Co-culture Experiments Research Articles

SCRATCH: A programmable, open-hardware, benchtop robot that automatically scratches cultured tissues to investigate cell migration, healing, and tissue sculpting.

Despite the widespread popularity of the ' scratch assay', where a pipette is dragged through cultured tissue to create an injury gap to study cell migration and healing, the manual nature of the assay carries significant drawbacks. So much of the process depends on individual manual technique, which can complicate quantification, reduce throughput, and limit the versatility and reproducibility of the approach. Here, we present a truly open-source, low-cost, accessible, and robotic scratching platform that addresses all of the core issues. Compatible with nearly all standard cell culture dishes and usable directly in a sterile culture hood, our robot makes highly reproducible scratches in a variety of complex cultured tissues with high throughput. Moreover, we demonstrate how scratching can be programmed to precisely remove areas of tissue to sculpt arbitrary tissue and wound shapes, as well as enable truly complex co-culture experiments. This system significantly improves the usefulness of the conventional scratch assay, and opens up new possibilities in complex tissue engineering and cell biological assays for realistic wound healing and migration research.

Kuratsuki Bacteria Interactions with Sake Yeast and Effect on Taste

Various microorganisms, referred to as kuratsuki microorganisms, inhabit each sake brewery. Previously, kuratsuki yeasts had been used for sake production in each sake brewery. Kuratsuki lactic acid bacteria have been used to produce kimoto, a fermentation starter. Kuratsuki non-lactic acid bacteria were examined to evaluate their potential roles and effects in sake production. The addition of kuratsuki bacteria to the sake-making process can change the flavor and taste of the sake. This change was observed in both the coculture experiments between sake yeast and kuratsuki bacteria and the sake making tests with and without kuratsuki bacteria. The comprehensive gene expression analysis of sake yeast cocultured with kuratsuki bacteria showed that 1.2% of the yeast genes were upregulated and 1.0% were downregulated following the addition of kuratsuki bacteria. This indicates that the change in flavor and taste of sake due to the addition of kuratsuki bacteria was caused by the interaction between sake yeast and kuratsuki bacteria. To understand the implications of kuratsuki bacteria in sake production, it is essential to study the interactions between sake yeast and kuratsuki bacteria.

Endothelial estrogen - myocardial cGMP axis critically determines angiogenesis and cardiac performance during pressure-overload.

Estrogen exerts beneficial cardiovascular effects by binding to specific receptors on various cells to activate nuclear and non-nuclear actions. Estrogen receptor α (ERα) non-nuclear signaling confers protection against heart failure remodeling, involving myocardial cyclic guanosine monophosphate (cGMP) - cGMP-dependent protein kinase G (PKG) activation; however, its tissue-specific role remains elusive. Herein, we examined the cell type-specific role of ERα non-nuclear signaling in estrogen-conferred protection against heart failure. We first assessed the tissue-specific impacts of ERα in estrogen's cardiac benefits, utilizing endothelial ERα deletion (ERαf/f/Tie2Cre+) and myocyte ERα deletion (ERαf/f/αMHCCre+) female mice. Female mice were ovariectomized and the effect of estradiol (E2) was assessed in hearts exposed to 3week pressure-overload (TAC). E2 failed to improve cardiac function in ERαf/f/Tie2Cre+ TAC hearts but provided benefits in ERαf/f/αMHCCre+ TAC hearts, indicating that endothelial ERα is essential. We next assessed the role of non-nuclear signaling in endothelial cells, employing animals with endothelial-specific inactivation of ERα non-nuclear signaling (ERαKI/KI/Tie2Cre+). Female OVX mice were supplemented with E2 and subjected to 3-week TAC. ERαKI/KI/Tie2Cre+ TAC hearts revealed exacerbated cardiac dysfunction and reduced myocardial PKG activity as compared to littermate TAC hearts, which was associated with attenuated myocardial induction of vascular endothelial growth factor (VEGF) and angiogenesis as assessed with CD31-stained capillary density. This phenotype of ERαKI/KI/Tie2Cre+ was rescued by myocardial PKG activation from chronic treatment with soluble guanylate cyclase (sGC) stimulator. We performed co-culture experiments to determine endothelial-cardiomyocyte interactions. VEGF induction by E2 in cardiac myocytes required co-existence of intact endothelial ERα signaling in a NOS-dependent manner. On the other hand, VEGF was induced in myocytes directly with an sGC stimulator in the absence of endothelial cells. An endothelial estrogen - myocardial cGMP axis stimulates angiogenic response and improves cardiac performance during pressure-overload.

