Search Results (723)

Organoids allow to model healthy and diseased human tissues. and have applications in developmental biology, drug discovery, and cell therapy. Traditionally cultured in immersion/suspension, organoids face issues like lack of standardization, fusion, hypoxia-induced necrosis, continuous agitation, and high media volume requirements. To address these issues, we developed an air–liquid interface (ALi) technology for culturing organoids, termed AirLiwell. It uses non-adhesive microwells for generating and maintaining individualized organoids on an air–liquid interface. This method ensures high standardization, prevents organoid fusion, eliminates the need for agitation, simplifies media changes, reduces media volume, and is compatible with Good Manufacturing Practices. We compared the ALi method to standard immersion culture for midbrain organoids, detailing the process from human pluripotent stem cell (hPSC) culture to organoid maturation and analysis. Air–liquid interface organoids (3D-ALi) showed optimized size and shape standardization. RNA sequencing and immunostaining confirmed neural/dopaminergic specification. Single-cell RNA sequencing revealed that immersion organoids (3D-i) contained 16% fibroblast-like, 23% myeloid-like, and 61% neural cells (49% neurons), whereas 3D-ALi organoids comprised 99% neural cells (86% neurons). Functionally, 3D-ALi organoids showed a striking electrophysiological synchronization, unlike the heterogeneous activity of 3D-i organoids. This standardized organoid platform improves reproducibility and scalability, demonstrated here with midbrain organoids. The use of midbrain organoids is particularly relevant for neuroscience and neurodegenerative diseases, such as Parkinson’s disease, due to their high incidence, opening new perspectives in disease modeling and cell therapy. In addition to hPSC-derived organoids, the method’s versatility extends to cancer organoids and 3D cultures from primary human cells. Full article

Ensuring bacterial safety of blood transfusions remains a critical focus in medicine. We investigated a novel pathogen reduction technology utilizing nylon functionalized with synthetic dyes (nylon affinity networks) to capture and remove bacteria from plasma. In the initial screening process, we spiked phosphate buffer solution (PBS) and human plasma (1 mL each) with 10 or 100 colony forming units (cfu) of either Escherichia coli or Staphylococcus epidermidis, exposed the suspensions to affinity networks and assessed the extent of bacterial reduction using agar plate cultures as the assay output. Nineteen synthetic dyes were tested. Among these, Alcian Blue exhibited the best performance with both bacterial strains in both PBS and plasma. Next, bacterial suspensions of approximately 1 and 2 cfu/mL in 10 and 50 mL, respectively, were treated with Alcian Blue affinity networks in three sequential capture steps. This procedure resulted in complete bacterial depletion, as demonstrated by the lack of bacterial growth in the remaining fraction. The viability of the captured bacteria was confirmed by plating the post-treatment affinity networks on agar. Alcian Blue affinity networks captured and sequestered a few plasma proteins identified by liquid chromatography tandem mass spectrometry. These findings support the potential applicability of nylon affinity networks to enhance transfusion safety, although additional investigations are needed. Full article

The growing demand for natural and sustainable skincare products has driven interest in plant-based active ingredients, especially from in vitro cultures. This placebo-controlled study investigated the impact of a facial cream containing 2% Kickxia elatine (L.) Dumort cell suspension culture extract on various skin biophysical parameters. The cream was applied to the cheek once daily for six weeks on 40 healthy female volunteers between the ages of 40 to 49. The evaluated skin parameters including skin hydration, transepidermal water loss (TEWL), erythema intensity (EI), melanin intensity (MI), skin surface pH, and skin structure, wrinkle depth, vascular lesions, and vascular discolouration. The results indicated that significant improvements were observed in skin hydration (from 40.36 to 63.00 AU, p < 0.001) and there was a decrease in TEWL score (14.82 to 11.76 g/h/m², p < 0.001), while the skin surface pH was maintained (14.82 to 11.76 g/h/m², p < 0.001). Moreover, the K. elatine cell extract significantly improved skin structure values (9.23 to 8.50, p = 0.028), reduced vascular lesions (2.72 to 1.54 mm², p = 0.011), and lowered skin discolouration (20.98% to 14.84%, p < 0.001), indicating its moisturising, protective, brightening, and soothing properties. These findings support the potential use of K. elatine cell extract in dermocosmetic formulations targeting dry, sensitive, or ageing skin. Full article

