7-day Culture Research Articles

Non-Canonical Wnt16 and microRNA-145 Mediate the Response of Human Bone Marrow Stromal Cells to Additively Manufactured Porous 3-Dimensional Biomimetic Titanium–Aluminum–Vanadium Constructs

Metal 3D printing is increasingly being used to manufacture titanium–aluminum–vanadium (Ti6Al4V) implants. In vitro studies using 2D substrates demonstrate that the osteoblastic differentiation of bone marrow stromal cells (MSCs) on Ti6Al4V surfaces, with a microscale/nanoscale surface topography that mimics an osteoclast resorption pit, involves non-canonical Wnt signaling; Wnt3a is downregulated and Wnt5a is upregulated, leading to the local production of BMP2 and semaphorin 3A (sema3A). In this study, it was examined whether the regulation of MSCs in a 3D environment occurs by a similar mechanism. Human MSCs from two different donors were cultured for 7, 14, or 21 days on porous (3D) or solid (2D) constructs fabricated by powder-bed laser fusion. mRNA and secretion of osteoblast markers, as well as factors that enhance peri-implant osteogenesis, were analyzed, with a primary focus on the Wnt family, sema3A, and microRNA-145 (miR-145) signaling pathways. MSCs exhibited greater production of osteocalcin, latent and active TGFβ1, sema3A, and Wnt16 on the 3D constructs compared to 2D, both of which had similar microscale/nanoscale surface modifications. Wnt3a was reduced on 2D constructs as a function of time; Wnt11 and Wnt5a remained elevated in the 3D and 2D cultures. To better understand the role of Wnt16, cultures were treated with rhWnt16; endogenous Wnt16 was blocked using an antibody. Wnt16 promoted proliferation and inhibited osteoblast differentiation, potentially by reducing production of BMP2 and BMP4. Wnt16 expression was reduced by exogenous Wnt16 in 3D cells. Addition of the anti-Wnt16 antibody to the cultures reversed the effects of exogenous Wnt16, indicating an autocrine mechanism. Wnt16 increased miR-145-5p, suggesting a potential feedback mechanism. The miR-145-5p mimic increased Wnt16 production and inhibited sema3A in a 3D porous substrate-specific manner. Wnt16 did not affect sema3A production, but it was reduced by miR-145-5p mimic on the 3D constructs and stimulated by miR-145-5p inhibitor. Media from 7-, 14-, and 21-day cultures of MSCs grown on 3D constructs inhibited osteoclast activity to a greater extent than media from the 2D cultures. The findings present a significant step towards understanding the complex molecular interplay that occurs in 3D Ti6Al4V constructs fabricated by additive manufacturing. In addition to enhancing osteogenesis, the 3D porous biomimetic structure inhibits osteoclast activities, indicating its role in modulating bone remodeling processes. Our data suggest that the pathway mediated by sema3A/Wnt16/miR145-5p was enhanced by the 3D surface and contributes to bone regeneration in the 3D implants. This comprehensive exploration contributes valuable insights to guide future strategies in implant design, customization, and ultimately aims at improving clinical outcomes and successful osseointegration.

Scaffolds fabricated via two-photon polymerisation for regulating cell morphology and differentiation

ABSTRACT Three-dimensional (3D) cell culture scaffolds play a key role in guiding cell fate. The fine-tunability of these scaffolds is essential for accurately replicating in vivo conditions and revealing cellular behaviours. In this study, two-photon polymerisation (TPP) technology was employed to create 3D scaffolds with woodpile and honeycomb architectures. The influence of scaffold geometry and pore dimensions on the proliferation, morphology, orientation, and differentiation of human mesenchymal stem cells (hMSCs) was investigated through multi-staining fluorescence and 3D confocal imaging technique. It was revealed that hMSCs cultured within these 3D scaffolds demonstrated higher density up to 15.04 ± 1.46 cells/100 × 100 μm2 after a 6-day culture compared to their 2D counterparts (7.47 ± 1.42 cells/100 × 100 μm2). Furthermore, hMSCs grown on the woodpile scaffolds displayed elongated morphology, with approximately 50% aligning along the columns. In contrast, hMSCs cultivated on honeycomb structures exhibited triangular and crescent cellular shapes, with a random orientation. Notably, compared to the control group, the cells on the scaffolds exhibited smaller nucleus areas, lower circularity, and aspect ratios, but these values increased as pore size increased. Furthermore, ALP staining showed a greater tendency for osteogenic differentiation with larger pore sizes. These TPP-fabricated 3D scaffolds hold immense promise for advancing understanding of cellular behaviour.

Leveraging the nanotopography of filamentous fungal chitin-glucan nano/microfibrous spheres (FNS) coated with collagen (type I) for scaffolded fibroblast spheroids in regenerative medicine.

