Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications.In vitro bacteria-host cell coculture models have enabled fundamental s...Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications.In vitro bacteria-host cell coculture models have enabled fundamental studies to characterize bacterial infection and host responses in a reductionist yet reproducible manner.However,existing in vitro coculture models fail to establish conditions that are suitable for the growth of both mammalian cells and anaerobes,thereby hindering a comprehensive understanding of their interactions.Here,we present an asymmetric gas coculture system that simulates the oral microenvironment by maintaining distinct normoxic and anaerobic conditions for gingival epithelial cells and anaerobic bacteria,respectively.Using a key oral pathobiont,Fusobacterium nucleatum,as the primary test bed,we demonstrate that the system preserves bacterial viability and supports the integrity of telomerase-immortalized gingival keratinocytes.Compared to conventional models,this system enhanced bacterial invasion,elevated intracellular bacterial loads,and elicited more robust host pro-inflammatory responses,including increased secretion of CXCL10,IL-6,and IL-8.In addition,the model enabled precise evaluation of antibiotic efficacy against intracellular pathogens.Finally,we validate the ability of the asymmetric system to support the proliferation of a more oxygen-sensitive oral pathobiont,Porphyromonas gingivalis.These results underscore the utility of this coculture platform for studying oral microbial pathogenesis and screening therapeutics,offering a physiologically relevant approach to advance oral and systemic health research.展开更多
Traditional agricultural systems have contributed to food and livelihood security. Rice-crab coculture （RC） is an important eco-agricultural process in rice production in northern China. Recognizing the soil fertili...Traditional agricultural systems have contributed to food and livelihood security. Rice-crab coculture （RC） is an important eco-agricultural process in rice production in northern China. Recognizing the soil fertility in RC may help develop novel sustainable agriculture. Soil carbohydrates are important factors in determining soil fertility in different culture modes. In this study, soil carbohydrates were analyzed under three different culture modes including rice monoculture （RM）, conventional rice-crab coculture （CRC） and organic rice-crab coculture （ORC）. Results showed that the contents of soil organic carbon and carbohydrates were significantly higher in the ORC than those in RM. The increasing effect was greater with increased organic manure. Similar tendency was found in CRC, but the overall effect was less pronounced compared with ORC. Carbohydrates were more Sensitive to RC mode and manure amendment than soil organic carbon. Compare to RM, the （Gal＋Man）/（Ara＋Xyl） ratio decreased in all the RC modes, indicating a relative enrichment in plant-derived carbohydrates due to the input of crab feed and manure. While the increasing （Gal＋Man）/（Ara＋Xyl） ratio in ORC modes with increased organic manure suggested that crab activity and metabolism induced microbially derived carbohydrates accumulation. The lower GluN/MurA ratio in ORC indicated an enhancement of bacteria contribution to SOM turnover in a short term. The findings reveal that the ORC mode could improve the quantity and composition of soil carbohydrates, effectively, to ensure a sustainable use of paddy soil.展开更多
Nitrogen(N)significantly affects rice yield and lodging resistance.Previous studies have primarily investigated the impact of N management on rice lodging in conventional rice monoculture(RM);however,few studies have ...Nitrogen(N)significantly affects rice yield and lodging resistance.Previous studies have primarily investigated the impact of N management on rice lodging in conventional rice monoculture(RM);however,few studies have performed such investigations in rice-crayfish coculture(RC).We hypothesized that RC would increase rice lodging risk and that optimizing N application practices would improve rice lodging resistance without affecting food security.We conducted a two-factor(rice farming mode and N management practice)field experiment from2021 to 2022 to test our hypothesis.The rice farming modes included RM and RC,and the N management practices included no nitrogen fertilizer,conventional N application,and optimized N treatment.The rice yield and lodging resistance characteristics,such as morphology,mechanical and chemical characteristics,anatomic structure,and gene expression levels,were analyzed and compared among the treatments.Under the same N application practice,RC decreased the rice yield by 11.1-24.4% and increased the lodging index by 19.6-45.6% compared with the values yielded in RM.In RC,optimized N application decreased the plant height,panicle neck node height,center of gravity height,bending stress,and lodging index by 4.0-4.8%,5.2-7.8%,0.5-4.5%,5.5-10.5%,and 1.8-19.5%,respectively,compared with those in the conventional N application practice.Furthermore,it increased the culm diameter,culm wall thickness,breaking strength,and non-structural and structural carbohydrate content by 0.8-4.9%,2.2-53.1%,13.5-19.2%,2.2-24.7%,and 31.3-87.2%,respectively.Optimized N application increased sclerenchymal and parenchymal tissue areas of the vascular bundle at the culm wall of the base second internode.Furthermore,optimized N application upregulated genes involved in lignin and cellulose synthesis,thereby promoting lower internodes on the rice stem and enhancing lodging resistance.Optimized N application in RC significantly reduced the lodging index by 1.8-19.5%and stabilized the rice yield(>8,570 kg ha~(-1)on average).This study systematically analyzed and compared the differences in lodging characteristics between RM and RC.The findings will aid in the development of more efficient practices for RC that will reduce N fertilizer application.展开更多
The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell emb...The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres（groups of at least 30 at the eight-cell stage） were separated into eight segments（to regard an eight-cell embryo as a tangerine, a blastomere as one segment） and one, two and four segments（blastomeres） were cultured respectively in microwells on the bottom of the four-well dish（Nunc, Denmark） with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics（cocultured with nothing, intact embryos, bovine cumulus cells（b CCs）, intact embryos ＆ b CCs） were applied to the group of four segments（blastomeres）. Finally, diameter and inner cell mass（ICM）：trophectoderm（TE） cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group（P〈0.05）. In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo ＆ b CCs（P〈0.05）. Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo（P〈0.05）, more important quality indicator of the blastocyst diameter and ICM：TE cell ratio was similar between our experimental group and the control group（P〉0.05）. Thus, these results suggest that combined with intact embryos ＆ b CCs coculture system, culturing four isolated segments（blastomeres） per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.展开更多