Sunflower (Helianthus annuus L.) is an important oilseed crop in Northwest China, exhibiting resistance to salt and drought. Mining its excellent tolerance genes can be used for breeding. However, the current platforms for identifying gene function in sunflower is inadequate. The transient transformation system, which can rapidly validate gene function, shows promising prospects in research. In this study, we established an efficient transient expression transformation system for sunflower using three methods: Agrobacterium-mediated infiltration, injection, and ultrasonic-vacuum. The detailed procedures were as follows: Agrobacterium GV3101 carrying a GUS reporter gene on the pBI121 vector with an OD₆₀₀ of 0.8 as the bacterial suspension and 0.02% Silwet L-77 as the surfactant were utilized in all three approaches. For the infiltration method, seedlings grown hydroponically for 3 days were immersed in a bacterial suspension containing 0.02% Silwet L-77 for 2 h; for the injection method, the same solution was injected into the cotyledons of seedlings grown in soil for 4 to 6 days. Subsequently, the seedlings were cultured in the dark at room temperature for three days; for the ultrasonic-vacuum method, seedlings cultured in Petri dishes for 3 days were first subjected to ultrasonication at 40 kHz for 1 min, followed by vacuum infiltration at 0.05 kPa for 5–10 min. Agrobacterium-mediated transient transformation efficiency achieved by the three methods exceeded 90%, with gene expression being sustained for at least 6 days. Next, we employed the infiltration-based sunflower transient transformation technology with the Arabidopsis stable transformation platform to confirm salt and drought stress tolerance of candidate gene HaNAC76 from sunflower responding to various abiotic stresses. Altogether, this study successfully established an Agrobacterium-mediated transient transformation system for sunflower using these three methods, which can rapidly identify gene function and explore the molecular mechanisms underlying sunflower’s resistance traits. Full article

Silver nanoparticles (AgNPs), iron oxide nanoparticles (Fe₂O₄NPs), and multiwalled carbon nanotubes (MWCNTs) are widely used in various applications, such as biomedicine, environmental remediation, and agriculture. In addition, these nanomaterials can affect the production of bioactive compounds in plants that have pharmacological activities. In the current study, the in vitro plant cultures of Chinese lobelia (Lobelia chinensis Lour.) were established in MS medium and treated with 0, 12.5, 25, 37.5, and 50 mg L⁻¹ AgNPs or Fe₂O₄NPs, or MWCNTs. Initially, plants were grown for four weeks without any elicitors, and after that, the cultures were treated with nano-elicitors for one week. After five weeks, the effects of nano-elicitors were estimated on growth, total phenolic, flavonoids, polyacetylenes, and ABTS/DPPH/FRAP antioxidant activity was investigated. The results showed that lower levels of AgNPs (25 mg L⁻¹), Fe₂O₄NPs (25 mg L⁻¹), and MWCNTs (12.5 mg L⁻¹) favored the accumulation of fresh and dry biomass. Whereas, 37.5 mg L⁻¹ AgNPs, 25 mg L⁻¹ Fe₂O₄NPs, and 37.5 mg L⁻¹ MWCNTs enhanced the accumulation of total phenolics, flavonoids, specific phenolic compounds including chlorogenic acid, catechin, phloretic acid, coumaric acid, salicylic acid, naringin, myricetin, linarin, and polyacetylenes viz. lobetylonin and lobetyolin in higher concentrations. The plant extracts elicited by nanomaterials also depicted very good antioxidant activities according to ABTS, DPPH, and FRAP assays. These results suggest that specific nanomaterials, and at specific levels, could be used for the production of bioactive compounds from shoot cultures of Chinese lobelia. Full article

Colonization of plant tissues by bacterial endophytes might lead to qualitative and quantitative changes in secondary metabolites (SMs). In this work, in vitro co-culture experiments were performed using cell suspensions of the medicinal plant Alkanna tinctoria and eight of its bacterial endophytes. An untargeted metabolomics approach using Ultra-High-Performance Liquid Chromatography High-Resolution Mass Spectrometry (UHPLC-HRMS) was employed to investigate plant–microbe interactions. Hierarchical clustering analysis and principal component analysis highlighted significant modifications of specific regulation patterns in SM production, caused by bacterial endophytes. The annotation step lead to the identification of 32 stimulated compounds in A. tinctoria cell suspensions. Among them, 3′-hydroxy-14-hydroxyshikonofuran H (5), 8′-decarboxy-rosmarinic acid (18), 8‴-decarboxy-salvianolic B (23), 8″-8‴-didecarboxy-salvianolic acid B (26) were putatively identified for the first time. Our findings highlight that employing selected microbial inoculants under controlled conditions can be an effective strategy for enhancing or stimulating the production of specific high-value metabolites. Full article