Numerous naturally occurring biological structures have inspired the development of innovative biomaterials for a wide range of applications. Notably, the nanotopographical architectures found in natural materials have been leveraged in biomaterial design to enhance cell adhesion and proliferation and improve tissue regeneration for biomedical applications. In this study, we fabricated three-dimensional (3D) chitin-glucan micro/nanofibrous fungal-based spheres coated with collagen (type I) to mimic the native extracellular matrix (ECM) microenvironment. These collagen-coated fungal nano/microfibrous spheres (C-FNS) were utilized to construct 3D scaffolded spheroids of human fibroblasts through suspension culture for tissue engineering and regenerative medicine. The particle sizes of C-FNS ranged from 1.4 to 3.25 µm (average: 2.27 ± 0.38 µm), with a porosity of 81.17 %. Field emission-scanning electron microscopy (FE-SEM) revealed that C-FNS comprised continuous chitin-glucan fibers with an average diameter of 363 ± 61 nm (range: 203-512 nm), exhibiting a highly interconnected structure. The reduced arithmetic average roughness (Ra) and root mean square roughness (Rq) values of C-FNS compared to uncoated FNS suggested that collagen coating reduced surface roughness, resulting in a smoother surface that enhanced hydrophilicity, crucial for mammalian cell adhesion and spheroid formation. Moreover, the in vitro cytocompatibility of C-FNS with fibroblasts was evaluated using a resazurin-based PrestoBlue assay, which demonstrated a time-dependent increase in the metabolic activity of C-FNS/fibroblast spheroids during suspension culture for up to 14 days. FE-SEM images of C-FNS/fibroblast spheroids further revealed enhanced adhesion and proliferation of fibroblasts on the nano/microfibrous mycelial architecture, accompanied by the secretion of ECM components and formation of multilayered cell sheets over the 14-day culture period. Similarly, an assessment of the hemocompatibility of C-FNS with erythrocytes revealed the non-hemolytic properties of the biomaterial. Overall, the interaction between collagen-coated fungal chitin-glucan nano/microfibrous structures and mammalian cells holds significant potential for the development of novel, sustainable biomaterials with tailored properties for a myriad of biomedical applications, including tissue engineering, regenerative medicine, drug screening, and wound healing.

Regulation of fibronectin and collagens type I, III and VI by TNF-α, TGF-β, IL-13, and tofacitinib

Understanding how inflammatory cytokines influence profibrogenic wound healing responses in fibroblasts is important for understanding the pathogenesis of fibrosis. TNF-α and IL-13 are key cytokines in Th1 and Th2 immune responses, respectively, while TGF-β1 is the principal pro-fibrotic mediator. We show that 12-day fibroblast culture with TNF-α or IL-13 induces fibrogenesis, marked by progressively increasing type III and VI collagen formation, and that TGF-β1 co-stimulation amplifies these effects. Tofacitinib substantially reduced the formation of ECM proteins in response to IL-13, while fibrogenesis in response to TNF-α or TGF-β1 was marginally inhibited. The in vitro findings were supported by clinical observations in patients with active rheumatoid arthritis, which had elevated serum type III collagen formation, indicating ongoing fibrogenesis during inflammation. After 48–60 weeks of tofacitinib treatment, type III collagen degradation, aswell as formation, were significantly decreased compared to baseline, highlighting dual anti-inflammatory and anti-fibrogenic effects of tofacitinib. In contrast, other anti-inflammatory treatments including methotrexate, adalimumab and tocilizumab demonstrated anti-inflammatory effects only. Our results highlight fibro-inflammatory profiles associated with TNF-α or IL-13 stimulation, both alone and in combination with TGF-β1, and support the use of tofacitinib as an anti-fibrogenic treatment in chronic inflammatory conditions.

Enhancing the efficiency of the cultivation of cladocerans (Moina sp. and Diaphanosoma sp.) through dietary diversification strategies.

The establishment of a feeding regimen for cladocerans is crucial in contemporary aquaculture due to their significance as nutrient-rich live feeds for various aquatic species. Three experiments were conducted to optimize the growth and reproduction rates of cladocerans (Moina sp. and Diaphanosoma sp.) using various stocking densities, types of food, and feeding regimens, emphasizing the importance of Haematococcus sp. in enhancing aquaculture productivity. In the first experiment, five different initial stocking densities: 1×103 indv. L-1 (T1), 3×103 indv. L-1 (T2), 6×103 indv. L-1 (T3), 9×103 indv. L-1 (T4), and 12×103 indv. L-1 (T5) of cladocerans were tested over a 10-day culture period, where T3 demonstrated the best growth. Using the best initial stocking density from experiment 1, the second experiment assessed varying concentrations of Haematococcus sp.: 1×105, 5×105, 10×105, 15×105, and 20×105cells mL-1 in T1, T2, T3, T4 and T5, respectively as feed of cladocerans; among them, T3 exhibited the highest population density. Experiment 3 evaluated the effects of three different diets including live Haematococcus sp. (T1), baker's yeast (Saccharomyces cerevisiae) (T2), and Haematococcus sp.+baker's yeast (Saccharomyces cerevisiae) (T3) on the growth of cladocerans over 10 days. The study employed the optimal initial stocking density of cladocerans and the feeding concentration of Haematococcus sp. as determined from the prior two experiments. Notably, T3 yielded the highest zooplankton density in this investigation. These findings indicate that elevating the initial stocking density of cladocerans and increasing the concentrations of Haematococcus sp. as feed up to an optimal level can effectively enhance the population growth of cladocerans. Both very low and excessively high initial stocking density, as well as suboptimal and excessively high concentrations of Haematococcus sp., negatively impact the growth of cladocerans. Moreover, using Haematococcus sp. alone or in combination with baker's yeast (Saccharomyces cerevisiae) enhances the growth and reproduction rates of cladocerans, indicating potential benefits for aquaculture systems.