Exosomal miR-155-5p drives ibrutinib resistance in B-cell lymphoma

Ibrutinib, a Bruton Tyrosine Kinase (BTK) inhibitor, has shown effectiveness against various B-cell lymphoid malignancies. However, prolonged usage can induce resistance, affecting treatment outcomes. The oncogenic microRNA, miR-155-5p, is associated with poor prognosis in B-cell lymphomas, prompting our investigation into the mechanism of acquired ibrutinib resistance in these cells. We generated ibrutinib-resistant OCI-Ly1 cells (OCI-Ly1-IbtR) through continuous exposure to 1 μM and 2 μM of ibrutinib. We conducted microRNA profiling of OCI-Ly1-IbtR and isolated exosomes via ultracentrifugation. Comparative studies of microRNA levels in cells and exosomes, as well as exploration of targets of up-regulated microRNAs in OCI-Ly1-IbtR, were performed. Target validation involved transfection of candidate microRNAs, and co-culture experiments utilized OCI-Ly1 cells with exosomes from OCI-Ly1-IbtR. Elevated levels of miR-155-5p were observed in OCI-Ly1-IbtR and its exosomes, correlating with AKT and NF-κB activation. Transfection of miR-155-5p induced AKT/NF-κB pathway activation in OCI-Ly1, resulting in ibrutinib resistance, enhanced colony formation, and sustained BTK activity. Primary cell lines from ibrutinib-refractory B-cell lymphoma patients exhibited similar signaling protein activation. Target evaluation identified KDM5B and DEPTOR as miR-155-5p targets, confirmed by downregulation after transfection. We observed KDM5B and DEPTOR enrichment in Ago2 during ibrutinib resistance and miR-155-5p transfection. Co-culture experiments demonstrated exosome-mediated transfer of miR-155-5p, inducing ibrutinib resistance and KDM5B/DEPTOR downregulation in OCI-Ly1. Our findings suggest that miR-155-5p overexpression is associated with AKT and NF-κB pathway activation in ibrutinib-resistant cells, proposing a potential role for acquired miR-155-5p upregulation in B-cell lymphoma ibrutinib resistance.

IDH2 regulates macrophage polarization and tumorigenesis by modulating mitochondrial metabolism in macrophages

BackgroundTargeting the tumor microenvironment represents an emerging therapeutic strategy for cancer. Macrophages are an essential part of the tumor microenvironment. Macrophage polarization is modulated by mitochondrial metabolism, including oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and reactive oxygen species content. Isocitrate dehydrogenase 2 (IDH2), an enzyme involved in the TCA cycle, reportedly promotes cancer progression. However, the mechanisms through which IDH2 influences macrophage polarization and modulates tumor growth remain unknown.MethodsIn this study, IDH2-deficient knockout (KO) mice and primary cultured bone marrow-derived macrophages (BMDMs) were used. Both in vivo subcutaneous tumor experiments and in vitro co-culture experiments were performed, and samples were collected for analysis. Western blotting, RNA quantitative analysis, immunohistochemistry, and flow cytometry were employed to confirm changes in mitochondrial function and the resulting polarization of macrophages exposed to the tumor microenvironment. To analyze the effect on tumor cells, subcutaneous tumor size was measured, and growth and metastasis markers were identified.ResultsIDH2-deficient macrophages co-cultured with cancer cells were found to possess increased mitochondrial dysfunction and fission than wild-type BMDM. Additionally, the levels of M2-associated markers decreased, whereas M1-associated factor levels increased in IDH2-deficient macrophages. IDH2-deficient macrophages were predominantly M1. Tumor sizes in the IDH2-deficient mouse group were significantly smaller than in the wild-type mouse group. IDH2 deficiency in macrophages was associated with inhibited tumor growth and epithelial–mesenchymal transition.ConclusionsOur findings suggest that IDH2 deficiency inhibits M2 macrophage polarization and suppresses tumorigenesis. This study underlines the potential contribution of IDH2 expression in macrophages and tumor microenvironment remodeling, which could be useful in clinical cancer research.

