BACKGROUND: Fungi and bacteria coexist in a wide variety of environments, and their interactions are now recognized as the norm in most agroecosystems. These microbial communities harbor keystone taxa, which facilitate connectivity between fungal and bacterial communities, influencing their composition and functions. The roots of most plants are associated with arbuscular mycorrhizal (AM) fungi, which develop dense networks of hyphae in the soil. The surface of these hyphae (called the hyphosphere) is the region where multiple interactions with microbial communities can occur, e.g., exchanging or responding to each other\'s metabolites. However, the presence and importance of keystone taxa in the AM fungal hyphosphere remain largely unknown.
RESULTS: Here, we used in vitro and pot cultivation systems of AM fungi to investigate whether certain keystone bacteria were able to shape the microbial communities growing in the hyphosphere and potentially improved the fitness of the AM fungal host. Based on various AM fungi, soil leachates, and synthetic microbial communities, we found that under organic phosphorus (P) conditions, AM fungi could selectively recruit bacteria that enhanced their P nutrition and competed with less P-mobilizing bacteria. Specifically, we observed a privileged interaction between the isolate Streptomyces sp. D1 and AM fungi of the genus Rhizophagus, where (1) the carbon compounds exuded by the fungus were acquired by the bacterium which could mineralize organic P and (2) the in vitro culturable bacterial community residing on the surface of hyphae was in part regulated by Streptomyces sp. D1, primarily by inhibiting the bacteria with weak P-mineralizing ability, thereby enhancing AM fungi to acquire P.
CONCLUSIONS: This work highlights the multi-functionality of the keystone bacteria Streptomyces sp. D1 in fungal-bacteria and bacterial-bacterial interactions at the hyphal surface of AM fungi. Video Abstract.

This study was envisaged to investigate the physiological reasons affecting the embryo development and abortion of seedless grapes on the basis of the previous embryo rescue breeding techniques of seedless grapes. Specifically, the relationship between the embryo rescue breeding of seedless grapes and the change of polyamine content was evaluated, in order to provide hybrid germplasm in the breeding of new seedless grape cultivars. Four ovules of 4 naturally pollinated Eurasian seedless grape cultivars, including \'Thompson Seedless\' grape (hereinafter referred to as \'Seedless White\' grape), \'Flame Seedless\' grape, \'Heshi Seedless\' grape and \'Ruby Seedless\' grape were employed for the study. Changes in the endogenous polyamine content, exogenous polyamine content, and the suitable combination of exogenous polyamines in the seedless grape berries and isolated ovules were determined during the best embryo rescue period. Furthermore, the effect of different exogenous polyamine contents on the germination and seedling rate of different seedless grape embryos was analyzed. In the best embryo rescue period, the number of ovules had different effects on the content of polyamines. For seedless grape cultivars with 4 ovules, a high content of polyamines was found to be more beneficial in the embryonic development. The existence of embryos had different effects on the development of embryos. In the ovules with embryo, an increase in the content of polyamine was beneficial to the growth and development of the ovule. Different ratios of exogenous polyamines had varying effects on the embryonic development. Putrescine (Put) exhibited the greatest effect on the embryonic development. Further, correlation analysis showed that different combinations of exogenous polyamines had varying effects on the embryonic development. A maximal ovule development was observed in the combination of exogenous polyamines of putrescine2+spermidine2+spermine1. For maximal embryo germination and seeding formation, the optimal combination was putrescine2+spermidine2+spermine2. Irrespective to the number of ovules or the existence of embryos, the results indicated that a high content of endogenous polyamines promoted the growth and development of embryos. The embryo rescue efficiency of different exogenous polyamines was different, and the appropriate combination of exogenous polyamines was beneficial to the growth and development of ovules, with a high development rate of the ovule and seedling.