Three-Dimensional Printing of Hydrogel Blend Tissue Engineering Scaffolds with In Situ Delivery of Anticancer Drug for Treating Melanoma Resection-Induced Tissue Defects.

Surgery is considered the gold standard for treating melanoma, but the high recurrence rate after surgery still remains as a major challenge. Therefore, using doxorubicin (DOX) as a model drug, this study investigated the 3D printing of anticancer drug-loaded hydrogel blend scaffolds for inhibiting post-operation melanoma recurrence and for promoting tissue regeneration. Three-dimensional printing could successfully produce methacrylate-modified chitosan (CSMA) and methylcellulose (MC) hydrogel blend scaffolds. Polymer blend inks exhibited satisfactory printability, and the printed porous scaffolds showed good biocompatibility and mechanical properties. Three-dimensionally printed DOX-loaded hydrogel scaffolds displayed controlled drug release, which may effectively prevent/impede tumor recurrence after surgery. Furthermore, combining 3D printing and bioprinting, DOX-loaded and rat bone marrow mesenchymal stem cell (rBMSC)-laden scaffolds were created for assessing local DOX delivery on healthy tissues. Within the 14-day culture period, rBMSCs encapsulated in multilayered scaffolds that were incorporated with DOX displayed rejuvenated cell viability. The 3D printed and bioprinted dual purpose hydrogel scaffolds have the promise of combating tumor recurrence and providing structural support for tissue regeneration.

First report of Phytopythium helicoides causing root and crown rot on Rhododendron in Ohio and the United States.

Rhododendron is a diverse genus of evergreen and deciduous species grown in gardens worldwide for their attractive flowers and foliage. In summer 2023, nine of 12 potted Rhododendron 'Nova Zembla' plants purchased from a wholesale nursery in Ohio exhibited wilting, leaf and stem discoloration, and severely darkened and softened roots, which eventually progressed into dieback and plant death. Roots tested positive with a Phytophthora Immunostrip® (Agdia, Inc.), a common serological test for oomycete root rots. Roots were surface disinfested by soaking in 10% bleach for 30 s, rinsed three rinses in sterile water, then sectioned into small pieces and plated on oomycete-selective PARP-CMA medium (Ivors, 2015). Pure cultures were obtained by transferring hyphal tips onto potato dextrose agar (PDA) and incubating at 23 ± 2 °C under fluorescent light (12 h) for one week. Colonies on PDA displayed radiate to petaloid radial growth with minimal aerial aseptate mycelia. Sporangia production was induced by flooding mycelial plugs with sterile 10% V8 broth and incubating in the dark for 48 h at 23 ± 2 °C. The resulting mycelial mat was rinsed three times with sterile water then mounted on glass slides. Sporangia were terminal, globose and rarely obovoid, papillate or non-papillate, and measured 22.24-46.38 µm in length and 19.80-33.70 µm in width (n=35). Oospores were not observed in culture after 1 week of incubation at room temperature. DNA was extracted using the QIAGEN DNEasy Plant Mini Kit from mycelial mats grown in potato dextrose broth for 7 days at 26°C with 70 rpm shaking followed by straining. PCR and bidirectional sequencing were conducted with primers OomCOILevup/FM85mod (mtDNA COX1 region; Robideau 2011), and NL1/NL4 (rDNA D1-D2 large subunit; O'Donnell 1993). NCBI BLAST searches of the LSU and COX1 consensus sequences had 100% (637/637 bp; PQ381277) and 98.33% (708/720 bp; PQ383368) match, respectively, to the type strain of Phytopythium helicoides PF-he2 (OM337587.1, GCA_024867545.1). Five of the nine plants yielded isolates identified as P. helicoides by sequencing. Isolate FPH2023-33 was selected for in-depth characterization. Pathogenicity tests were conducted once using young (<6" height and diameter) Rhododendron 'PJM Elite Star' (n=6), and once using 'Cherries and Merlot' (n=6) plants. Plants were inoculated by placing five, 5-mm agar plugs taken from 3-day old V8 agar cultures or sterile V8 agar plates (control) into the root area of each plant. Plants were maintained in flood trays under saturated soil conditions for 30 days in the greenhouse at an average of 24°C and 70% RH. Uninoculated plants were kept in separate trays to prevent cross-contamination. At 30 DPI, 75% of inoculated 'PJM Elite Star' plants showed severe leaf curl and wilting, and 100% had darkened and weakened root systems, while 50% of inoculated 'Cherries & Merlot' plants showed stunting, and 75% showed mild discoloration of the roots and crown. Control plants of both cultivars remained asymptomatic. Phytopythium helicoides was re-isolated from all inoculated plants and confirmed to be identical to the original isolate using the same methods described above. No Phytopythium was recovered from the control plants. This is the first report of this pathogen causing root rot on Rhododendron in the United States. As chemical and cultural control practices for this recently described pathogen are not yet well-studied, its presence may have a significant impact in both nursery production and landscape settings.