DYRK1B blockade promotes tumoricidal macrophage activity in pancreatic cancer

ObjectiveHighly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments...

SP6.7 - Exploring the Mechanistic Basis of the Adipose Derived Stem Cell Secretome on Extracellular Matrix Remodelling in Radiation Induced Fibrosis

Abstract Background Radiation-induced fibrosis (RIF) is a debilitating complication following radiotherapy, characterised by fibroblast proliferation and excessive extracellular matrix (ECM) deposition, leading to functional and aesthetic impairments. Although autologous fat grafting (AFG) has shown promise in reversing RIF, the mechanisms, largely attributed to adipose-derived stem cells (ADSCs), remain elusive. This study aimed to investigate ADSCs’ and their secretome on ECM remodelling in RIF management. Methods The anti-fibrotic potential of the ADSC secretome on HDFs subjected to ionising radiation was assessed via a series of in vitro experiments. Control HDFs were compared with irradiated HDFs to assess any phenotypical changes. Irradiated HDFs were treated with ADSC pre-conditioned media (CM) or ADSC co-culture (CC). Gene expression (COL1A1, TGF-β1, CCN-2, MMP-1, MMP-3, TIMP-1, ACTA2, TNC) was assessed via real-time quantitative PCR (RT-qPCR). ProCollagen1a1 and CCN2 protein concentrations were assessed by ELISA. Results Irradiated HDFs demonstrated changes in fibrotic gene expression. ADSC-CM treatment significantly reduced COL1A1 expression, although protein levels remained unchanged. TGF-β1, CCN2, MMP-3, TIMP-1 and ACTA-2 exhibited reduced expression post-ADSC-CM treatment whereas TNC expression was found to be significantly increased. Co-culture with ADSCs led to a general, but not statistically significant, decrease in pro-fibrotic gene expression. Notably, CCN-2 protein expression significantly increased. Conclusion This study demonstrates that ionising radiation results in a pathological pro-fibrotic phenotype in HDFs. The ADSC secretome potentially mitigates this, reducing pro-fibrotic gene expression. Unexpectedly, co-culture experiments highlighted a complex interaction with upregulation of fibrosis-associated genes. Further research is needed to clarify these interactions to improve therapeutic interventions for RIF.

Abstract P128: Endothelial Nitric Oxide Determines Hematopoietic Cell Fate and Osteoclastogenesis By Modulating CSF1 During Hypertension

Introduction: Loss of endothelial nitric oxide (NO) is common in hypertension (HTN). The bone marrow niche contains endothelial cells; however, how endothelial NO affects hematopoiesis and bone in HTN remains undefined. Hypothesis: We hypothesized that NO loss in HTN drives hematopoiesis in the bone marrow, later favoring osteoclastogenesis and bone resorption. Methods and Results: By microcomputed tomography, eNOS deficient mice (eNOS -/- ) had reduced femoral cortical area (Figure 1A) and trabecular bone volume fraction (Figure 1B) compared to wildtype mice (WT). Flow cytometry showed that granulocyte-monocyte (Figure 1C) and common lymphoid progenitors (Figure 1D) were markedly higher in eNOS -/- than in WT. mRNA analysis revealed loss of endothelial NO increased expression of cathepsin K (Ctsk), colony stimulating factor-1 receptor (CSF1R), and tartrate resistant acid phosphatase (TRAP) in the bone marrow. We exposed murine bone-marrow endothelial cells (BMECs) to 5% (normotension), 5% with L-NAME, or 10% (HTN) cyclic strain. Compared to 5% strain, BMECs exposed to L-NAME or 10% strain exhibited markedly increased CSF1 production, an essential cytokine for myelopoiesis and osteoclastogenesis (Figure 1E and F). In coculture experiments, we also found that macrophages cultured with BMECs undergoing 5% strain with L-NAME or 10% strain exhibited higher expression of Ctsk, CSF1R, and TRAP, indicating osteoclast formation. Conclusion: Endothelial NO likely inhibits osteoclastogenesis and bone resorption. The loss of NO through mechanical stretch or inhibiting eNOS increases CSF1 production and hematopoiesis. This shows a potential mechanism by which NO loss leads to pathological hematopoiesis and bone resorption during HTN.