Zinc, an essential trace mineral, exerts a pivotal influence in various biological processes. Through zinc concentration analysis, we found that the zinc concentration in the bovine embryo in vitro culture (IVC) medium was significantly lower than that in bovine follicular fluid. Therefore, this study explored the impact of zinc sulfate on IVC bovine embryo development and investigated the underlying mechanism. The results revealed a significant decline in zygote cleavage and blastocyst development rates when zinc deficiency was induced using zinc chelator N, N, N\', N\'-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) in culture medium during embryo in vitro culture. The influence of zinc-deficiency was time-dependent. Conversely, supplementing 0.8 μg/mL zinc sulfate to culture medium (CM) increased the cleavage and blastocyst formation rate significantly. Moreover, this supplementation reduced reactive oxygen species (ROS) levels, elevated the glutathione (GSH) levels in blastocysts, upregulated the mRNA expression of antioxidase-related genes, and activated the Nrf2-Keap1-ARE signaling pathways. Furthermore, 0.8 μg/mL zinc sulfate enhanced mitochondrial membrane potential, maintained DNA stability, and enhanced the quality of bovine (in vitro fertilization) IVF blastocysts. In conclusion, the addition of 0.8 μg/mL zinc sulfate to CM could enhance the antioxidant capacity, activates the Nrf2-Keap1-ARE signaling pathways, augment mitochondrial membrane potential, and stabilizes DNA, ultimately improving blastocyst quality and in vitro bovine embryo development.

Wide hybridizations across species and genera have been employed to enhance agriculturally important traits in crops. Within the tribe Maleae of the Rosaceae family, different genera and species exhibit several traits useful for increasing diversity and gene pool through hybridization. This study aimed to develop and characterize intergeneric hybrid individuals between Malus and Pyrus. Through seed germination, shoot multiplication, and rooting in vitro, acclimatized seedlings showing vegetative growth on their own roots were obtained from crosses of Malus × domestica pollinated by Pyrus communis, P. bretschneideri, and the Pyrus interspecific hybrid (P. communis × P. pyrifolia). Comparative analysis of leaf morphology, flow cytometry, and molecular genotyping confirmed the hybrid status of the individuals. Genome-wide genotyping revealed that all the hybrid individuals inherited genomic fragments symmetrically from the Malus and Pyrus parents. To the best of our knowledge, this is the first report on the development of intergeneric hybrid seedlings between Malus × domestica and P. bretschneideri. Furthermore, the Pyrus interspecific hybrid individual served as a bridge plant for introducing the genetic background of P. pyrifolia into Malus × domestica. The results of this study provided a crucial foundation for breeding through intergeneric hybridization between Malus and Pyrus, facilitating the incorporation of valuable traits from diverse gene pools.

OBJECTIVE: Does a matrix-free culture system supplemented with neurotrophic factor 4 (NT4) improve human in vitro follicular development and meiotic maturation, ultimately resulting in fertilizable oocytes?
CONCLUSIONS: NT4 supplementation of in vitro culture significantly enhances the growth, steroid hormone production, and maturity potential of human secondary follicles derived from fresh ovarian medulla (from post- and pre-pubertal patients), thereby yielding fertilizable oocytes.
BACKGROUND: Reconstituting folliculogenesis in vitro is of paramount importance in the realms of fertility preservation, reproductive biology research, and reproductive toxicity assessments. However, the efficiency of in vitro culture systems remains suboptimal, as the attainment of fertilizable oocytes from in vitro growth (IVG) of human follicles remains unachieved, with the data being particularly scant regarding follicles from prepubertal girls. We have previously found that mouse oocytes from secondary follicles derived from IVG are deficient in neuroendocrine regulation. NT4 and its corresponding receptor have been identified in human follicles. Significantly, the addition of NT4 during the IVG process markedly enhances both follicle growth and oocyte maturation rates in mice.
UNASSIGNED: Fresh medulla tissue obtained during tissue preparation for ovarian tissue cryopreservation (OTC) were collected from 10 patients aged from 6 to 21 years old, all of whom had undergone unilateral oophorectomy as a means of fertility preservation. Isolated secondary follicles were individually cultured in vitro with or without NT4 in a matrix-free system.
METHODS: Secondary follicles, extracted via enzymatic digestion and mechanical disruption from each patient, were randomly allocated to either a control group or an NT4-supplemented group (100 ng/ml), followed by individual culture on an ultra-low attachment plate. Follicle growth and viability were assessed by microscopy. Levels of anti-Müllerian hormone (AMH), estradiol, and progesterone in the medium were quantified. An oocyte-specific marker was identified using confocal fluorescence microscopy following DEAD box polypeptide 4 (DDX4) staining. The competence of individual oocytes for maturation and fertilization were assessed after IVM and ICSI with donated sperm samples.
RESULTS: Overall, isolated follicles from both groups survived up to 6 weeks with increasing diameters over the duration (P < 0.05), reaching terminal diameters of almost 1 mm with confirmed steroidogenesis and expression of oocyte marker (DDX4), and producing morphologically normal MII oocytes. When compared with the control group, the NT4 group had a similar initial follicular diameter (206 ± 61.3 vs 184 ± 93.4 μm) but exhibited a significant increase in follicular diameter from the ninth day of culture onwards (P < 0.05). From Week 3, estradiol and progesterone production were significantly increased in the NT4 group, while no significant difference was observed in AMH production between groups. The proportion of \'fast-growth\' follicles in the NT4 group was significantly higher than that in the control group (13/23 vs 6/24, P < 0.05). An increased efficiency of MII oocyte maturation per live follicle in the NT4 group was also observed (control group vs NT4 group, 4/24 vs 10/23, P < 0.05). It is noteworthy that an MII oocyte obtained from the control group exhibited abnormal fertilization after ICSI. In contrast, an MII oocyte acquired from the NT4 group progressed to the blastocyst stage and showed potential for transfer.
METHODS: N/A.
CONCLUSIONS: The cohort examined in this study was all patients diagnosed with beta-thalassemia major. Whether this culture system is effective for patients with other diseases remains unknown. Since the chosen dose of NT4 was established based on dose finding in mice, the optimal dose for use in a human IVG system needs further confirmation. The oocytes and embryos procured from this study have not been quantified for ploidy status or epigenetic signatures.
CONCLUSIONS: Fresh medulla tissue obtained during tissue preparation for OTC may serve as a precious source of fertilizable oocytes for female fertility preservation, even for pre-pubertal girls, without the threat of tumour reintroduction. After further characterization and optimization of the system, this culture system holds the potential to provide a powerful future research tool, for the comprehensive exploration of human follicular development mechanisms and for conducting reproductive toxicity evaluations.
BACKGROUND: This work was supported by the National Key R&D Program of China (grant number 2022YFC2703000) and National Natural Science Foundation of China (grant numbers 82271651 and 81871214). The medium used in human follicle in vitro culture in this study has been applied for a national invention patent in China (No. 202211330660.7). The inventors of the patent, in order, are: Y.G., C.F., and X.L.