Polysaccharide with anticancer activity from Grifola frondosa cultured in industrial wastewater of Agaricus bisporus.

Culture conditions for Grifola frondosa using Agaricus bisporus industrial wastewater as a medium were optimized using Plackett-Burman and Box-Behnken methodologies. Plackett-Burman screening identified culture temperature, shaking speed, and wastewater solubility as the key factors influencing G. frondosa biomass. The Box-Behnken design then established optimal fermentation conditions: 23.7°C, 202rpm shaking speed, 10.4% wastewater solubility, pH6, 150mL culture volume in a 250mL flask, 9-day culture duration, and 10% inoculation volume. Polysaccharides were isolated and purified from both the mycelia and the culture medium. UV and molecular weight analysis determined that the polysaccharides AIPGF01, NEPGF01, AEPGF01, and NIPGF01 were homogeneous, with molecular weights of 234.7kDa, 125.2kDa, 80.0kDa, and 48.6kDa, respectively. GC revealed that NIPGF01 and AIPGF01 contained only glucose, while NEPGF01 and AEPGF01 comprised both mannose and glucose in ratios of 12.37:83.69 and 10.06:85.78, respectively. FT-IR confirmed the presence of β-glycosidic linkages in all polysaccharides. NIPGF01 exhibited strong anticancer activity, significantly inhibiting human gastric cancer (MGC80-3) and liver cancer (Hep G2) cells. Apoptosis rates at 50μg/mL, 200μg/mL, and 800μg/mL of NIPGF01 were 38.5±3.5%, 61.4±4.3%, and 79.2±5.5%, respectively. Flow cytometry indicated that NIPGF01 induced apoptosis in MGC80-3 cells by arresting them in the S and G2 phases, leading to a marked reduction in G1 phase cells.

Evaluation of a direct lymphocyte proliferation test for the diagnosis of canine food allergies with delayed reactions after oral food challenge.

In humans, food allergies (FAs) are divided into those with immunoglobulin (Ig) E-mediated (immediate FA), cell-mediated (delayed FA) or both mechanisms (mixed FA). In dogs, lymphocyte stimulation tests have the highest concordance with oral food challenges (OFCs). To report the evaluation of a lymphocyte proliferation test (LPT) in dogs with FA and delayed reactions (≥6 h) after OFC. Thirty-five healthy and 28 dogs with delayed FA. Peripheral blood mononuclear cells (PBMCs) were isolated and automatically counted before and after a 5-day culture with food allergens. Stimulation indices (SIs) were then calculated. Food allergen-specific IgE was quantified using the Pet Allergy Xplorer (PAX). None of the 10 healthy laboratory beagles and 25 healthy pet dogs had an SI greater than the 3.0 cut-off, indicating a specificity of 100%. All 28 dogs with delayed FA had at least one positive stimulation to a food item that induced delayed flares after OFC; the sensitivity of this LPT for the identification of delayed canine FA was 100%. The LPT correctly identified 57 of 68 food items causing delayed flares after OFC (84%). The PAX was negative for food-specific IgE in 18 of the 28 dogs (64%), as expected for delayed FA. In three dogs (11%), PAX results overlapped with those of the LPT, suggesting a mixed FA. Food allergies with delayed reactions after OFC-those suspected of having a cell-mediated mechanism-seemed to be the most common type of FA in the studied dogs. The LPT was helpful in identifying such cases.

Neurotrophic extracellular matrix proteins promote neuronal and iPSC astrocyte progenitor cell- and nano-scale process extension for neural repair applications.

The extracellular matrix plays a critical role in modulating cell behaviour in the developing and adult central nervous system influencing neural cell morphology, function and growth. Neurons and astrocytes, play vital roles in neural signalling and support respectively and respond to cues from the surrounding matrix environment. However, a better understanding of the impact of specific individual extracellular matrix proteins on both neurons and astrocytes is critical for advancing the development of matrix-based scaffolds for neural repair applications. This study aimed to provide an in-depth analysis of how different commonly used extracellular matrix proteins- laminin-1, Fn, collagen IV, and collagen I-affect the morphology and growth of trophic induced pluripotent stem cell (iPSC)-derived astrocyte progenitors and mouse motor neuron-like cells. Following a 7-day culture period, morphological assessments revealed that laminin-1, fibronectin, and collagen-IV, but not collagen I, promoted increased process extension and a stellate morphology in astrocytes, with collagen-IV yielding the greatest increases. Subsequent analysis of neurons grown on the different extracellular matrix proteins revealed a similar pattern with laminin-1, fibronectin, and collagen-IV supporting robust neurite outgrowth. fibronectin promoted the greatest increase in neurite extension, while collagen-I did not enhance neurite growth compared to poly-L-lysine controls. Super-resolution microscopy highlighted extracellular matrix-specific nanoscale changes in cytoskeletal organization, with distinct patterns of actin filament distribution where the three basement membrane-associated proteins (laminin-1, fibronectin, and collagen-IV) promoted the extension of fine cellular processes. Overall, this study demonstrates the potent effect of laminin-1, fibronectin and collagen-IV to promote both iPSC-derived astrocyte progenitor and neuronal growth, yielding detailed insights into the effect of extracellular matrix proteins on neural cell morphology at both the whole cell and nanoscale levels. The ability of laminin-1, collagen-IV and fibronectin to elicit strong growth-promoting effects highlight their suitability as optimal extracellular matrix proteins to incorporate into neurotrophic biomaterial scaffolds for the delivery of cell cargoes for neural repair.