Phenomenological investigation of organic modified cements as biocompatible substrates interfacing model marine organisms.

Organic modification can generally endow inorganic materials with novel and promotional characteristics to fit into new functionalities. In this paper, new cement-based composite materials, with Portland cement as the substrate and polyacrylamide (PAM, alone) and PAM/chitosan as the functional components mixed with cement (bulk modified) or served as the surface coating (surface modified), were prepared and engineered as sampling substrates for biofilm and coral co-culture. In comparison to the bulk modified substrate and pure cement material, the surface modified substrate showed a balanced mechanical property, considering both bending and compressive strengths and distinctive surface features toward facilitating biofilm and coral growth, as characterized by spectroscopic, morphological, mechanical, and biofilm and coral co-culture experiments. We, thus, believe that the as-prepared surface modified substrate has the very potential to be applied as a substitute/alternative for the conventional cement material in the construction and engineering of artificial facilities with ecological protection functions.

Tuning the B-CLL microenvironment: evidence for BAG3 protein- mediated regulation of stromal fibroblasts activity

The Bcl2-associated athanogene-3 (BAG3) protein, a critical regulator of cellular survival, has been identified as a potential therapeutic target in various malignancies. This study investigates the role of BAG3 within stromal fibroblasts and its interaction with B-cell chronic lymphocytic leukemia (B-CLL) cells. Previous research demonstrated that BAG3 maintains the active state of pancreatic stellate cells (PSCs) and aids pancreatic ductal adenocarcinoma (PDAC) spread via cytokine release. To explore BAG3’s role in bone marrow-derived stromal fibroblasts, BAG3 was silenced in HS-5 cells using siRNA. In co-culture experiments with PBMCs from B-CLL patients, BAG3 silencing in HS-5 cells increased apoptosis and decreased phosphorylation of BTK, AKT, and ERK in B-CLL cells, thus disrupting their pro-survival key signaling pathways. The observation of fibroblast-activated protein (FAP) positive cells in infiltrated bone marrow specimens co-expressing BAG3 further support the involvement of the protein in fibroblast-mediated tumor survival. Additionally, BAG3 appears to support B-CLL survival by modulating cytokine networks, including IL-10 and CXCL12, which are essential for leukemic cell survival and proliferation. A robust correlation between BAG3 expression and the levels of CXCL12 and IL-10 was observed in both co-cultures and patient specimens. These findings point out the need for a more in-depth comprehension of the intricate network of interactions within the tumor microenvironment and provide valuable insights for the selection of new potential therapeutic targets in the medical treatment of CLL.

Interleukin-4-Loaded Heparin Hydrogel Regulates Macrophage Polarization to Promote Osteogenic Differentiation.