Shellfish and seaweed aquaculture substantially influence marine carbon sinks.However,comprehensive comparisons and analyses of dissolved inorganic carbon(DIC) uptake,dissolved organic carbon(DOC) release,and CO_(2) s...Shellfish and seaweed aquaculture substantially influence marine carbon sinks.However,comprehensive comparisons and analyses of dissolved inorganic carbon(DIC) uptake,dissolved organic carbon(DOC) release,and CO_(2) source-sink dynamics under both monoculture and coculture regimes remain limited.This study utilized a custom-built closed system to monitor CO_(2) in the water and air,and the dynamics of seawater DIC,DOC,and overlying atmospheric CO_(2) concentrations in mono-and co-cultures of seaweed(Gracilaria lemaneiformis) and oysters(Crassostrea gigas).The monoculture of G.lemaneiformis apparently demonstrated significant carbon sequestration capacity,effectively reducing both seawater DIC and overlying atmospheric CO_(2) concentrations.The absorption rates were 0.026 mg/(g h) for atmospheric CO_(2) and 1.081 mg/(g h) for seawater DIC(both calculated as CO_(2) equivalent).In contrast,oyster monoculture had minimal impact on seawater DIC but significantly elevated overlying atmospheric CO_(2) levels,functioning as a CO_(2) source with a release rate of 0.110 mg/(g d).Notably,in G.lemaneiformis–oyster cocultures,the system not only reduced seawater DIC concentrations—often more effectively than G.lemaneiformis monoculture alone—but also substantially mitigated the CO_(2) release associated with oysters.Furthermore,cocultures with a high G.lemaneiformis-to-oyster ratio facilitated a net shift from CO_(2) emission to sequestration.At a G.lemaneiformis-to-oyster weight ratio of 1:8,the water-air CO_(2) exchange approached equilibrium.Regarding organic carbon,DOC release rates also differed significantly among the three cultivation modes.G.lemaneiformis monoculture produced a notably higher DOC release rate than oyster monoculture,while their coculture exhibited an approximately additive effect on DOC release.Furthermore,the photosynthetic activity of G.lemaneiformis was highly responsive to light-dark cycles.During the light phase,seawater p H,dissolved oxygen,and DOC levels increased,while DIC concentrations decreased;these trends reversed during the dark phase.Among these parameters,p H was identified as a critical environmental factor regulating seawater partial pressure of CO_(2) and,consequently,the water-air CO_(2) exchange.These findings provide valuable insights and a robust scientific basis for assessing the carbon source-sink dynamics of shellfish and seaweed aquaculture systems.展开更多
A team of researchers from Nagoya, Tokyo and Hamilton developed a unique technique for studying neuroimmune interaction with confocal laser scanning fluorescence microscopy several years ago. It relies on guiding immu...A team of researchers from Nagoya, Tokyo and Hamilton developed a unique technique for studying neuroimmune interaction with confocal laser scanning fluorescence microscopy several years ago. It relies on guiding immune and nerve cell interaction by creating an adhesive environment using an in vitro coculture dish. With their technique, they are able to study details of the mechanism of how nerve cells communicate with immune cells （mast cells and T lymphocytes） and vice versa. They showed that nerve-mast cell communication could occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating v/a NK-1 receptors, was a soluble factor of this communication. In addition, recently, they showed that ATP which was released from activated mast cells mediated the activation of nerve cells. Further, with their technique, Nagoya＇s group was able to study details of the molecular mechanism of nerve-mast cell interaction. N-cadherin and CADM1 （cell adhesion molecule 1） appeared to mediate attachment and promoted the communication between mast cells and nerves predominantly. It would lead to new therapeutic modalities for diseases based on neuroimmune interaction such as neurogenic inflammation, intestinal bowel diseases, asthma, and autoimmune disorders. Cellular ＆ Molecular Immunology. 2008;5（4）：249-259.展开更多
Resveratrol,a valuable plant-derived polyphenolic compound with various bioactivities,has been widely used in nutraceutical industries.Microbial production of resveratrol suffers from metabolic burden and low malonyl-...Resveratrol,a valuable plant-derived polyphenolic compound with various bioactivities,has been widely used in nutraceutical industries.Microbial production of resveratrol suffers from metabolic burden and low malonyl-CoA availability,which is a big challenge for synthetic biology.Herein,we took advantage of coculture engineering and divided the biosynthetic pathway of resveratrol into the upstream and downstream strains.By enhancing the supply of malonyl-CoA via CRISPRi system and fine-tuning the expression intensity of the synthetic pathway genes,we significantly improved the resveratrol productivity of the downstream strain.Furthermore,we developed a resveratrol addiction circuit that coupled the growth of the upstream strain and the resveratrol production of the downstream strain.The bidirectional interaction stabilized the coculture system and increased the production of resveratrol by 74%.Moreover,co-utilization of glucose and arabinose by the coculture system maintained the growth advantage of the downstream strain for production of resveratrol throughout the fermentation process.Under optimized conditions,the engineered E.coli coculture system produced 204.80 mg/L of resveratrol,12.8-fold improvement over monoculture system.This study demonstrates the promising potential of coculture engineering for efficient production of natural products from biomass.展开更多
Extracellular matrix(ECM)with mimetic tissue niches was attractive to facilitate tissue regeneration in situ via recruitment of endogenous cells and stimulation of self-healing process.However,how to engineer the comp...Extracellular matrix(ECM)with mimetic tissue niches was attractive to facilitate tissue regeneration in situ via recruitment of endogenous cells and stimulation of self-healing process.However,how to engineer the complicate tissue specific ECM with unique matrisome in vitro was a challenge of ECM-based biomaterials in tissue engineering and regenerative medicine.Here,we introduced coculture system to engineer bone mimetic ECM niche guided by cell-cell communication.In the cocultures,fibroblasts promoted osteogenic differentiation of osteoblasts via extracellular vesicles.The generated ECM(MN-ECM)displayed a unique appearance of morphology and biological components.The advantages of MN-ECM were demonstrated with promotion of multiple cellular behaviors(proliferation,adhesion and osteogenic mineralization)in vitro and bone regeneration in vivo.Moreover,proteomic analysis was used to clarify the molecular mechanism of MN-ECM,which revealed a specific matrisome signature.The present study provides a novel strategy to generate ECM with tissue mimetic niches via cell-cell communication in a coculture system,which forwards the development of tissue-bioactive ECM engineering along with deepening the understanding of ECM niches regulated by cells for bone tissue engineering.展开更多
Vanillyl alcohol is a precursor of vanillin,which is one of the most widely used flavor compounds.Currently,vanillyl alcohol biosynthesis still encounters the problem of low efficiency.In this study,coculture engineer...Vanillyl alcohol is a precursor of vanillin,which is one of the most widely used flavor compounds.Currently,vanillyl alcohol biosynthesis still encounters the problem of low efficiency.In this study,coculture engineering was adopted to improve production efficiency of vanillyl alcohol in E.coli.First,two pathways were compared for biosynthesis of the immediate precursor 3,4-dihydroxybenzyl alcohol in monocultures,and the 3-dehydroshikimate-derived pathway showed higher efficiency than the 4-hydroxybenzoate-derived pathway.To enhance the efficiency of the last methylation step,two strategies were used,and strengthening S-adenosylmethionine(SAM)regeneration showed positive effect while strengthening SAM biosynthesis showed negative effect.Then,the optimized pathway was assembled in a single cell.However,the biosynthetic efficiency was still low,and was not significantly improved by modular optimization of pathway genes.Thus,coculturing engineering strategy was adopted.At the optimal inoculation ratio,the titer reached 328.9 mg/L.Further,gene aroE was knocked out to reduce cell growth and improve 3,4-DHBA biosynthesis of the upstream strain.As a result,the titer was improved to 559.4 mg/L in shake flasks and to 3.89 g/L in fed-batch fermentation.These are the highest reported titers of vanillyl alcohol so far.This work provides an effective strategy for sustainable production of vanillyl alcohol.展开更多