Developing a workflow for the isolation of hybridoma cells producing fully human antigen-specific antibodies using a surface IgG detection method

The antigen-mediated B cell isolation method, based on the detection of surface IgG (sIgG), has increased the efficiency of therapeutic antibody (Ab) discovery. However, the reduction in sIgG expression on B cells during plasma cell differentiation presents challenges as it enables Ab production from only a small subset of B cells (e.g., memory B cells). The present study aimed to addressed this problem by developing a workflow to isolate human-IgG-secreting hybridoma cells produced by cell fusion, the majority of which express sIgG. We showed that our sIgG-based antigen-coated bead separation method efficiently enriched hybridoma cells expressing antigen-specific Abs with a yield of 83.5% (from the cell fusion pool) and a positive rate of 73.2%. Furthermore, because the separation could be performed after only a short (1–2-day) culture period following cell fusion, diverse hybridoma clones could be obtained, minimizing clonal selection and the incidence of duplicates. Given that the expression of membrane-bound IgG and sIgG are regulated by different splicing mechanisms, we speculate that the cell fusion step potentially attenuated the suppression of human sIgG expression. Overall, our proposed method is expected to markedly improve the efficiency of therapeutic Ab candidate production, which will have important clinical implications.

Anti-Müllerian hormone signaling in the ovary involves stromal fibroblasts: a study in humans and mice provides novel insights into the role of ovarian stroma.

What is the involvement of ovarian stroma in the anti-Müllerian hormone (AMH) signaling pathway and which stromal cells are involved? Mouse and human ovaries show high expression of AMH receptor II (AMHR2) in the stromal fibroblasts surrounding the follicles and activation of the post-AMHR2 pathway by recombinant AMH was evidenced by increased phosphorylation of SMAD1,5 and 9, increased expression AMHR2 and upregulation of αSMA, suggesting fibroblast activation to initiate myofibroblast differentiation. AMH secreted by small growing follicles, regulates ovarian activity. It suppresses initial primordial follicle (PMF) recruitment and FSH-dependent growth. AMH signal transduction is mediated by AMHR2, activating intracellular SMAD proteins and other signaling cascades to induce target-gene expression. Although AMHR2 expression has been reported within the follicle unit, there is evidence suggesting it may be identified in the stroma as well. Fresh murine ovaries were extracted from BALB/c mice (6 weeks old; n = 12 and 21 days old; n = 56). Frozen-thawed ovarian fragments were obtained from 10 women, aged 18-35, who had undergone ovarian tissue cryopreservation and donated frozen ovarian tissue for research. Murine (6 weeks old) and human donor ovaries were immunostained for AMHR2 and Collagen 1α/αSMA/VCAM1, with additional vimentin staining in mice. Murine (21 days old) and human donor ovaries were used for fibroblast isolation and subsequent 7-day cultures. Prior to assessing AMH effects on isolated fibroblast culture, purity validation tests were implemented to ensure the absence of epithelial, immune, endothel, granulosa, and theca ovarian cell populations. The fibroblast culture's homogeneity was validated by RT-qPCR and western-blot assays, confirming negativity for E-cadherin, CD31, aromatase, CYP17A1, and positivity for αSMA and vimentin. Fibroblasts were then subjected to rAMH treatment in vitro (200 ng/ml) for 0-72h, with an additional time point of 96 h for human samples, followed by RT-qPCR, western blot, and immunocytochemistry (ICC) for AMHR2 expression. AMHR2 post-receptor signaling was examined by pSMAD1,5,9 levels via western blot. Activated fibroblast marker, αSMA, was assessed via western blot and ICC. Immunostaining of mouse and human ovarian tissue showed that stromal cells around follicles at all developmental stages exhibit high AMHR2 expression, while granulosa cells of growing follicles show considerably lower levels. The majority of these AMHR2-positive stromal cells were identified as fibroblasts (Collagen1α in mice and human; vimentin in mice). RT-qPCR, western blot, and immunostaining were performed on cultured mouse and human fibroblasts, confirming that they consisted of a pure fibroblast population (αSMA/vimentin positive and negative for other cell-type markers). A total of 99.81% (average 28.94 ± 1.34 cells/field in mice) and 100% (average 19.20 ± 1.39 cells/field in human samples) of these fibroblasts expressed AMHR2 (ICC). rAMH treated cultured fibroblasts showed increased pSMAD1,5 and 9 levels, demonstrating the effects of AMH on its downstream signaling pathway. pSMAD1,5 and 9 expression increased, as detected by western blot: 1.92-fold in mice (48 h, P = 0.026) and 2.37-fold in human samples (48 h, P = 0.0002). In addition, rAMH treatment increased AMHR2 protein expression, as observed in ICC (human): a 2.57-fold upregulation of AMHR2 Mean Fluorescence Intensity (MFI) (96 h, P = 0.00036), and western blot, showing a 4.2-fold time-dependent increase (48 h, P = 0.026) in mice and 2.4-fold change (48 h, P = 0.0003) in human donors. Exposure to rAMH affected AMHR2 transcription upregulation, with a 6.48-fold change (72 h, P = 0.0137) in mice and a 7.87-fold change (72 h, P < 0.0001) in humans. rAMH treatment induced fibroblast activation (αSMA positive), demonstrating the dynamic effects of AMH on fibroblast behavior. αSMA expression elevation was detected in ICC with a 2.28-fold MFI increase in humans (96 h, P = 0.000067), and in western blot with a 5.12-fold increase in mice (48 h, P = 0.0345) and a 2.69-fold increase in humans (48 h, P ≤ 0.0001). Activated AMHR2-positive stained fibroblast fractions were solely located around growing follicles, in both human and mice. In addition, a small population of AMHR2-positive stained theca cells (VCAM1 positive) was observed. N/A. Ex vivo, fibroblast gene expression might be changed by adhesion to the tissue-culture plate. Nevertheless, cultured fibroblasts (with and without rAMH) are subjected to the same conditions. Observations or significant differences can therefore be considered reliable. In addition, the presented effect of rAMH on fibroblasts is not directly linked to the known inhibitory effect of AMH on follicle activation. Clarifying the populations of AMH-responsive cells in the ovary provides a foundation for further investigation of the complex AMH signaling across the ovary. The composition of AMH-releasing and -responsive cells can shed light on the communication network between follicles and their environment, which may elucidate the mechanisms behind the AMH inhibitory effect on PMF activation. This work was financially supported by grants from the Kahn Foundation. There are no competing interests in this study. N/A.