In bone tissue engineering, biological scaffolds are designed with structural and functional properties that closely resemble the extracellular environment, aiming to establish a microenvironment conducive to osteogenesis. Macrophages hold significant potential for promoting osteogenesis and modulating the biological behavior of tumor cells. Multiple coculture experiments of macrophages and osteoblasts have demonstrated that macrophage polarization significantly impacts osteogenesis. Therefore, exploring bone biomaterials that can modulate macrophage polarization holds great clinical significance. In this study, heparin was modified with maleimide and was used as a raw material to form a hydrogel with 4-am-PEG-SH. The compound was used to polarize macrophages and promote osteogenesis after combining with interleukin 4 (IL-4) by taking advantage of the electronegativity of heparin. The results revealed overexpressed M2 macrophage-related phenotypic genes and cocultivation with MC3T3-E1 cells demonstrated the osteogenesis-promoting effect of the loaded IL-4 heparin hydrogel. Previous research reported that hydrogel loaded with IL-4 can be used as a biomaterial for osteogenesis promotion. Heparin materials used in this paper are derived from clinically anticoagulant drugs and feature a simple operation. The synthesized hydrogel effectively binds cytokines, regulates macrophages to induce osteogenesis and has many potential clinical applications.

Endothelium-Inspired Hemocompatible Silicone Surfaces: An Elegant Balance between Antifouling Properties and Endothelial Cell Selectivity.

To address the adverse reactions caused by the implantation of blood-contacting materials, researchers have developed different strategies, of which mimicking multiple key features of endothelial cells is the most effective. However, simultaneously immobilizing multiple chemical components on a single material surface and maintaining the effects of individual components are challenging. In this work, endothelium-mimicking silicone surfaces were developed by incorporating the antifouling polymer poly(oligo(ethylene glycol) methacrylate), the glycosaminoglycan analog poly(sodium 4-vinyl-benzenesulfonate) and a nitric oxide catalyst (selenocystamine dihydrochloride). Through the rational regulation of multiple chemical components, the surfaces harmoniously resisted nonspecific protein adsorption, platelet adhesion and activation and smooth muscle cell hyperproliferation while promoting endothelial cell proliferation and migration. The coculture experiment with HUVECs and HUVSMCs showed that the optimum selectivity of HUVECs/HUVSMCs was ∼1.7. This work contributes insight into the control of antifouling properties and endothelial selectivity, providing a new avenue for the development of blood-contacting materials.

Impact of swine influenza A virus on porcine reproductive and respiratory syndrome virus infection in alveolar macrophages.

Porcine respiratory disease complex represents a major challenge for the swine industry, with swine influenza A virus (swIAV) and porcine reproductive and respiratory syndrome virus (PRRSV) being major contributors. Epidemiological studies have confirmed the co-circulation of these viruses in pig herds, making swIAV-PRRSV co-infections expected. A couple of in vivo co-infection studies have reported replication interferences between these two viruses. Herein, using a reductionist in vitro model, we investigated the potential mechanisms of these in vivo interferences. We first examined the impact of swIAV on porcine alveolar macrophages (AMs) and its effects on AMs co-infection by PRRSV. This was done either in monoculture or in co-culture with respiratory tracheal epithelial cells to represent the complexity of the interactions between the viruses and their respective target cells (epithelial cells for swIAV and AMs for PRRSV). AMs were obtained either from conventional or specific pathogen-free (SPF) pigs. SwIAV replication was abortive in AMs, inducing cell death at high multiplicity of infections. In AMs from three out of four conventional animals, swIAV showed no impact on PRRSV replication. However, inhibition of PRRSV multiplication was observed in AMs from one animal, accompanied by an early increase in the expression of interferon (IFN)-I and IFN-stimulated genes. In AMs from six SPF pigs, swIAV inhibited PRRSV replication in all animals, with an early induction of antiviral genes. Co-culture experiments involving tracheal epithelial cells and AMs from either SPF or conventional pigs all showed swIAV-induced inhibition of PRRSV replication, together with early induction of antiviral genes. These findings highlight the complex interactions between swIAV and PRRSV in porcine AMs, and would suggest a role of host factors, such as sanitary status, in modulating viral propagation. Our co-culture experiments demonstrated that swIAV inhibits PRRSV replication more effectively in the presence of respiratory tracheal epithelial cells, suggesting a synergistic antiviral response between AMs and epithelial cells, consistent with in vivo experiments.

