnological processes is traditionally limited by time-consuming trial-and-error approaches and the complexity of simultaneously optimizing multiple, often conflicting objectives. This applies particularly to plant tissue culture medium design, which therefore serves as the application case in this study. Recent advances in machine learning and evolutionary algorithms offer powerful alternatives, yet 80% of published studies rely on licensed software, and systematic data-driven optimization frameworks remain scarce. This creates significant barriers to adoption in both academic and commercial plant biotechnology. We introduce ADAM (Advanced Design and AI-Driven Modeling for Plant Tissue Culture Media Optimization), an open-access, web-based platform that transforms protocol development into a data-driven computational process. ADAM implements a complete ML-EA workflow through five integrated modules: 1. Design of Experiments (five different concepts) for systematic parameter exploration, 2. Data Preparation with automated quality control, and 3. Model Building using nine machine learning algorithms with automated selection. The platform enables Optimization (4.) through four advanced evolutionary algorithms (genetic algorithm, particle swarm optimization, NSGA-II, SMS-EMOA) for single- and multi-objective problems, with Evaluation (5.) tools to compare original versus optimized solutions. Validation across two plant tissue culture applications showed that ADAM's models matched or exceeded the predictive performance of manually optimized approaches in the original studies. The platform successfully identified multiple optimal culture conditions balancing conflicting objectives, providing experimentally testable predictions that reduce the trial-and-error cycle. Deployed as a browser-based application requiring neither specialized hardware nor software licenses, ADAM democratizes advanced AI optimization for plant biotechnology, eliminating traditional barriers to entry while maintaining the rigor and flexibility required for scientific research.

Bu araştırma, 3 adet yerli ve 60 adet yabancı orijinli Achillea millefolium L. genotiplerinin Isparta ekolojik koşullarında verim ve kalite özelliklerini belirlemek amacıyla 2023 yılında yürütülmüştür. Çalışmada çiçeklenme süresi 10–41 gün, bitki boyu 21.8–72.0 cm, çiçek çapı 4.3–15.5 cm ve çiçekçik sayısı 26.0–419.0 adet/kapitulum arasındadır. Taze çiçek verimi 246.9–2829.7 kg/da, kuru çiçek verimi ise 82.6–1017.7 kg/da aralığında ölçülmüş ve PI 439888, PI 661151 ve PI 439892 genotipleri yüksek verim potansiyeliyle dikkat çekmiştir. Uçucu yağ oranları %0.05-0.50 arasında değişmiştir. Taze herba ve uçucu yağ verimi ile uçucu yağ oranı ve rengine göre seçilen 10 genotip ve yerli genotiplerde; 1,8-sineol (%3.1–22.4), borneol (%2.5–18.3), kamfen (%1.8–12.7), α-pinen (%4.2–28.9), β-pinen (%3.7–19.5), sabinen (%1.2–9.8), mirsen (%0.9–7.4), β-karyofilen (%2.1–10.6) ve karyofilen oksit (%1.5–8.3) ana bileşenler olarak belirlenmiştir. Sonuçlar, A. millefolium genotiplerinin kültür koşullarında başarıyla yetiştirilebildiğini ve doğal popülasyonlardan kültüre alınan materyalin sürdürülebilir ve ekonomik bir üretim potansiyeline sahip olduğunu göstermektedir. Bu çalışma, civanperçeminin endüstriyel kullanımı, kalite standardizasyonu ve gelecekte yürütülecek adaptasyon ve ıslah çalışmalarına bilimsel temel oluşturmaktadır.

Spermatogenesis relies on the highly specialized interaction between germ cells and a supportive somatic niche, yet replicating this environment in vitro remains a major challenge. Primary human Sertoli cells (hSCs), key architects of this niche, often lose their phenotype under conventional culture conditions, limiting the establishment of a stable blood-testis barrier (BTB)-associated phenotype and their ability to support germ cells. Here, we present a structurally organized, human ECM-derived connective tissue equivalent (CTE) designed to support long-term maintenance and organization of hSCs in vitro. The CTE, generated from fibroblast-secreted matrix enriched in laminin, fibronectin, and collagen IV, reproduces key biochemical and physical features of a supportive microenvironment. hSCs introduced into the CTE as single cells or pre-formed spheroids were evaluated for survival, structural organization, phenotypic stability, and ECM remodeling. Both configurations supported progressive expression and organization of BTB-associated proteins (ZO-1, OCLDN) together with upregulation of Sertoli cell-associated markers, including SOX9 and ABP, with the spheroid-based model showing improved structural cohesion, integration within the construct, and more evident junctional organization over time. Overall, this bioactive human-derived platform supports long-term maintenance of hSC phenotype and barrier-associated features in vitro, providing a promising basis for future human testis models and co-culture studies.