This study assessed the viable and culturable microbial diversity that remained on equipment surfaces after hygiene procedures in Brazilian poultry and fish slaughterhouses. Food-contact surface samples were collected using sterile swabs in poultry (n = 50) and fish (Oreochromis niloticus, n = 50) slaughterhouses. The swab samples were used to prepare culture plates to recover viable and culturable cells. The grown plates were washed, and the total DNA of the cell suspension was extracted with a commercial kit. Sequencing of the total DNA extracted from cultures was targeted at the V3 and V4 regions of the 16S rRNA. DNA reads were analyzed by QIIME2 software, with results expressed in relative frequency (%RF). Alpha and beta diversity indexes were analyzed considering the spots of sample collection, type of industry, surfaces (smooth or modular), and materials (polypropylene, stainless steel, or polyurethane). The results showed that in the poultry slaughterhouse, the most abundant genera were Acinetobacter (27.4%), Staphylococcus (7.7%), and Pseudomonas (5.3%), while for the fish slaughterhouse, there was a higher abundance of Staphylococcus (27.7%), Acinetobacter (17.2%), and Bacillus (12.5%). Surface characteristics influenced the microbial diversity, with Acinetobacter spp. dominating modular surfaces and Staphylococcus spp. prevailing on smooth surfaces. The results obtained indicate there is an important resident microbiota that persists even after hygiene processes, and surface-specific cleaning strategies should be developed. Full article

Background/Objectives: Antimicrobial stewardship programs (ASPs) optimize antimicrobial use, improving outcomes and reducing resistance. This study assessed the impact of a ward-specific ASP. Methods: A pre/post quasi-experimental study was conducted in an internal medicine ward at a tertiary hospital in Padua, Italy. During the intervention year (September 2023–August 2024), a multidisciplinary team (infectious disease consultants, pharmacists, microbiologists, nurses, and hygienists) held bi-weekly ward-based audits, reviewing antimicrobial prescriptions and performing bedside assessments. Therapy adjustments followed guidelines and local epidemiology. Educational sessions and infection prevention and control (IPC) protocols were also reinforced. Outcomes were compared to the previous year, considering patient characteristics. The primary outcome was antimicrobial consumption (DDD/100 patient days, DDD/100PD); secondary outcomes included cost savings, length of stay (LOS), and mortality. Results: Fifty audits assessed 1074 patients and 1401 antimicrobial treatments. Patient characteristics were similar. Antibiotic suspension or de-escalation occurred in 37.9% and 22% of patients, respectively. AWARE ACCESS class use increased (+17.5%), while carbapenem (−54.4%) and fluoroquinolone (−42.0%) use significantly declined (p < 0.05). IPC and microbiological culture guidance were provided in 12.1% of cases. Antimicrobial consumption dropped from 107.7 to 84.4 DDD/100PD (p < 0.05). No significant changes in LOS or mortality were observed. Antimicrobial costs fell by 48.8% (with EUR 57,100 saved). Conclusions: ASP reduced antimicrobial consumption, improved prescription quality, and cut costs without compromising patient outcomes. Multidisciplinary collaboration, audits, and education proved essential. Future studies should assess long-term resistance trends and integrate rapid diagnostics for enhanced stewardship. Full article

Three-dimensional (3D) spheroid models have revolutionized in vitro cancer research by offering more physiologically relevant alternatives to traditional two-dimensional (2D) cultures. A systematic search identifies English-language studies on patient-derived cancer spheroids for drug screening, using defined inclusion and exclusion criteria, with data extracted on cancer type, culture methods, spheroid characteristics, and therapeutic responses. This manuscript evaluates the methods for spheroid formation and the cellular sources used, highlighting the diverse applications and preferences in this field. The five most investigated cancer origins for spheroid seeding are breast, colon, lung, ovary, and brain cancers, reflecting their clinical importance and research focus. Among seeding methodologies, forced-floating and scaffold-based methods predominate, demonstrating reliability and versatility in spheroid generation. Other techniques, including microfluidics, bioprinting, hanging drop, and suspension culture also play significant roles, each with distinct advantages and limitations. This review underscores the increasing use of spheroid models and the need for standardization in methodologies to enhance the reproducibility and translational potential in cancer research. Full article