Male germline stem cells (mGSCs), also known as spermatogonial stem cells (SSCs), are the fundamental seed cells of male animal reproductive physiology. However, environmental influences, drugs, and harmful substances often pose challenges to SSCs, such as population reduction and quality decline. With advancements in bioengineering technology and biomaterial technology, an increasing number of novel cell culture methods and techniques have been employed for studying the proliferation and differentiation of SSCs in vitro. This paper provides a review on recent progress in 3D culture techniques for SSCs in vitro; we summarize the microenvironment of SSCs and spermatocyte development, with a focus on scaffold-based culture methods and 3D printing cell culture techniques for SSCs. Additionally, decellularized testicular matrix (DTM) and other biological substrates are utilized through various combinations and approaches to construct an in vitro culture microenvironment suitable for SSC growth. Finally, we present some perspectives on current research trends and potential opportunities within three areas: the 3D printing niche environment, alternative options to DTM utilization, and advancement of the in vitro SSC culture technology system.

Nitrate is a primary nitrogen source for plant growth, and previous studies have indicated a correlation between nitrogen and browning. Nitrate transporters (NRTs) are crucial in nitrate allocation. Here, we utilized a genome-wide approach to identify and analyze the expression pattern of 74 potential GbNRTs under nitrate treatments during calluses browning in Ginkgo, including 68 NITRATE TRANSPORTER 1 (NRT1)/PEPTIDE TRANSPORTER (PTR) (NPF), 4 NRT2 and 2 NRT3. Conserved domains, motifs, phylogeny, and cis-acting elements (CREs) were analyzed to demonstrate the evolutionary conservation and functional diversity of GbNRTs. Our analysis showed that the NPF family was divided into eight branches, with the GbNPF2 and GbNPF6 subfamilies split into three groups. Each GbNRT contained 108-214 CREs of 19-36 types, especially with binding sites of auxin and transcription factors v-myb avian myeloblastosis viral oncogene homolog (MYB) and basic helix-loop-helix (bHLH). The E1X1X2E2R motif had significant variations in GbNPFs, indicating changes in the potential dynamic proton transporting ability. The expression profiles of GbNRTs indicated that they may function in regulating nitrate uptake and modulating the signaling of auxin and polyphenols biosynthesis, thereby affecting browning in Ginkgo callus induction. These findings provide a better understanding of the role of NRTs during NO3- uptake and utilization in vitro culture, which is crucial to prevent browning and develop an efficient regeneration and suspension production system in Ginkgo.