Xenogenous implanted dental follicle stem cells promote periodontal regeneration through inducing the N2 phenotype of neutrophils

BackgroundPeriodontal tissue loss is the main reason for tooth mobility and loss caused by periodontal disease. Dental follicle stem cells (DFSCs) have significant therapeutic potential in periodontal regeneration, which maybe mainly depends on their potent immunomodulatory capacity. Consequently, this study aims to elucidate the impact of implanted xenogenous DFSCs on innate immune responses during early and late stages in the periodontal defect repair period.MethodsTo trace and investigate the immunomodulation mechanisms of DFSCs in vivo, DFSCs were engineered (E-DFSCs) using lentiviral vectors expressing CD63-enhanced green fluorescent protein (CD63-EGFP) and β-Actin-mCherry protein (ACTB-mCherry) to exhibit green and red fluorescence. The biological characteristics and functions of E-DFSCs were verified by proliferation, differentiation, and co-culture experiments in vitro. In vivo, the periodontal regeneration capacity of E-DFSCs was detected by implantation of murine periodontal defect model, and the response of innate immune cells was detected at the 1st, 3rd, and 5th days (early stage) and 4th week (late stage) after implantation.ResultsIn vitro assessments showed that E-DFSCs retain similar properties to their non-engineered counterparts but exhibit enhanced macrophage immunomodulation capability. In mice models, four-week micro-CT and histological evaluations indicated that E-DFSCs have equivalent efficiency to DFSCs in periodontal defect regeneration. At the early stage of repair in mice periodontal defect, fluorescence tracking showed that implanted E-DFSCs might primarily activate endogenous cells through direct contact and indirect actions, and most of these cells are myeloperoxidase-positive neutrophils. Additionally, compared with the control group, the neutrophilic infiltration and conversion of N2-type were significantly increased in the E-DFSC group. At the late stage of defect regeneration, more M2-type macrophages, fewer TRAP + osteoclasts, and an upregulated OPG/RANKL ratio were detected in the E-DFSC group compared to the control group, which indicated that immune balance tilts towards healing and bone formation.ConclusionThe xenogenous implanted DFSCs can induce the N2 phenotype of neutrophils in the early stage, which can activate the innate immune mechanism of the host to promote periodontal tissue regeneration.

Mesenchymal Stem Cells and Secretome as a New Possible Approach to Treat Cartilage Damage: An In Vitro Study.

Introduction: Osteoarthritis is a degenerative condition of the cartilage, often common among the population and occurs frequently with aging. Many factors are decisive for the development of its pathogenesis such as age, obesity, trauma, mechanical load, and modification of synovial biology. The main features of osteoarthritis are chondrocytes and cartilage matrix loss, which lead to pain, loss of function of the whole joint, and disability, representing a relevant health problem. Recently, a new therapeutic approach based on cell therapy has been studying the regenerative ability of mesenchymal stem cells for osteoarthritic chondrocytes. Aim: This in vitro study clarifies the regenerative effects of multipotent adipose-derived stem cells and the pluripotent amniotic epithelial stem cells on arthrosis chondrocytes by performing co-culture experiments. Methods: We studied the regenerative potential of secretome (soluble factors and extracellular vesicles), mesenchymal stem cells, and the adipose stromal vascular fraction. The regenerative effects were evaluated by gene and protein expression analysis of articular cartilage-specific genes and proteins like col2a1, acan, and sox9. Results: Mesenchymal stem cells, secretome, and adipose stromal vascular fractions influenced the cartilage genes and protein expression. Conclusions: The results indicate that the treatment with mesenchymal stem cells could be the best biological approach for cartilage regenerative medicine.