Piriformospora indica is a beneficial endophytic fungus that promotes plant growth and root development by colonizing plant roots. In order to investigate whether P. indica could promote the growth of tissue-cultured Cerasus humilis seedlings, in this study, we co-cultivated P. indica colony segments (P+) and P. indica spore suspensions (P++) in the rooting medium, and plant biomass as well as chlorophyll and root hormone contents of ‘3-19-3’ tissue-cultured C. humilis seedlings were determined under P+, P++, and CK (without fungus inoculation) treatments. The results showed that above-ground biomass and chlorophyll content of P+-and P++-treated tissue-cultured seedlings were significantly increased, and root peroxidase (POD), indole-3-acetic-acid (IAA) content, and root activities were significantly enhanced, while jasmonic acid (JA) and 1-aminocyclopropane-1-carboxylic acid (ACC) contents were reduced. Moreover, the growth-promoting effects of P++ treatment were found to be stronger than those of P+ treatment. Our results confirmed that P. indica was able to promote the growth of tissue-cultured C. humilis seedlings and effectively promoted root development by regulating hormone content. Therefore, the application of P. indica in the production of C. humilis is promising, especially in the cultivation of elite varieties. Full article

Passiflora caerulea L., commonly known as the blue passionflower, is traditionally grown as an ornamental plant, but has a diverse chemical composition resulting in a wide range of biological activities that determine its pharmacological properties and use in medicine. Traditional propagation methods, including seed germination and vegetative cuttings, are often inefficient due to low germination rates, susceptibility to pathogens, and slow growth. In particular, P. caerulea presents significant challenges in germination due to its slow development. In this context, in vitro cultivation is used to enable rapid, large-scale plant production while maintaining genetic fidelity. The study of Passiflora tissue cultures began in 1966 and has since attracted increasing attention from researchers around the world. However, despite growing interest, studies specifically focused on the in vitro propagation of P. caerulea remain limited. This review aims to summarize existing knowledge on the main techniques used for in vitro culturing and propagation of P. caerulea, including organogenesis, somatic embryogenesis, and callogenesis. Particular attention is paid to the key factors that influence the initiation, growth, and regeneration of cultures, including the type of explant, the composition of the media, and the environmental conditions. Advances in the in vitro cultivation of P. caerulea have greatly improved the understanding and propagation of this species. Although in vitro cultivation offers several advantages, it is crucial to conduct thorough research on the selection of explants, their age, and the appropriate culture media to ensure optimal growth and development. Full article

Weeds compete with crops for resources, posing multiple negative impacts for agricultural production systems and triggering degradation of ecosystem services (e.g., alterations in the soil microbial community structure). Under the guidance of green plant protection, the development of efficient biocontrol strains with environmentally friendly characteristics has become a crucial research direction for sustainable agriculture. This study aimed to develop a fungal bioherbicide by isolating and purifying a pathogenic fungal strain (JZ-5) from infected redroot pigweed (Amaranthus retroflexus L.). The strain exhibited pathogenicity rates ranging from 23.46% to 86.25% against six weed species, with the most pronounced control efficacy observed against henbit deadnettle (Lamium amplexicaule L.), achieving a pathogenicity rate of 86.25%. Through comprehensive characterization of cultural features, morphological observations, and molecular biological identification, the strain was taxonomically classified as Fusarium oxysporum. Scanning electron microscopy revealed that seven days post-inoculation, F. oxysporum JZ-5 formed dense mycelial networks on the leaf surfaces of cluster mallow (Malva verticillata L.), causing severe tissue damage. Safety assessments demonstrated that the spore suspension (10⁴ spores/mL) had no adverse effects on three crops: hulless barley (Hordeum vulgare var. coeleste L.), wheat (Triticum aestivum L.), and potato (Solanum tuberosum L.). These findings suggest that F. oxysporum strain JZ-5 warrants further investigation as a potential bioherbicide for controlling three problematic weed species—Chenopodium album L. (common lambsquarters), Elsholtzia densa Benth. (dense-flowered elsholtzia), and Lamium amplexicaule L. (henbit deadnettle)—in cultivated fields of hulless barley (Hordeum vulgare var. coeleste L.), wheat (Triticum aestivum L.), and potato (Solanum tuberosum L.). This discovery provides valuable fungal resources for ecologically sustainable weed management strategies, contributing significantly to the advancement of sustainable agricultural practices. Full article