First report of Rhizoctonia blight caused by Rhizoctonia solani on Chinese cabbage (Brassica rapa subsp. chinensis) in India.

During January and February 2021, foliar blight symptoms were observed on the leaves of Chinese cabbage (Pak choi) at Lembucherra research farm, College of Agriculture, Tripura, India. The incidence of disease symptoms ranged from 5 to 10% of the plants observed in the field. The symptomatic leaves showed grayish colored water-soaked lesions with an irreguar shape and size. A total of 10 symptomatic leaves (1 leaf per plant) from Chinese cabbage infected plant were sampled, surface decontaminated with 1% NaOCl, washed twice in sterile water, plated on 2% water agar, and incubated at 25 ± 2°C. Hyphal tips from mycelium of 7-day old culture (2 isolates from two different plants) with right-angled branching were transferred to potato dextrose agar (PDA) media (SRL, India). Cream or light brown hyphae that branched at right angles, with septa near the point of the origin of hyphae, and a slight constriction at the base of the branch) were visible under a microscope. Olive-brown sclerotia were observed after 5 days of incubation. Multiple nuclei per cell were visible after staining with 4', 6-diamidino-2-phenylindole (Estandarte et al. 2016). Based on morphological characteristics (Parmeter et al. 1970) the isolates TP36 and TP37 were identified as Rhizoctonia solani. The internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were amplified with ITS1& ITS4 (White et al. 1990) and (GAPDH F-5'- CAAGGAGAACCCAGGTGTTAAG-3' and GAPDH R- 5'-GGCGTCGAAGATAGAAGAGTGT-3') respectively for both isolates and sequenced (accession #. PP458158, PP458159, PP425343, PP425344). BLASTn analysis showed 99.26%( 668/673 nt) to 99.46% (659/664 nt) identity with R. solani sequences (GenBank MG397062.1 and KX674524.1) for ITS and 98.42% (552/562 nt) to 100% 540/540 nt)identity with R. solani sequences (GenBank HQ425709.1 and CP102644.1) for GAPDH. Isolates TP36 and TP37 were deposited in the Indian Type Culture Collection (ITCC), New Delhi as R. solani (nos. 9154 and 9319, respectively). Both isolates were amplified using (anastomosis group) AG1 subgroup specific primers (Matsumoto 2002; Prashantha et al. 2021) to identify their AG. The presence of a 265 bp amplicon for both isolates suggested that they belong to AG1-IA. A multilocus analysis of R. solani isolates from different host plants with concatenated sequences ITS and GAPH showed that TP36 and TP37 are closely related to rice isolate RS107. A pathogenicity test on five plants per treatment was conducted and repeated twice on one month old Chinese cabbage plants (hybrid, TOKITA, India) grown under glasshouse conditions in a sterilized mixture of soil and sand (3:1) at 27-28oC during January 2024 at ICAR-IARI, New Delhi. R. solani isolates TP36 and TP37 were grown on PDA and plants were inoculated by placing single sclerotia of 10-day old colony on different plant parts and covering it with moist cotton. After 7 day, typical lesions of R. solani infection were visible. No symptoms were observed on the control plants. The fungus was reisolated from the inoculated plants and identified as R. solani based on morphology. R. solani has previously been reported to cause disease on some members of Brassicaceae in different countries (Budge et al. 2009; Hua et al. 2014). Based on literature available this is the first report of R. solani infecting Chinese cabbage in India.

Isolates of Acanthamoeba species in the marine environment in the Philippines.