To develop efficient and economical direct ethanol production from fine rice straw crashed mechanically, two high-performing fungi, which can secret hyperactive cellulases and/or ferment effectively various sugars, we...To develop efficient and economical direct ethanol production from fine rice straw crashed mechanically, two high-performing fungi, which can secret hyperactive cellulases and/or ferment effectively various sugars, were selected from some strains belong to Mucor circinelloides preserved in our laboratory. The simultaneous saccharification and fermentation （SSF） by coculture with these fungi was investigated. The screening of high- performing fungi resulted in the selection of NBRC 4572 as an ethanol-producing fungus and NBRC 5398 as a cellulase-secreting fungus. The strain 4572 produced ethanol aerobically from glucose and xylose in high yields of 0.420 g/g at 36 h and 0.478 g/g at 60 h, respectively, but secreted fairly low cellulases. On the other hand, the strain 5398 also produced ethanol from glucose in yield of 0.340 g/g though it had a little growth in xylose culture. However, it secreted hyperactive cellulases that are essential for hydrolysis of rice straw in culture and the maximum activities of endo-β-glucanase and β-glucosi- dase were 2.11 U/L and 1.47 U/L, respectively. In SSF of rice straw by coculture with two fungi selected, the ethanol production reached 1.28 g/L after 96 h when the inoculation ratio of the strain 5398 to the strain 4572 was 9.展开更多
Studies on simultaneous saccharification and fermentation(SSF)of wheat bran flour,a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and...Studies on simultaneous saccharification and fermentation(SSF)of wheat bran flour,a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation.Experi-ments based on central composite design(CCD)were conducted to maximize the glucose yield and to study the effects of substrate concentration,pH,temperature,and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungalα-amylase and the above parameters were optimized using response surface methodology(RSM).The optimum values of substrate concentration,pH,temperature,and enzyme concentration were found to be 200g/L,5.5,65℃ and 7.5IU,respectively,in the starch saccharification step.The effects of pH,temperature and substrate concentration on ethanol concentration,biomass and reducing sugar concentration were also investigated.The optimum temperature and pH were found to be 30℃ and 5.5,respectively.The wheat bran flour solution equivalent to 6%(w/V)initial starch concentration gave the highest ethanol concentrationof 23.1g/Lafter 48hoffermentation at optimum conditions of pH and temperature.The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model.Simultaneous saccharificiation and fermenta-tion of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A.niger and nonamylolytic sugar fermenting K.marxianus.展开更多
Cellular strategies remain a crucial component in bone tissue engineering （BTE）. So far, the outcome of cell-based strategies from initial clinical trials is far behind compared to animal studies, which is suggested...Cellular strategies remain a crucial component in bone tissue engineering （BTE）. So far, the outcome of cell-based strategies from initial clinical trials is far behind compared to animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the Ussue-engineered constructs. Cocultures, by introducing angiogenic cells into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, pre-clinical studies demonstrated that cocultures enhance vascularization and bone formation compared to monocultures. However, there has been no report on the application of cocultures in clinics. Therefore, this mini-review aims to provide an overview regarding （i） critical parameters in cocultures and the outcomes of cocultures compared to monocultures in the currently available pre-clinical studies using human mesenchymal stem cells implanted in orthotopic animal models; and （ii） the usage of monocultures in clinical application in BTE.展开更多
Most of the biological processes,including cell signaling,cancer invasion,embryogenesis,or neural development,are dependent on and guided by the complex architecture and composition of cellular microenvironments.Mimic...Most of the biological processes,including cell signaling,cancer invasion,embryogenesis,or neural development,are dependent on and guided by the complex architecture and composition of cellular microenvironments.Mimicking such microenvironments in cell coculture models is crucial for fundamental and applied biology investigations.The ability to combine different cell types grown as both two‐dimensional(2D)monolayers and three‐dimensional(3D)spheroids in specific defined location inside a microculture environments is a key towards in vitro tissue modeling and towards mimicking complex in vivo cellular processes.In this study,we introduce and investigate a method to create in vitro models of 2D cell monolayers cocultured with 3D spheroids in defined preorganization.We demonstrate the possibility of creating such complex cellular microenvironments in a high‐throughput and automated manner by creating arrays of such droplets containing prearranged 2D and 3D cellular microcolonies.Furthermore,we demonstrate an application of this approach to study paracrine propagation of Wnt signaling between 2D and 3D cellular colonies.This method provides a general approach for the miniaturized,high‐throughput,and automated formation of complex coculture cellular microarchitectures that will be useful for mimicking various in vivo complex cellular structures and for studying complex biological processes in vitro.展开更多
Pediococcus pentosaceus has a promising potential to improve the quality and flavor of Chinese steamed bread(CSB),but its synergistic effect with Saccharomyces cerevisiae is still unclear.In this study,P.pentosaceus w...Pediococcus pentosaceus has a promising potential to improve the quality and flavor of Chinese steamed bread(CSB),but its synergistic effect with Saccharomyces cerevisiae is still unclear.In this study,P.pentosaceus was cocultured with three S.cerevisiae isolates separately,which had different leavening ability.The times required for the fermentation of dough by 2 times the volume of S.cerevisiae isolates ACX 0089,ACX 0490 and ACX 0078 were 46,88 and 120 min,respectively.The results showed that the leavening ability of S.cerevisiae had a significant impact on the quality of CSBs.Compared other S.cerevisiae isolates,the ACX 0490 cocultured with P.pentosaceus increased the contents of bound water and free water in CSB,decreased the content of semibound water,and produced larger and regular porosity,thus endowed a greater specific volume and softer texture.A total of 36 volatile organic compounds(VOCs)were found in CSBs,with alcohols accounting for the main component.The coculture of P.pentosaceus with the intermediate leavening ability of S.cerevisiae ACX 0490 produced the highest proportion of esters among all CSBs.Moreover,S.cerevisiae ACX 0490 endowed the CSB unique VOCs,including cyclodecanol,ethylacetate,and 3,5-dimethylbenzaldehyde.The results of sensory evaluation also showed that S.cerevisiae ACX0490 cocultured CSB obtained the highest scores among all the test samples.The results provided a reference for selecting S.cerevisiae isolates with different leavening ability and the combination of P.pentosaceus to achieve the best quality and flavor of CSB.展开更多
In Myanmar,the advancement of the integrated rice-fish farming system legs behind rice monoculture farming,and there exists limited awareness of its advantages.Ecosystem services(ES)valuation plays a crucial role in i...In Myanmar,the advancement of the integrated rice-fish farming system legs behind rice monoculture farming,and there exists limited awareness of its advantages.Ecosystem services(ES)valuation plays a crucial role in integrated environmental decision-making,promoting sustainable agriculture practices,facilitating land-use planning,and ensuring food security in rural areas.Assessing the ES value in Delta region of Myanmar where rice-fish coculture is extensively practiced is essential for understanding the level of ES benefits derived from this farming system.The objective of this study is to promote the development of the rice-fish coculture system in delta region by estimating its ES value.We conducted a comprehensive examination of the Direct,Indirect,Option and Existence ES value of the rice-fish and rice monoculture in Maubin District,an area where rice-fish development research is being actively carried out within the delta region.The results revealed that the ES value of rice-fish coculture ecosystems in the study area was amounted to 28,588 US$/hm^(2)/year.This value was 2.82%higher than rice monoculture system.Additionally,the rice-fish coculture system yielded product provisional values averaging 1,275 US$/hm^(2)/year,representing a significant increase of 40.3%compared to rice monoculture farming.Our study shows that the adoption of rice-fish coculture farming system not only improves the ES value of the delta region,but also supports food security and socio-economic well-being.Furthermore,it provides valuable insights for policymakers on effective management policies for future development of the rice-fish coculture ecosystem.展开更多