Culturing cerebrovascular smooth muscle cells (CVSMCs) in vitro can provide a model for studying many cerebrovascular diseases. This study describes a convenient and efficient method to obtain mouse CVSMCs by enzyme digestion.
Mouse circle of Willis was isolated, digested, and cultured with platelet-derived growth factor-BB (PDGF-BB) to promote CVSMC growth, and CVSMCs were identified by morphology, immunofluorescence analysis, and flow cytometry. The effect of PDGF-BB on vascular smooth muscle cell (VSMC) proliferation was evaluated by cell counting kit (CCK)-8 assay, morphological observations, Western blotting, and flow cytometry.
CVSMCs cultured in a PDGF-BB-free culture medium had a typical peak-to-valley growth pattern after approximately 14 days. Immunofluorescence staining and flow cytometry detected strong positive expression of the cell type-specific markers alpha-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain 11 (SMMHC), smooth muscle protein 22 (SM22), calponin, and desmin. In the CCK-8 assay and Western blotting, cells incubated with PDGF-BB had significantly enhanced proliferation compared to those without PDGF-BB.
We obtained highly purified VSMCs from the mouse circle of Willis using simple methods, providing experimental materials for studying the pathogenesis and treatment of neurovascular diseases in vitro. Moreover, the experimental efficiency improved with PDGF-BB, shortening the cell cultivation period.

Cryptosporidiosis is a significant diarrheal disease in humans and animals. Immunodeficient mice are the primary small animal models, but their high costs and specialized breeding/housing requirements limit in vivo drug testing. Numerous anticryptosporidial lead compounds identified in vitro remain untested in vivo.
Cryptosporidium tyzzeri, a natural mouse parasite closely related to Cryptosporidium parvum and Cryptosporidium hominis, was isolated to establish an infection model in immunocompetent mice. The model was validated using classic anticryptosporidial drugs (paromomycin and nitazoxanide) and then employed to assess the efficacy of 3 new leads (vorinostat, docetaxel, and baicalein). An in vitro culture of C. tyzzeri was also developed to complement the animal model.
Chronic C. tyzzeri infection was established in chemically immunosuppressed wild-type mice. Paromomycin (1000 mg/kg/d) and nitazoxanide (100 mg/kg/d) demonstrated efficacy against C. tyzzeri. Vorinostat (30 mg/kg/d), docetaxel (25 mg/kg/d), and baicalein (50 mg/kg/d) were highly effective against C. tyzzeri infection. In vitro, nitazoxanide, vorinostat, docetaxel, and baicalein exhibited low to submicromolar efficacy against C. tyzzeri.
Novel in vivo and in vitro models have been developed for cost-effective anticryptosporidial drug testing. Vorinostat, docetaxel, and baicalein show potential for repurposing and/or optimization for developing new anticryptosporidial drugs.

Three-dimensional (3D) cell culture model is a system that co-culture carriers with 3D structural materials and different types of cells in vitro to simulate the microenvironment in vivo. This novel cell culture model has been proved to be close to the natural system in vivo. In the process of cell attachment, migration, mitosis and apoptosis, it could produce biological reactions different from that of monolayer cell culture. Therefore, it can be used as an ideal model to evaluate the dynamic pharmacological effects of active substances and the metastasis process of cancer cells. This paper compared and analyzed the different characteristics of cell growth and development under two-dimensional (2D) and 3D model culture and introduced the establishment method of 3D cell model. The application progress of 3D cell culture technology in tumor model and intestinal absorption model was summarized. Finally, the application prospect of 3D cell model in the evaluation and screening of active substance was revealed. This review is expected to provide reference for the development and application of new 3D cell culture models.
三维（3D）细胞培养模型是将具有3D结构的载体与不同类型的细胞在体外共培养，以模拟体内微环境的系统。这种新型细胞培养模型经证明与体内的自然系统接近，在细胞附着、迁移、有丝分裂和凋亡等过程产生不同于单层细胞培养的生物学反应，因此可作为评价活性物质动态药理作用和癌细胞转移过程的理想模型。本文对比分析了二维（2D）和3D模型培养下细胞生长发育的不同特点，介绍了3D细胞培养模型的建立方法，重点总结了3D细胞培养技术在肿瘤模型和肠道吸收模型中的应用进展，并揭示了3D细胞培养模型在活性物质评估和筛选中的应用前景。通过本文综述，期望可为新型3D细胞培养模型的开发和应用提供参考。.