Organoid models have transformed experimental biology by enabling three-dimensional systems that recapitulate tissue architecture, cellular heterogeneity, and metabolic activity patterns more faithfully than conventional in vitro cultures. In parallel, metabolomics has emerged as a systems-level approach to interrogate the biochemical processes integrating genetic programs, environmental cues, and phenotypic outcomes. Despite this promise, the application of metabolomics to organoids remains analytically fragile, challenged by low sample biomass, complex extracellular matrices, heterogeneous culture conditions, and substantial variability in experimental and computational workflows. This systematic review critically examines metabolomics and lipidomics applications across intestinal, hepatic, renal, cerebral, vascular, and tumor-derived organoids, spanning development, disease modeling, toxicology, and drug response. We synthesize how metabolic profiling provides functional insights often inaccessible to transcriptomic or morphological analyses alone. Particular emphasis is placed on analytical design and quality control, highlighting how matrix-aware strategies, normalization choices, and QC-driven preprocessing critically shape metabolite recovery, reproducibility, and biological interpretability. By comparing targeted and untargeted approaches, mass spectrometry- and NMR-based platforms, and extracellular matrix mitigation strategies, we identify recurring sources of analytical variability and interpretative bias. We further propose a minimal, context-aware QC framework tailored to the specific constraints of organoid-based metabolomics. Collectively, this work provides a critical analytical reference to strengthen reproducibility, comparability, and translational robustness in 3D organoid metabolomics.

Influence of culture conditions and dietary regimes on survival, population dynamics and naupliar production of the copepod Paramphiascella fulvofasciata (Rosenfield & Coull, 1974)

Myxofibrosarcoma (MFS) is a rare soft-tissue sarcoma with limited systemic therapy options, necessitating preclinical platforms that better simulate clinical drug responses. We investigated how 2D monolayers versus 3D spheroids shape the baseline transcriptome and doxorubicin (DOX)-responsive programs across six patient-derived MFS cell lines. RNA sequencing revealed that 3D culture induces a distinct transcriptomic state characterized by the enrichment of microenvironment-associated stress programs, such as hypoxia, inflammatory/NF-κB signaling, and glycolysis, alongside the suppression of proliferation-related pathways. Although the global DOX-induced transcriptional response was highly environment-dependent, we identified a robust core of six regulators-MCRIP1, FGF12, HGF, EMSY, FZD2, and SECISBP2-whose transcriptional changes consistently correlated with cell survival rates across both 2D and 3D geometries. These genes are involved in transcriptional plasticity, redox homeostasis, and bypass survival signaling, providing a mechanistic basis for DOX resistance that transcends culture conditions. Our findings demonstrate that while culture geometry is a critical determinant of the MFS transcriptome, a robust set of environment-agnostic regulators dictates DOX efficacy. Integrating 3D systems with these specific transcriptomic readouts enhances the interpretability of drug screenings and supports the prioritization of rational therapeutic combinations for this rare sarcoma.

The cultivation of Pleurotus ostreatus mushrooms has gained popularity in the Philippines because of its ability to grow across a wide range of temperatures and to utilize diverse lignocellulosic substrates. However, optimizing culture conditions is essential for improving the growth efficiency of sporeless strains, which are preferred due to reduced health risks and better quality of mushroom products. This study aimed to determine the optimum cultural conditions for the mycelial growth of P. ostreatus (sporeless strain) by evaluating the effects of media type, pH level, aeration (sealed vs. unsealed plates), illumination (dark, light, or alternating dark/light), and incubation temperature. Mycelial growth was assessed across five different culture media and varying environmental conditions. Results revealed that rice bran decoction gulaman supported the most favorable growth, with near-neutral to slightly basic pH, under sealed or non-aerated conditions. Growth was consistent across dark, light, or alternating illumination, and was optimal at both room temperature and air-conditioned settings. These findings demonstrate that the sporeless strain exhibits strong adaptability to diverse conditions, reducing cultivation constraints compared with spore-forming counterparts. This research establishes the specific cultural requirements for the sporeless strain, which has been less studied despite its advantages. Optimizing its growth conditions can enhance mushroom production efficiency, minimize spore-related health risks for growers, and promote sustainable utilization of local agro-industrial byproducts, contributing to food security and rural livelihood development. This research supports SDGs by contributing to SDGs 2 (Zero Hunger) through improved mushroom production efficiency and food security; SDGs 3 (Good Health and Well-being) by reducing spore-related occupational health risks; SDGs 8 (Decent Work and Economic Growth) by strengthening livelihood opportunities in small-scale enterprises; SDGs 12 (Responsible Consumption and Production) through the sustainable use of agro-industrial byproducts; and SDGs 13 (Climate Action) by promoting resource-efficient cultivation practices aligned with the sustainability framework of the United Nations Sustainable Development Goals.