Objective To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs). Methods The samples were divided into three groups. In Group One,...Objective To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs). Methods The samples were divided into three groups. In Group One, NSCs were cultured in DMED/F12 in which Schwann cells had grown for one day. In Group Two, NSCs and Schwann cells were co-cultured. In Group Three, NSCs were cultured in DMEM/F12. The morphology of NSCs was checked and β-tubulin, GalC, hoechst 33342 and GFAP labellings were detected. Results In Group One, all neural spheres were attached to the bottom and differentiated. The majority of them were p-tubulin positive while a few of cells were GFAP or GalC positive. In Group Two, neural spheres remained undifferentiatied and their proliferation was inhibited in places where Schwann cells were robust. In places where there were few Schwann cells, NSCs performed in a similar manner as in Group One. In Group Three, the cell growth state deteriorated day after day. On the 7th day, most NSCs died. Conclusion The secretion of rat Schwann cells has a growth supportive and differentiation-inducing effect on human NSCs.展开更多
Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein...Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.展开更多
AIM： The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments reveale...AIM： The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stern cell cultures. We investigated the potential of rat mesenchymal stem cells （MSC） from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS： Mesenchymal stem cells were marked with green fluorescent protein （GFP） by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP＋ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thyl and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS： Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression, CONCLUSION： The results indicate that （1） rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and （2） that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.展开更多
The single cell gel electrophoresis （SCGE） technique was used to detect DNA damage in germ cells of rats, which was induced by five tested nitroaromatic compounds. Mixed cultures of Sertoli and germ cells were prepa...The single cell gel electrophoresis （SCGE） technique was used to detect DNA damage in germ cells of rats, which was induced by five tested nitroaromatic compounds. Mixed cultures of Sertoli and germ cells were prepared from the rats testis and their responses to rn-dinitrobenzene （ m-DNB）, 2,4-dinitrotoluene （ 2,4-DNT）, 2,6-dinitroto-luene（2,6-DNT）, 4-nitrotoluene（4-NT） and 2,4-dinitroaniline（2,4-DNAn） were studied. The results show that all the five chemicals have a reproductive toxicity. Each dose group and the control group were significantly different （ P 〈 0. 01 ）. All of them can lead to the damage to DNA in the germ cells of Kunming male rats in the definite range of concentration（m-DNB ： 0. 04-25μmol/L; 2,4-DNT, 2,6-DNT and 4-NT： 0. 032-500μmol/L; 2,4-DNAn ：0. 8-20μmol/L）. When the concentration increases, the damage rate will become higher, which shows an evident logarithm dose-effect relationship.展开更多
Prostate cancer （PCa） is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and o...Prostate cancer （PCa） is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and old tissue are still obscure. We established primary cultured stromal cells from normal prostatic peripheral zone （PZ） of donors of varying ages and found that cultured stromal cells from old donors （PZ-old） were more enlarged and polygonal than those from young donors （PZ-young）. Furthermore, based on immunocytochemical and ultrastructural analysis, the components of stromal cells changed from a majority of fibroblasts to a mixture of fibroblasts and myofibroblasts with increasing donor age. Using a three-dimensional in vitro culture system, we found that PZ-old stromal cells could enhance the proliferation, migration and invasion of cocultured benign BPH-1 and PC-3 cells. Using an in vivo tissue recombination system, we also found that PZ-old stromal cells are more effective than PZ-young cells in promoting tumour formation by BPH-1 cells of high passage（〉100） and PC-3 cells. To probe the possible mechanism of these effects, we performed cDNA microarray analysis and profiled 509 upregulated genes and 188 downregulated genes in PZ-old cells. Among the changed genes, we found genes coding for a subset of paracrine factors that are capable of influencing adjacent epithelial cells; these include hepatocyte growth factor （HGF）, fibroblast growth factor 5 （FGF5）, insulin-like growth factor 2 （IGF2）, insulin-like growth factor-binding protein 4 （IGFBP4）, IGFBP5 and matrix metallopeptidase 1 （MMP1）. Changes in the expression of these genes were further confirmed by quantitative real-time polymerase chain reaction （PCR）, Western blotting and enzyme-linked immunosorbent assays. Overall, our findings indicate that stromal cells from prostate PZ of old donors are more active than similar cells from young donors in promoting the malignant process of adjacent epithelial cells. This finding hints at a new potential strategy for the prevention of PCa.展开更多
基金supported by National Science Foundation CAREER (2238972)National Institute of Dental and Craniofacial Research awards (R03DE031329 and R01DE030943)The Translational Tissue Modeling Laboratory is supported by the University of Michigan (Center for Gastrointestinal Research,Office of the Dean, Comprehensive Cancer Center, and the Departments of Pathology, Pharmacology, and Internal Medicine) with additional funding from the Endowment for Basic Sciences
文摘Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications.In vitro bacteria-host cell coculture models have enabled fundamental studies to characterize bacterial infection and host responses in a reductionist yet reproducible manner.However,existing in vitro coculture models fail to establish conditions that are suitable for the growth of both mammalian cells and anaerobes,thereby hindering a comprehensive understanding of their interactions.Here,we present an asymmetric gas coculture system that simulates the oral microenvironment by maintaining distinct normoxic and anaerobic conditions for gingival epithelial cells and anaerobic bacteria,respectively.Using a key oral pathobiont,Fusobacterium nucleatum,as the primary test bed,we demonstrate that the system preserves bacterial viability and supports the integrity of telomerase-immortalized gingival keratinocytes.Compared to conventional models,this system enhanced bacterial invasion,elevated intracellular bacterial loads,and elicited more robust host pro-inflammatory responses,including increased secretion of CXCL10,IL-6,and IL-8.In addition,the model enabled precise evaluation of antibiotic efficacy against intracellular pathogens.Finally,we validate the ability of the asymmetric system to support the proliferation of a more oxygen-sensitive oral pathobiont,Porphyromonas gingivalis.These results underscore the utility of this coculture platform for studying oral microbial pathogenesis and screening therapeutics,offering a physiologically relevant approach to advance oral and systemic health research.