Acanthamoebae spp. are considered the most commonly occurring free-living amoebae (FLA) in the environment. Their high resilience enables them to thrive in different types of environments. Using purposive sampling, 80 surface water samples were collected from identified coastal sites in Mariveles, Bataan, and Lingayen Gulf (40 water samples for each). Nineteen (23.75%) of the 80 water samples yielded positive amoebic growth during the 14-day culture and microscopic examination. The polymerase chain reaction confirmed Acanthamoeba spp. DNA in isolates MB1, A3, A4, A7, C5, and D3 using JDP1 and JDP2 primer sets. Further sequencing revealed that the isolates belonged to Acanthamoeba sp., Acanthamoeba culbertsoni, Acanthamoeba castellani, and Acanthamoeba genotype T4. The sequences were deposited in GenBank and registered under accession numbers PP741651, PP767364, PP741728, PP741729, PP767365, and PP767366, respectively. Potential risk factors such as waste disposal, expansion of human settlements to coastal locations, and soil runoffs in these environments should be controlled to mitigate the proliferation of potentially pathogenic strains of FLAs.

Three-dimensional culture in a bioengineered matrix and somatic cell complementation to improve growth and survival of bovine preantral follicles.

Here we explored poly(ethylene glycol) (PEG) bioengineered hydrogels for bovine preantral follicle culture with or without ovarian cell co-culture and examined the potential for differentiation of bovine embryonic stem cells (bESCs) towards gonadal somatic cells to develop a system more similar to the ovarian microenvironment. Bovine preantral follicles were first cultured in two-dimensional (2D) control or within PEG hydrogels (3D) and then co-cultured within PEG hydrogels with bovine ovarian cells (BOCs) to determine growth and viability. Finally, we tested conditions to drive differentiation of bESCs towards the intermediate mesoderm and bipotential gonad fate. Primary follicles grew over the 10-day culture period in PEG hydrogels compared to 2D control. Early secondary follicles maintained a similar diameter within the PEG while control follicles decreased in size. Follicles lost viability after co-encapsulation with BOCs; BOCs lost stromal cell signature over the culture period within hydrogels. Induction of bESCs towards gonadal somatic fate under WNT signaling was sufficient to upregulate intermediate mesoderm ( LHX1 ) and early coelomic epithelium/bipotential gonad markers ( OSR1 , GATA4 , WT1 ). Higher BMP4 concentrations upregulated the lateral plate mesoderm marker FOXF1 . PAX3 expression was not induced, indicating absence of the paraxial mesoderm lineage. Culture of primary stage preantral follicles in PEG hydrogels promoted growth compared to controls; BOCs did not maintain identity in the PEG hydrogels. Collectively, we demonstrate that PEG hydrogels can be a potential culture system for early preantral follicles pending refinements, which could include addition of ESC-derived ovarian somatic cells using the protocol described here. We demonstrate that three-dimensional bioengineered hydrogels could aid in the survival and growth of small bovine preantral follicles. Moreover, bovine embryonic stem cells have the potential to differentiate towards precursors of somatic gonadal cell types, presenting an alternative cell source for preantral follicle co-culture.

Pulsed electromagnetic fields potentiate bone marrow mesenchymal stem cell chondrogenesis by regulating the Wnt/β-catenin signaling pathway

BackgroundPulsed electromagnetic fields (PEMFs) show promise as a treatment for knee osteoarthritis (KOA) by reducing inflammation and promoting chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs).PurposeTo identify the efficacy window of PEMFs to induce BMSCs chondrogenic differentiation and explore the cellular mechanism under chondrogenesis of BMSCs in regular and inflammatory microenvironments.MethodsBMSCs were exposed to PEMFs (75 Hz, 1.6/2/3/3.8 mT) for 7 and 14 days. The histology, proliferation, migration and chondrogenesis of BMSCs were assessed to identify the optimal parameters. Using these optimal parameters, transcriptome analysis was performed to identify target genes and signaling pathways, validated through immunohistochemical assays, western blotting, and qRT-PCR, with or without the presence of IL-1β. The therapeutic effects of PEMFs and the effective cellular signaling pathways were evaluated in vivo.ResultsBMSCs treated with 3 mT PEMFs showed the optimal chondrogenesis on day 7, indicated by increased expression of ACAN, COL2A, and SOX9, and decreased levels of MMP3 and MMP13 at both transcriptional and protein levels. The advantages of 3 mT PEMFs diminished in the 14-day culture groups. Transcriptome analysis identified sFRP3 as a key molecule targeted by PEMF treatment, which competitively inhibited Wnt/β-catenin signaling, regardless of IL-1β presence or duration of exposure. This inhibition of the Wnt/β-catenin pathway was also confirmed in a KOA mouse model following PEMF exposure.ConclusionsPEMFs at 75 Hz and 3 mT are optimal in inducing early-stage chondrogenic differentiation of BMSCs. The induction and chondroprotective effects of PEMFs are mediated by sFRP3 and Wnt/β-catenin signaling, irrespective of inflammatory conditions.

Characterization of a human thyroid microtissue model for testing thyroid disrupting chemicals.