Background/Objectives: More than 33 billion chickens are industrially raised for meat and egg production globally and vaccinated against Marek’s disease virus (MDV). The antigenically related herpesvirus of turkey (HVT) is used as a live-attenuated vaccine, commonly provided as a recombinant vector to protect chickens against additional unrelated pathogens. Because HVT replicates in a strictly cell-associated fashion to low levels of infectious units, adherent primary chicken or duck embryo fibroblasts are infected, dislodged from the cultivation surface and distributed as cryocultures in liquid nitrogen to the site of application. Although viable cells are complex products, application of infected cells in ovo confers protection even in presence of maternal antibodies. Methods/Results: The aim of our study was to determine whether a continuous cell line in a scalable cultivation format can be used for production of HVT-based vaccines. The AGE1.CR cell line (from Muscovy duck) was found to be highly permissive in adherent cultures. Propagation in suspension, however, initially gave very low yields. The induction of cell-to-cell contacts in carrier-independent suspensions and a metabolic shock improved titers to levels suitable for vaccine production (>10⁵ infectious units/mL after infection with multiplicity of 0.001). Conclusions: Production of HVT is challenging to scale to large volumes and the reliance on embryonated eggs from biosecure facilities is complex. We demonstrate that a cell-associated HVT vector can be propagated in a carrier-independent suspension culture of AGE1.CR cells in chemically defined medium. The fed-batch production is independent of primary cells and animal-derived material and can be scaled to large volumes. Full article

Castanea mollissima Bl. is rich in nutrition and strong in stress resistance, and has nutritional, economic, and ecological values. A protoplast is impactful in somatic fusion and germplasm creation. Here, we propose an effective scheme for the construction of an embryonic suspension cell, protoplast isolation, and fusion. Studies have shown that when 1.0 g yellow loose embryonic callus was inoculated into MS + 1.5 mg∙L⁻¹ 6-BA + 0.2 mg∙L⁻¹ NAA + 0.5 mg∙L⁻¹ 2, 4-D liquid medium, a stable suspension cell line can be obtained. After further culturing for 2–4 days, protoplast isolation was performed. First, single-factor screening was conducted on the four enzymes, and then a two-factor random block was further set up to screen the enzyme combinations based on the results. We found that 1.0%cellulase R-10 + 0.5%pectolase Y-23 led to the highest protoplast yield (9.27 × 10⁶/g FW) and the highest activity (92.49%). Furthermore, the protoplast yield could be increased to 9.47 × 10⁶/g FW by adding 0.4 M mannitol and shaking for 8 h. The protoplasts were purified by centrifuging at 40× g for 4 min and then mixed with 30% PEG 6000 at a volume ratio of 1.5:1 for 25 min. The fusion rate could reach 70.00%. This study laid a foundation for the creation of new germplasm by Castanea mollissima Bl. Full article

E. coli attaches to, and forms biofilms on various surfaces, including latex and polystyrene, contributing to nosocomial spread. E. coli responds to both exogenous and endogenous insulin, which induces behavioral changes. Human insulin, a quorum signal surrogate for microbial insulin, may affect the ability of E. coli to interact with latex and polystyrene in the presence of various sugars. E. coli ATCC 25923 was grown in peptone (1%) yeast nitrogen base broth to either the logarithmic or stationary growth phase. Adherence to latex was determined using 6 × 6 mm latex squares placed in a suspension of washed cells (10³ CFU/mL; 30 min; 37 °C) in buffer containing insulin at 2, 20, and 200 µU/mL (Humulin^® R; Lilly) with and without mannose, galactose, fructose, sorbose, arabinose, xylose, lactose, maltose, melibiose, glucose-6-phosphate, glucose-1-phosphate, and glucosamine at concentrations reported to affect behavioral response. Attachment levels to latex were determined by the press plate method. Biofilm levels were measured in a similar fashion but with overnight cultures in flat bottom uncoated polystyrene plates. Controls were media, insulin, sugar, or buffer alone. Glucose served as the positive control. Overall, the stationary phase cells’ adherence to latex was greater, regardless of the test condition, than was measured for the logarithmic phase cells. The effect of insulin on adherence to latex was insulin and sugar concentration dependent. The addition of insulin (200 µU/mL) resulted in a significantly (p < 0.05) increased adherence to latex and biofilm formation on polystyrene compared with sugar alone for 12 of the 13 sugars tested with stationary phase bacteria and 10 of the 13 sugars tested with logarithmic phase bacteria. Adherence in response to sorbose was the only sugar tested that was unaffected by insulin. These findings show that insulin enhances E. coli’s association with materials in common usage in medical environments in a nutrition-dependent manner. Full article