基金supported by the National Natural Science Foundation of China (41101274 and 41101275)
文摘Traditional agricultural systems have contributed to food and livelihood security. Rice-crab coculture （RC） is an important eco-agricultural process in rice production in northern China. Recognizing the soil fertility in RC may help develop novel sustainable agriculture. Soil carbohydrates are important factors in determining soil fertility in different culture modes. In this study, soil carbohydrates were analyzed under three different culture modes including rice monoculture （RM）, conventional rice-crab coculture （CRC） and organic rice-crab coculture （ORC）. Results showed that the contents of soil organic carbon and carbohydrates were significantly higher in the ORC than those in RM. The increasing effect was greater with increased organic manure. Similar tendency was found in CRC, but the overall effect was less pronounced compared with ORC. Carbohydrates were more Sensitive to RC mode and manure amendment than soil organic carbon. Compare to RM, the （Gal＋Man）/（Ara＋Xyl） ratio decreased in all the RC modes, indicating a relative enrichment in plant-derived carbohydrates due to the input of crab feed and manure. While the increasing （Gal＋Man）/（Ara＋Xyl） ratio in ORC modes with increased organic manure suggested that crab activity and metabolism induced microbially derived carbohydrates accumulation. The lower GluN/MurA ratio in ORC indicated an enhancement of bacteria contribution to SOM turnover in a short term. The findings reveal that the ORC mode could improve the quantity and composition of soil carbohydrates, effectively, to ensure a sustainable use of paddy soil.
基金supported by the National Natural Science Foundation of China(32301961)the Natural Science Foundation of Jiangsu Province,China(BK20210791)+3 种基金the General Project of Philosophy and Social Science Research in Colleges and Universities in Jiangsu Province,China(2023SJYB2057)the Priority Academic Program Development of Jiangsu Higher Education Institutions,China(PAPD)the Qinglan Project of Yangzhou University,Chinathe Lv Yang Jin Feng Talent Plan of Yangzhou City,China(YZLYJF2020PHD100)。
文摘Nitrogen(N)significantly affects rice yield and lodging resistance.Previous studies have primarily investigated the impact of N management on rice lodging in conventional rice monoculture(RM);however,few studies have performed such investigations in rice-crayfish coculture(RC).We hypothesized that RC would increase rice lodging risk and that optimizing N application practices would improve rice lodging resistance without affecting food security.We conducted a two-factor(rice farming mode and N management practice)field experiment from2021 to 2022 to test our hypothesis.The rice farming modes included RM and RC,and the N management practices included no nitrogen fertilizer,conventional N application,and optimized N treatment.The rice yield and lodging resistance characteristics,such as morphology,mechanical and chemical characteristics,anatomic structure,and gene expression levels,were analyzed and compared among the treatments.Under the same N application practice,RC decreased the rice yield by 11.1-24.4% and increased the lodging index by 19.6-45.6% compared with the values yielded in RM.In RC,optimized N application decreased the plant height,panicle neck node height,center of gravity height,bending stress,and lodging index by 4.0-4.8%,5.2-7.8%,0.5-4.5%,5.5-10.5%,and 1.8-19.5%,respectively,compared with those in the conventional N application practice.Furthermore,it increased the culm diameter,culm wall thickness,breaking strength,and non-structural and structural carbohydrate content by 0.8-4.9%,2.2-53.1%,13.5-19.2%,2.2-24.7%,and 31.3-87.2%,respectively.Optimized N application increased sclerenchymal and parenchymal tissue areas of the vascular bundle at the culm wall of the base second internode.Furthermore,optimized N application upregulated genes involved in lignin and cellulose synthesis,thereby promoting lower internodes on the rice stem and enhancing lodging resistance.Optimized N application in RC significantly reduced the lodging index by 1.8-19.5%and stabilized the rice yield(>8,570 kg ha~(-1)on average).This study systematically analyzed and compared the differences in lodging characteristics between RM and RC.The findings will aid in the development of more efficient practices for RC that will reduce N fertilizer application.
基金supported by the China Agriculture Research System (CARS-37)the Agricultural Science and Technology Innovation Program, China (ASTIP-IAS06-2015)
文摘The objective of this study was to establish an efficient system of producing early monozygotic twin bovine embryos in vitro using the blastomere separation and coculture technique. In this study, early eight-cell embryos were chosen to optimize the separation method, and multi-coculture tactics were applied to improve the efficiency of this production system. Bovine embryo blastomeres（groups of at least 30 at the eight-cell stage） were separated into eight segments（to regard an eight-cell embryo as a tangerine, a blastomere as one segment） and one, two and four segments（blastomeres） were cultured respectively in microwells on the bottom of the four-well dish（Nunc, Denmark） with 400 μL of culture medium under paraffin oil. Four different types of coculture tactics（cocultured with nothing, intact embryos, bovine cumulus cells（b CCs）, intact embryos ＆ b CCs） were applied to the group of four segments（blastomeres）. Finally, diameter and inner cell mass（ICM）：trophectoderm（TE） cell ratio was measured as a criterion to assess the quality of the twin embryos which were derived from bovine separated blastomeres. Our results showed that rate of blastocyst formation of the four segments group was significantly greater than one or two group（P〈0.05）. In addition, rate of blastocyst formation was significantly increased when the four segments were cocultured with intact embryo ＆ b CCs（P〈0.05）. Although the ICM, TE and total cells of blastocysts derived from separated blastomeres was less than the control group from intact embryo（P〈0.05）, more important quality indicator of the blastocyst diameter and ICM：TE cell ratio was similar between our experimental group and the control group（P〉0.05）. Thus, these results suggest that combined with intact embryos ＆ b CCs coculture system, culturing four isolated segments（blastomeres） per microwell is an efficient system of producing early monozygotic twin bovine embryos. Furthermore, our results also indicate that the quality of blastocysts derived from separated blastomere may be similar to those derived from intact eight-cell embryos.
基金supported by the Ocean Negative Carbon Emissions(ONCE) Programthe Special Funds Program for Promoting High-Quality Development of Marine and Fishery Industry in Fujian Province,China(Grant Nos.FJHYF-TH-2023-1&FJHYF-TH-2023-4)+2 种基金the National Natural Science Foundation of China(Grant No.42188102)the National Key Research and Development Program of China(Grant No.2020YFA0607603)the Science and Technology Plan Program of Fujian Province,China(Grant No.2022Y0068)。
文摘Shellfish and seaweed aquaculture substantially influence marine carbon sinks.However,comprehensive comparisons and analyses of dissolved inorganic carbon(DIC) uptake,dissolved organic carbon(DOC) release,and CO_(2) source-sink dynamics under both monoculture and coculture regimes remain limited.This study utilized a custom-built closed system to monitor CO_(2) in the water and air,and the dynamics of seawater DIC,DOC,and overlying atmospheric CO_(2) concentrations in mono-and co-cultures of seaweed(Gracilaria lemaneiformis) and oysters(Crassostrea gigas).The monoculture of G.lemaneiformis apparently demonstrated significant carbon sequestration capacity,effectively reducing both seawater DIC and overlying atmospheric CO_(2) concentrations.The absorption rates were 0.026 mg/(g h) for atmospheric CO_(2) and 1.081 mg/(g h) for seawater DIC(both calculated as CO_(2) equivalent).In contrast,oyster monoculture had minimal impact on seawater DIC but significantly elevated overlying atmospheric CO_(2) levels,functioning as a CO_(2) source with a release rate of 0.110 mg/(g d).Notably,in G.lemaneiformis–oyster cocultures,the system not only reduced seawater DIC concentrations—often more effectively than G.lemaneiformis monoculture alone—but also substantially mitigated the CO_(2) release associated with oysters.Furthermore,cocultures with a high G.lemaneiformis-to-oyster ratio facilitated a net shift from CO_(2) emission to sequestration.At a G.lemaneiformis-to-oyster weight ratio of 1:8,the water-air CO_(2) exchange approached equilibrium.Regarding organic carbon,DOC release rates also differed significantly among the three cultivation modes.G.lemaneiformis monoculture produced a notably higher DOC release rate than oyster monoculture,while their coculture exhibited an approximately additive effect on DOC release.Furthermore,the photosynthetic activity of G.lemaneiformis was highly responsive to light-dark cycles.During the light phase,seawater p H,dissolved oxygen,and DOC levels increased,while DIC concentrations decreased;these trends reversed during the dark phase.Among these parameters,p H was identified as a critical environmental factor regulating seawater partial pressure of CO_(2) and,consequently,the water-air CO_(2) exchange.These findings provide valuable insights and a robust scientific basis for assessing the carbon source-sink dynamics of shellfish and seaweed aquaculture systems.