Dietary sodium modulates mTORC1-dependent trained immunity in macrophages to accelerate CKD development

Chronic Kidney Disease (CKD) is a significant global health issue linked to dietary habits, especially high salt intake. However, the precise mechanisms driving this progression remain incompletely understood. This study reveals that a high-salt diet intensifies macrophage trained immunity, leading to a marked pro-inflammatory response upon repeated pathogenic exposures, as evidenced by increased renal damage and fibrosis. Under high-salt conditions, there was an induction of CD45+F4/80+ macrophage infiltration into the renal tissue, accompanied by heightened production of inflammatory cytokines. Distinct responses were observed between circulating and resident renal macrophages to a high-salt diet, with a notable upsurge in the migration of pro-inflammatory macrophages, driven by CCL2-CCR2 signaling and aberrant mTORC1 pathway activation. Treatment with rapamycin-liposome effectively reduced this inflammatory cascade by mitigating mTORC1 signaling. Transplantation of monocytes from CKD mice with a high-salt diet significantly exacerbates renal inflammatory damage in the host mice, showing increased migratory tendency and inflammatory activity. The cell co-culture experiment further confirmed that macrophages derived from CKD mice, particularly those under conditions of high salt exposure, significantly induced apoptosis and inflammatory responses in renal tubular cells. Taken together, recurrent exposure to LPS elicits the activation of trained immunity, consequently augmenting inflammatory response of monocytes/macrophages in the involved kidneys. The high-salt diet exacerbates this phenomenon, attributable at least in part to the overactivation of the mTORC1 pathway. This research emphasizes the importance of dietary modulation and targeted immunological interventions in slowing CKD progression, providing new insights into mTORC1-mediated pathophysiological mechanisms and potential management strategies for CKD.

In vitro characterization of probiotic potential of Lactobacillus plantarum CM49 against selected cattle mastitogens

BackgroundBovine mastitis results in significant economic losses for the dairy industry globally due to milk production losses and decreased herd efficiency. This research aimed to isolate, select, and characterize indigenous lactobacilli with probiotic properties. A total of 40 lactobacilli were isolated from healthy milk samples of cattle and identified at the species level through 16S rDNA sequencing. All isolates were initially screened for antimicrobial activity, and selected isolates underwent in vitro assessment of probiotic properties.ResultsAmong the lactobacilli isolates, varying levels of activity (9 to 19 mm) against cattle mastitogens; Stapylococcus aureus (Staph. aureus), Escherichia coli (E. coli) and Streptococcus dysgalactiae (Strep. dysgalactiae) were observed in the well diffusion assay. These isolates demonstrated auto-aggregation (ranging from 14.29 ± 0.96% to 62.11 ± 1.09%) and co-aggregate (ranging from 9.21 ± 0.14% to 55.74 ± 0.74%) with mastitogens after 2 h. Lactobacillus (Lb.) plantarum CM49 showed sensitivity to most antibiotics tested and exhibited strong inhibitory effects, with mean log10 reductions of 3.46 for Staph. aureus, 2.82 for E. coli, and 1.45 for Strep. dysgalactiae in co-culture experiments. Furthermore, Lb. plantarum CM49 significantly decreased the adhesion rate of mastitogens on the bovine mammary cell line and mouse model, demonstrating its potential effectiveness in preventing mastitis.ConclusionIt is concluded that Lb. plantarum CM49 has remarkable probiotic potential with activity against cattle mastitogens in the laboratory and cell culture and competitively excludes mastitogens from bovine mammary cells and ameliorates Staph. aureus-induced mastitis in mice.

Unexpected methane oxidation acceleration by two species of bacteria from the rainwater promoting Methylomonas sp. in the soil

Altering the function of a mixed microbial community through specific microorganisms is a challenge. In this study, we constructed different co-culture systems to not only elucidate the mechanisms of microbial interactions but also explain the significance to the environment. Metagenomic analysis indicated that Methylomonas sp. was the main contributor to aerobic methane oxidation in the soils. Co-culture experiments showed that the introduction of Methylophilus sp. and Rhizobium sp., isolated from rainwater, significantly enhanced aerobic methane oxidation rates, on an hourly scale, representing maximum increases of 50.49 % and 24.62 %, respectively. Furthermore, metatranscriptomic analysis revealed that Methylophilus sp. and Rhizobium sp. could recolonize in the soil with abundance increasing to 1.23 % and 0.02 %. The genes of hxlA (methanol metabolism gene) and cobC (cobalamin synthesis gene) were upregulated, leading to the upregulation of methane oxidation genes such as pmoA and mmoX. The findings uncover the mechanisms of microbial interactions that improve aerobic methane oxidation and highlight the importance of these interactions in maintaining carbon cycle equilibrium in wetlands.