Perturbation of thyroid hormone (T4) synthesis is known to cause numerous developmental, metabolic, and cognitive disorders in humans. Due to species differences in sensitivity to chemical exposures, there is a need for human-based in vitro approaches that recapitulate thyroid cellular architecture and T4 production when screening. To address these limitations, primary human thyrocytes, isolated from healthy adult donor tissues and cryopreserved at passage one (p'1) were characterized for cellular composition, 3D follicular architecture, and thyroglobulin (TG)/T4 expression and inhibition by prototype thyroid disrupting chemicals (TDC). Flow analysis of the post-thaw cell suspension showed >80% EpCAM-positive cells with 10%-50% CD90-positive cells. When seeded onto 96-well Matrigel®-coated plates and treated with bovine thyroid stimulating hormone (TSH), thyrocytes formed 3D microtissues during the initial 4-5 days of culture. The microtissues exhibited a stable morphology and size over a 14-day culture period. TG and T4 production were highest in microtissues when the proportion of CD90-positive cells, seeding density and thyroid stimulating hormone concentrations were between 10%-30%, 6K-12Kcells per well, and 0.03-1 mIU/mL, respectively. At maximal TG and T4 production levels, average microtissue diameters ranged between 50 and 200µm. The T4 IC50 values for two prototype TPO inhibitors, 6-propyl-2-thiouracil and methimazole, were ∼0.7µM and ∼0.5 µM, respectively, in microtissue cultures treated between days 9 and 14. Overall, p'1 cryopreserved primary human thyrocytes in 3D microtissue culture represent a promising new model system to prioritize potential TDC acting directly on the thyroid as part of a weight-of-evidence hazard characterization.

Spatiotemporal analysis of multi-scale cell structure in spheroid culture reveals hypertrophic chondrocyte differentiation

3D cell culture has emerged as a promising approach to replicate the complex behaviors of cells within living organisms. This study aims to analyze spatiotemporal behavior of the morphological characteristics of cell structure at multiscale in 3D scaffold-free spheroids using chondrogenic progenitor ATDC5 cells. Over a 14-day culture period, it exhibited cell hypertrophy in the spheroids regarding cellular and nuclear size as well as changes in morphology. Moreover, biological analysis indicated a signification up-regulation of normal chondrocyte as well as hypertrophic chondrocyte markers, suggesting early hypertrophic chondrocyte differentiation. Cell nuclei underwent changes in volume, sphericity, and distribution in spheroid over time, indicating alterations in chromatin organization. The ratio of chromatin condensation volume to cell nuclear volume decreased as the cell nuclei enlarged, potentially signifying changes in chromatin state during hypertrophic chondrocyte differentiation. Our image analysis techniques in this present study enabled detailed morphological measurement of cell structure at multi-scale, which can be applied to various 3D culture models for in-depth investigation.

The Influence of Sulfation Degree of Glycosaminoglycan-Functionalized 3D Collagen I Networks on Cytokine Profiles of In Vitro Macrophage-Fibroblast Cocultures.

Cell-cell interactions between fibroblasts and immune cells, like macrophages, are influenced by interaction with the surrounding extracellular matrix during wound healing. In vitro hydrogel models that mimic and modulate these interactions, especially of soluble mediators like cytokines, may allow for a more detailed investigation of immunomodulatory processes. In the present study, a biomimetic extracellular matrix model based on fibrillar 3D collagen I networks with a functionalization with heparin or 6-ON-desulfated heparin, as mimics of naturally occurring heparan sulfate, was developed to modulate cytokine binding effects with the hydrogel matrix. The constitution and microstructure of the collagen I network were found to be stable throughout the 7-day culture period. A coculture study of primary human fibroblasts/myofibroblasts and M-CSF-stimulated macrophages was used to show its applicability to simulate processes of progressed wound healing. The quantification of secreted cytokines (IL-8, IL-10, IL-6, FGF-2) in the cell culture supernatant demonstrated the differential impact of glycosaminoglycan functionalization of the collagen I network. Most prominently, IL-6 and FGF-2 were shown to be regulated by the cell culture condition and network constitution, indicating changes in paracrine and autocrine cell-cell communication of the fibroblast-macrophage coculture. From this perspective, we consider our newly established in vitro hydrogel model suitable for mechanistic coculture analyses of primary human cells to unravel the role of extracellular matrix factors in key events of tissue regeneration and beyond.

Effects of microplastics on reproductive characteristics and mechanisms of the marine rotifer Brachionus plicatilis

Microplastic pollution, especially secondary microplastics (MPs), poses a significant threat to marine ecosystems. Despite its prevalence, the impact of natural-aged MPs on marine organisms, hindered by collection challenges, remains poorly understood. This study focused on 1–3 μm natural-aged MPs collected from Japan's coastal sea, investigating their effects on the rotifer Brachionus plicatilis sensu stricto and its reproductive mechanisms. Rotifers exposed to varying MP concentrations (0, 20, and 200 particles/mL) over 14-day batch cultures exhibited reduced population growth and fertilization rates. Down-regulation of reproductive genes and up-regulation of oxidative stress-related genes were observed, indicating MP-induced disruptions. Enhanced activities of superoxide dismutase and acetylcholinesterase and elevated malondialdehyde levels further emphasized oxidative stress. These findings underscore the detrimental impact of MPs on rotifer reproductivity, shedding light on the underlying mechanisms.