文摘A team of researchers from Nagoya, Tokyo and Hamilton developed a unique technique for studying neuroimmune interaction with confocal laser scanning fluorescence microscopy several years ago. It relies on guiding immune and nerve cell interaction by creating an adhesive environment using an in vitro coculture dish. With their technique, they are able to study details of the mechanism of how nerve cells communicate with immune cells （mast cells and T lymphocytes） and vice versa. They showed that nerve-mast cell communication could occur in the absence of an intermediary transducing cell and that the neuropeptide substance P, operating v/a NK-1 receptors, was a soluble factor of this communication. In addition, recently, they showed that ATP which was released from activated mast cells mediated the activation of nerve cells. Further, with their technique, Nagoya＇s group was able to study details of the molecular mechanism of nerve-mast cell interaction. N-cadherin and CADM1 （cell adhesion molecule 1） appeared to mediate attachment and promoted the communication between mast cells and nerves predominantly. It would lead to new therapeutic modalities for diseases based on neuroimmune interaction such as neurogenic inflammation, intestinal bowel diseases, asthma, and autoimmune disorders. Cellular ＆ Molecular Immunology. 2008;5（4）：249-259.
基金This work was supported by the Key-Area Research and Development Program of Guangdong Province(2020B0303070002)the National Natural Science Foundation of China(31870077).
文摘Resveratrol,a valuable plant-derived polyphenolic compound with various bioactivities,has been widely used in nutraceutical industries.Microbial production of resveratrol suffers from metabolic burden and low malonyl-CoA availability,which is a big challenge for synthetic biology.Herein,we took advantage of coculture engineering and divided the biosynthetic pathway of resveratrol into the upstream and downstream strains.By enhancing the supply of malonyl-CoA via CRISPRi system and fine-tuning the expression intensity of the synthetic pathway genes,we significantly improved the resveratrol productivity of the downstream strain.Furthermore,we developed a resveratrol addiction circuit that coupled the growth of the upstream strain and the resveratrol production of the downstream strain.The bidirectional interaction stabilized the coculture system and increased the production of resveratrol by 74%.Moreover,co-utilization of glucose and arabinose by the coculture system maintained the growth advantage of the downstream strain for production of resveratrol throughout the fermentation process.Under optimized conditions,the engineered E.coli coculture system produced 204.80 mg/L of resveratrol,12.8-fold improvement over monoculture system.This study demonstrates the promising potential of coculture engineering for efficient production of natural products from biomass.
基金supported by the National Natural Science Foundation of China(Grant No.31300800 , 81702625)the Zhejiang Province Welfare Technology Application Research Project(Grant No.2017C33135)+1 种基金the Natural Science Foundation of Ningbo(Grant No.2018A610202 , 2019A610309)the K.C.Wong Magna Fund in Ningbo University.
文摘Extracellular matrix(ECM)with mimetic tissue niches was attractive to facilitate tissue regeneration in situ via recruitment of endogenous cells and stimulation of self-healing process.However,how to engineer the complicate tissue specific ECM with unique matrisome in vitro was a challenge of ECM-based biomaterials in tissue engineering and regenerative medicine.Here,we introduced coculture system to engineer bone mimetic ECM niche guided by cell-cell communication.In the cocultures,fibroblasts promoted osteogenic differentiation of osteoblasts via extracellular vesicles.The generated ECM(MN-ECM)displayed a unique appearance of morphology and biological components.The advantages of MN-ECM were demonstrated with promotion of multiple cellular behaviors(proliferation,adhesion and osteogenic mineralization)in vitro and bone regeneration in vivo.Moreover,proteomic analysis was used to clarify the molecular mechanism of MN-ECM,which revealed a specific matrisome signature.The present study provides a novel strategy to generate ECM with tissue mimetic niches via cell-cell communication in a coculture system,which forwards the development of tissue-bioactive ECM engineering along with deepening the understanding of ECM niches regulated by cells for bone tissue engineering.
基金supported by National Key Research and Development Program of China(2018YFA0901800)National Natural Science Foundation of China(21978015)Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-009).
文摘Vanillyl alcohol is a precursor of vanillin,which is one of the most widely used flavor compounds.Currently,vanillyl alcohol biosynthesis still encounters the problem of low efficiency.In this study,coculture engineering was adopted to improve production efficiency of vanillyl alcohol in E.coli.First,two pathways were compared for biosynthesis of the immediate precursor 3,4-dihydroxybenzyl alcohol in monocultures,and the 3-dehydroshikimate-derived pathway showed higher efficiency than the 4-hydroxybenzoate-derived pathway.To enhance the efficiency of the last methylation step,two strategies were used,and strengthening S-adenosylmethionine(SAM)regeneration showed positive effect while strengthening SAM biosynthesis showed negative effect.Then,the optimized pathway was assembled in a single cell.However,the biosynthetic efficiency was still low,and was not significantly improved by modular optimization of pathway genes.Thus,coculturing engineering strategy was adopted.At the optimal inoculation ratio,the titer reached 328.9 mg/L.Further,gene aroE was knocked out to reduce cell growth and improve 3,4-DHBA biosynthesis of the upstream strain.As a result,the titer was improved to 559.4 mg/L in shake flasks and to 3.89 g/L in fed-batch fermentation.These are the highest reported titers of vanillyl alcohol so far.This work provides an effective strategy for sustainable production of vanillyl alcohol.
文摘To develop efficient and economical direct ethanol production from fine rice straw crashed mechanically, two high-performing fungi, which can secret hyperactive cellulases and/or ferment effectively various sugars, were selected from some strains belong to Mucor circinelloides preserved in our laboratory. The simultaneous saccharification and fermentation （SSF） by coculture with these fungi was investigated. The screening of high- performing fungi resulted in the selection of NBRC 4572 as an ethanol-producing fungus and NBRC 5398 as a cellulase-secreting fungus. The strain 4572 produced ethanol aerobically from glucose and xylose in high yields of 0.420 g/g at 36 h and 0.478 g/g at 60 h, respectively, but secreted fairly low cellulases. On the other hand, the strain 5398 also produced ethanol from glucose in yield of 0.340 g/g though it had a little growth in xylose culture. However, it secreted hyperactive cellulases that are essential for hydrolysis of rice straw in culture and the maximum activities of endo-β-glucanase and β-glucosi- dase were 2.11 U/L and 1.47 U/L, respectively. In SSF of rice straw by coculture with two fungi selected, the ethanol production reached 1.28 g/L after 96 h when the inoculation ratio of the strain 5398 to the strain 4572 was 9.