Sturgeons (order Acipenseriformes) represent one of the most ancient lineages of Actinopterygii. Presently, most sturgeon species are classified as critically endangered. In this study, a novel ovarian cell line which contained germline cells derived from the sterlet (Acipenser ruthenus) was established, designated as the ARO cell line. Germline cells in the cell line were identified using marker gene analysis, alkaline phosphatase activity assay and immunofluorescence staining. Transmission electron microscope observation showed that previtellogenic oocytes existed in the ARO cell line. Flow cytometry analysis indicated the cell line maintained meiotic competence to generate DNA reduced ploidy (diploid) oocytes. Under 3D culture condition, germinal vesicle (GV) stage, germinal vesicle breakdown (GVBD) stage and meiosis I oocytes were found. Furthermore, the cell line exhibited sustained proliferation and self-organization into reconstituted ovaries (rOvaries). Transcriptome analysis of rOvaries revealed that rOvaries had formed cell junctions and normal intercellular signaling, and possessed normal endocrine function and oocyte-producing capacity. In summary, the established ARO cell line exhibited stable passaging over multiple generations in vitro, was capable of sustainably generating oocytes, and was expected to be applicable to the establishment of ovarian organoids.

Mechanical confinement plays an important role in regulating tumor growth and invasion; however, the quantitative, time-resolved, three-dimensional evaluation of confined tumor spheroids remains technically challenging. In this study, we developed a custom-built selective plane illumination microscopy (SPIM)-based monitoring platform for long-term volumetric imaging of tumor spheroids under mechanically confined conditions. This system integrates a culture housing unit and a transparent cuvette-based spheroid culture method optimized for SPIM observation. Colorectal adenocarcinoma-derived cell spheroids were embedded in agarose gels with defined concentrations to modulate the stiffness of the surrounding matrix. Bright-field imaging and viability analyses confirmed sustained spheroid growth without necrotic core formation over a 4-day culture period, demonstrating that the SPIM-based system maintained the physiological culture conditions. Three-dimensional imaging using SPIM enabled a quantitative evaluation of spheroid growth and anisotropic invasion. Volumetric expansion was observed under all confinement conditions. Notably, increasing the matrix stiffness enhanced both the volumetric growth rate and anisotropic invasion, indicating stiffness-dependent directional growth under mechanical confinement. The developed SPIM-based platform has the potential to serve as a practical tool for the time-resolved three-dimensional analysis of tumor spheroid growth and may provide a useful approach for investigating the mechanobiological regulation of tumor progression in confined microenvironments.

Leptolyngbya JSC-1 is a thermophilic and siderophilic cyanobacterium inhabiting iron-rich hot springs. Response surface method (RSM) is being reported for the first time for optimizing growth conditions of this thermophilic and siderophilic cyanobacterium. Using response surface quadratic model of Box-Behnken design, optimal culture conditions (A: temperature, 45°C; B: Fe concentration, 42 µM; and C: light intensity, 2000lx which is equivalent to 27 µmol photons m⁻² s⁻¹ intensity of cool white fluorescent lamp) were determined. The significant model terms were found to be B, AB, A2, B2, and C2. The model R2 value (coefficient of determination) was 0.939, suggesting that the fitted model could explain 93.9% of the total variation. Both the predicted response (OD730 = 2.133) and experimental response (OD730 = 2.1) were in proximity, suggested the appropriateness of the model and RSM. Moreover, an unusual inverse proportion was observed between the Fe concentration and ROS generation with the least ROS generation in JSC-1 grown with 42 µM Fe concentration. Hence, RSM allows evaluating the effects of multiple factors and their interactions on one or more response variables and is recommended to be used for multifactorial optimization studies.

Embryonic stem cells (ESCs) derived from the inner cell mass of embryos possess unlimited self-renewal and pluripotency, offering a powerful system to study early development and enable genetic and biotechnological innovation. Although several livestock ESC lines have been reported in recent years, defining culture conditions that support stable long-term self-renewal and controlled transitions across pluripotent states remains challenging. Here, we report the de novo derivation of sheep (ovine) embryonic stem cells (oESCs) from in vivo blastocysts using a chemically defined culture system. The derived cells exhibit morphological and molecular features of primed pluripotency and can be propagated under both feeder-dependent and feeder-free conditions without loss of identity or karyotypic stability. Building on this foundation, we developed enhancer-driven reporter lines that faithfully reflect (Octamer-binding transcription factor 4) OCT4 and (SRY-box transcription factor 2) SOX2 transcriptional activity, enabling dynamic visualization of pluripotency and differentiation in live cultures. These reporter systems revealed the responsiveness of oESCs to signaling modulation and provided a functional readout of pluripotency state transitions. When cultured in defined media previously shown to stabilize naïve pluripotency in human ESCs, oESCs adopted dome-shaped colony morphology, maintained OCT4, SOX2, and NANOG expression, retained differentiation potential, and exhibited a transcriptomic profile consistent with resetting to an intermediate pluripotent state with naïve-like morphological features. These findings establish stable ovine ESC lines and demonstrate their plasticity across the pluripotency spectrum, providing a valuable platform for investigating ruminant stem cell biology and advancing livestock biotechnology.