Root-secreted (-)-loliolide mediates chemical defense in rice and wheat against pests.

Plant chemical defense can be elicited by signaling chemicals. As yet, the elicitation is mainly known from volatile aboveground signals. Root-secreted belowground signals and their underlying mechanisms are largely unknown. This study examined a root-secreted signaling (-)-loliolide to trigger chemical defense in rice and wheat against pests by means of cocultivation and incubation experiments. Wild-type Arabidopsis (WT) and its root exudates with (-)-loliolide induced the production of defensive metabolites of rice and wheat and reduced the performance of weeds, pathogens and herbivores, while a carotenoid-deficient mutant (szl1-1) and its root exudates without (-)-loliolide had no similar effects. However, the induction and reduction occurred in the szl1-1 root exudates by (-)-loliolide supplementation with the level equal to that of WT. RNA-sequencing analysis revealed a significant change in the transcript level of defense-related genes in rice exposure to (-)-loliolide. Furthermore, (-)-loliolide enhanced rice resistance against Rhizoctonia solani through changing reactive oxygen species (ROS) system, and mediating jasmonic acid, salicylic acid and abscisic acid biosynthesis. Root-secreted signaling (-)-loliolide can trigger chemical defense in rice and wheat against their pests. Such perception-dependent chemical defenses provide an intriguing possibility for ecological pest management to increase crop productivity and sustainability. © 2024 Society of Chemical Industry.

Linarin inhibits microglia activation-mediated neuroinflammation and neuronal apoptosis in mouse spinal cord injury by inhibiting the TLR4/NF-κB pathway

To investigate the mechanism underlying the neuroprotective effect of linarin (LIN) against microglia activation-mediated inflammation and neuronal apoptosis following spinal cord injury (SCI). Fifty C57BL/6J mice (8- 10 weeks old) were randomized to receive sham operation, SCI and linarin treatment at 12.5, 25, and 50 mg/kg following SCI (n=10). Locomotor function recovery of the SCI mice was assessed using the Basso Mouse Scale, inclined plane test, and footprint analysis, and spinal cord tissue damage and myelination were evaluated using HE and LFB staining. Nissl staining, immunofluorescence assay and Western blotting were used to observe surviving anterior horn motor neurons in injured spinal cord tissue. In cultured BV2 cells, the effects of linarin against lipopolysaccharide (LPS)‑induced microglia activation, inflammatory factor release and signaling pathway changes were assessed with immunofluorescence staining, Western blotting, RT-qPCR, and ELISA. In a BV2 and HT22 cell co-culture system, Western blotting was performed to examine the effect of linarin against HT22 cell apoptosis mediated by LPS-induced microglia activation. Linarin treatment significantly improved locomotor function (P < 0.05), reduced spinal cord damage area, increased spinal cord myelination, and increased the number of motor neurons in the anterior horn of the SCI mice (P < 0.05). In both SCI mice and cultured BV2 cells, linarin effectively inhibited glial cell activation and suppressed the release of iNOS, COX-2, TNF-α, IL-6, and IL-1β, resulting also in reduced neuronal apoptosis in SCI mice (P < 0.05). Western blotting suggested that linarin-induced microglial activation inhibition was mediated by inhibition of the TLR4/NF- κB signaling pathway. In the cell co-culture experiments, linarin treatment significantly decreased inflammation-mediated apoptosis of HT22 cells (P < 0.05). The neuroprotective effect of linarin is medicated by inhibition of microglia activation via suppressing the TLR4/NF‑κB signaling pathway, which mitigates neural inflammation and reduce neuronal apoptosis to enhance motor function of the SCI mice.