文摘Studies on simultaneous saccharification and fermentation(SSF)of wheat bran flour,a grain milling residue as the substrate using coculture method were carried out with strains of starch digesting Aspergillus niger and nonstarch digesting and sugar fermenting Kluyveromyces marxianus in batch fermentation.Experi-ments based on central composite design(CCD)were conducted to maximize the glucose yield and to study the effects of substrate concentration,pH,temperature,and enzyme concentration on percentage conversion of wheat bran flour starch to glucose by treatment with fungalα-amylase and the above parameters were optimized using response surface methodology(RSM).The optimum values of substrate concentration,pH,temperature,and enzyme concentration were found to be 200g/L,5.5,65℃ and 7.5IU,respectively,in the starch saccharification step.The effects of pH,temperature and substrate concentration on ethanol concentration,biomass and reducing sugar concentration were also investigated.The optimum temperature and pH were found to be 30℃ and 5.5,respectively.The wheat bran flour solution equivalent to 6%(w/V)initial starch concentration gave the highest ethanol concentrationof 23.1g/Lafter 48hoffermentation at optimum conditions of pH and temperature.The growth kinetics was modeled using Monod model and Logistic model and product formation kinetics using Leudeking-Piret model.Simultaneous saccharificiation and fermenta-tion of liquefied wheat bran starch to bioethanol was studied using coculture of amylolytic fungus A.niger and nonamylolytic sugar fermenting K.marxianus.
文摘Cellular strategies remain a crucial component in bone tissue engineering （BTE）. So far, the outcome of cell-based strategies from initial clinical trials is far behind compared to animal studies, which is suggested to be related to insufficient nutrient and oxygen supply inside the Ussue-engineered constructs. Cocultures, by introducing angiogenic cells into osteogenic cell cultures, might provide a solution for improving vascularization and hence increasing bone formation for cell-based constructs. So far, pre-clinical studies demonstrated that cocultures enhance vascularization and bone formation compared to monocultures. However, there has been no report on the application of cocultures in clinics. Therefore, this mini-review aims to provide an overview regarding （i） critical parameters in cocultures and the outcomes of cocultures compared to monocultures in the currently available pre-clinical studies using human mesenchymal stem cells implanted in orthotopic animal models; and （ii） the usage of monocultures in clinical application in BTE.
基金Deutsche Forschungsgemeinschaft(DFG,German Research Foundation),Grant/Award Numbers:406232485,LE 2936/9‐1,331351713–SFB1324Heidelberg Karlsruhe Strategic Partnership(HeiKa,Germany)Impuls‐und Vernetzungsfonds der Helmholtz‐Gemeinschaft。
文摘Most of the biological processes,including cell signaling,cancer invasion,embryogenesis,or neural development,are dependent on and guided by the complex architecture and composition of cellular microenvironments.Mimicking such microenvironments in cell coculture models is crucial for fundamental and applied biology investigations.The ability to combine different cell types grown as both two‐dimensional(2D)monolayers and three‐dimensional(3D)spheroids in specific defined location inside a microculture environments is a key towards in vitro tissue modeling and towards mimicking complex in vivo cellular processes.In this study,we introduce and investigate a method to create in vitro models of 2D cell monolayers cocultured with 3D spheroids in defined preorganization.We demonstrate the possibility of creating such complex cellular microenvironments in a high‐throughput and automated manner by creating arrays of such droplets containing prearranged 2D and 3D cellular microcolonies.Furthermore,we demonstrate an application of this approach to study paracrine propagation of Wnt signaling between 2D and 3D cellular colonies.This method provides a general approach for the miniaturized,high‐throughput,and automated formation of complex coculture cellular microarchitectures that will be useful for mimicking various in vivo complex cellular structures and for studying complex biological processes in vitro.
基金funded by the National Key R&D Program of China(2021YFD2100200/2021YFD2100201)the Program for Science&Technology Innovation Talents in Universities of Henan Province(22HASTIT034)+1 种基金the Key Project of Henan Natural Science Foundation(242300421201)the Science and Technology Department of Henan Province(232103810025).
文摘Pediococcus pentosaceus has a promising potential to improve the quality and flavor of Chinese steamed bread(CSB),but its synergistic effect with Saccharomyces cerevisiae is still unclear.In this study,P.pentosaceus was cocultured with three S.cerevisiae isolates separately,which had different leavening ability.The times required for the fermentation of dough by 2 times the volume of S.cerevisiae isolates ACX 0089,ACX 0490 and ACX 0078 were 46,88 and 120 min,respectively.The results showed that the leavening ability of S.cerevisiae had a significant impact on the quality of CSBs.Compared other S.cerevisiae isolates,the ACX 0490 cocultured with P.pentosaceus increased the contents of bound water and free water in CSB,decreased the content of semibound water,and produced larger and regular porosity,thus endowed a greater specific volume and softer texture.A total of 36 volatile organic compounds(VOCs)were found in CSBs,with alcohols accounting for the main component.The coculture of P.pentosaceus with the intermediate leavening ability of S.cerevisiae ACX 0490 produced the highest proportion of esters among all CSBs.Moreover,S.cerevisiae ACX 0490 endowed the CSB unique VOCs,including cyclodecanol,ethylacetate,and 3,5-dimethylbenzaldehyde.The results of sensory evaluation also showed that S.cerevisiae ACX0490 cocultured CSB obtained the highest scores among all the test samples.The results provided a reference for selecting S.cerevisiae isolates with different leavening ability and the combination of P.pentosaceus to achieve the best quality and flavor of CSB.
基金supported by the Asian Cooperation Fund from Ministry of Foreign Affairs of the People’s Republic of China:Technology cooperation and poverty reduction through integrated rice-fish farming in Lancang-Mekong countries(125A0607)the earmarked fund for China Agriculture Research System(CARS)[CARS-48].
文摘In Myanmar,the advancement of the integrated rice-fish farming system legs behind rice monoculture farming,and there exists limited awareness of its advantages.Ecosystem services(ES)valuation plays a crucial role in integrated environmental decision-making,promoting sustainable agriculture practices,facilitating land-use planning,and ensuring food security in rural areas.Assessing the ES value in Delta region of Myanmar where rice-fish coculture is extensively practiced is essential for understanding the level of ES benefits derived from this farming system.The objective of this study is to promote the development of the rice-fish coculture system in delta region by estimating its ES value.We conducted a comprehensive examination of the Direct,Indirect,Option and Existence ES value of the rice-fish and rice monoculture in Maubin District,an area where rice-fish development research is being actively carried out within the delta region.The results revealed that the ES value of rice-fish coculture ecosystems in the study area was amounted to 28,588 US$/hm^(2)/year.This value was 2.82%higher than rice monoculture system.Additionally,the rice-fish coculture system yielded product provisional values averaging 1,275 US$/hm^(2)/year,representing a significant increase of 40.3%compared to rice monoculture farming.Our study shows that the adoption of rice-fish coculture farming system not only improves the ES value of the delta region,but also supports food security and socio-economic well-being.Furthermore,it provides valuable insights for policymakers on effective management policies for future development of the rice-fish coculture ecosystem.
基金This research is supported by grants from the Chinese Postdoctoral Foundation and the Beijing Young Scientist Culture Foundation.
文摘Objective To investigate the effect of rat Schwann cell secretion on the proliferation and differentiation of human embryonic neural stem cells (NSCs). Methods The samples were divided into three groups. In Group One, NSCs were cultured in DMED/F12 in which Schwann cells had grown for one day. In Group Two, NSCs and Schwann cells were co-cultured. In Group Three, NSCs were cultured in DMEM/F12. The morphology of NSCs was checked and β-tubulin, GalC, hoechst 33342 and GFAP labellings were detected. Results In Group One, all neural spheres were attached to the bottom and differentiated. The majority of them were p-tubulin positive while a few of cells were GFAP or GalC positive. In Group Two, neural spheres remained undifferentiatied and their proliferation was inhibited in places where Schwann cells were robust. In places where there were few Schwann cells, NSCs performed in a similar manner as in Group One. In Group Three, the cell growth state deteriorated day after day. On the 7th day, most NSCs died. Conclusion The secretion of rat Schwann cells has a growth supportive and differentiation-inducing effect on human NSCs.
基金supported by the Natural Science Foundation of Guangdong Province,No.2020A1515010090(to ZLZ)the Science and Technology Project Foundation of Guangzhou City,No.202002030004(to HZ).
文摘Astrocytes and microglia play an orchestrated role following spinal cord injury;however,the molecular mechanisms through which microglia regulate astrocytes after spinal cord injury are not yet fully understood.Herein,microglia were pharmacologically depleted and the effects on the astrocytic response were examined.We further explored the potential mechanisms involving the signal transducers and activators of transcription 3(STAT3)pathway.For in vivo experiments,we constructed a contusion spinal cord injury model in C57BL/6 mice.To deplete microglia,all mice were treated with colony-stimulating factor 1 receptor inhibitor PLX3397,starting 2 weeks prior to surgery until they were sacrificed.Cell proliferation was examined by 5-ethynyl-2-deoxyuridine(EdU)and three pivotal inflammatory cytokines were detected by a specific Bio-Plex Pro^(TM) Reagent Kit.Locomotor function,neuroinflammation,astrocyte activation and phosphorylated STAT3(pSTAT3,a maker of activation of STAT3 signaling)levels were determined.For in vitro experiments,a microglia and astrocyte coculture system was established,and the small molecule STA21,which blocks STAT3 activation,was applied to investigate whether STAT3 signaling is involved in mediating astrocyte proliferation induced by microglia.PLX3397 administration disrupted glial scar formation,increased inflammatory spillover,induced diffuse tissue damage and impaired functional recovery after spinal cord injury.Microglial depletion markedly reduced EdU+proliferating cells,especially proliferating astrocytes at 7 days after spinal cord injury.RNA sequencing analysis showed that the JAK/STAT3 pathway was downregulated in mice treated with PLX3397.Double immunofluorescence staining confirmed that PLX3397 significantly decreased STAT3 expression in astrocytes.Importantly,in vitro coculture of astrocytes and microglia showed that microglia-induced astrocyte proliferation was abolished by STA21 administration.These findings suggest that microglial depletion impaired astrocyte proliferation and astrocytic scar formation,and induced inflammatory diffusion partly by inhibiting STAT3 phosphorylation in astrocytes following spinal cord injury.
基金Supported by the "Rudoff Bartling Foundation" and "Foerdergemeinschaft Kinder-Krebs-Zentrum Hamburg e.V."
文摘AIM： The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stern cell cultures. We investigated the potential of rat mesenchymal stem cells （MSC） from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS： Mesenchymal stem cells were marked with green fluorescent protein （GFP） by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP＋ or GFP- cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thyl and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS： Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression, CONCLUSION： The results indicate that （1） rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and （2） that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.
文摘The single cell gel electrophoresis （SCGE） technique was used to detect DNA damage in germ cells of rats, which was induced by five tested nitroaromatic compounds. Mixed cultures of Sertoli and germ cells were prepared from the rats testis and their responses to rn-dinitrobenzene （ m-DNB）, 2,4-dinitrotoluene （ 2,4-DNT）, 2,6-dinitroto-luene（2,6-DNT）, 4-nitrotoluene（4-NT） and 2,4-dinitroaniline（2,4-DNAn） were studied. The results show that all the five chemicals have a reproductive toxicity. Each dose group and the control group were significantly different （ P 〈 0. 01 ）. All of them can lead to the damage to DNA in the germ cells of Kunming male rats in the definite range of concentration（m-DNB ： 0. 04-25μmol/L; 2,4-DNT, 2,6-DNT and 4-NT： 0. 032-500μmol/L; 2,4-DNAn ：0. 8-20μmol/L）. When the concentration increases, the damage rate will become higher, which shows an evident logarithm dose-effect relationship.
基金ACKNOWLEDGMENTS This work was supported by the Innovation Program of the Shanghai Municipal Education Commission （No. 102216） and by the National Natural Science Foundation of China （No. 81072096）.
文摘Prostate cancer （PCa） is an age-related disease, and the stromal microenvironment plays an important role in prostatic malignant progression. However, the differences in prostate stromal cells present in young and old tissue are still obscure. We established primary cultured stromal cells from normal prostatic peripheral zone （PZ） of donors of varying ages and found that cultured stromal cells from old donors （PZ-old） were more enlarged and polygonal than those from young donors （PZ-young）. Furthermore, based on immunocytochemical and ultrastructural analysis, the components of stromal cells changed from a majority of fibroblasts to a mixture of fibroblasts and myofibroblasts with increasing donor age. Using a three-dimensional in vitro culture system, we found that PZ-old stromal cells could enhance the proliferation, migration and invasion of cocultured benign BPH-1 and PC-3 cells. Using an in vivo tissue recombination system, we also found that PZ-old stromal cells are more effective than PZ-young cells in promoting tumour formation by BPH-1 cells of high passage（〉100） and PC-3 cells. To probe the possible mechanism of these effects, we performed cDNA microarray analysis and profiled 509 upregulated genes and 188 downregulated genes in PZ-old cells. Among the changed genes, we found genes coding for a subset of paracrine factors that are capable of influencing adjacent epithelial cells; these include hepatocyte growth factor （HGF）, fibroblast growth factor 5 （FGF5）, insulin-like growth factor 2 （IGF2）, insulin-like growth factor-binding protein 4 （IGFBP4）, IGFBP5 and matrix metallopeptidase 1 （MMP1）. Changes in the expression of these genes were further confirmed by quantitative real-time polymerase chain reaction （PCR）, Western blotting and enzyme-linked immunosorbent assays. Overall, our findings indicate that stromal cells from prostate PZ of old donors are more active than similar cells from young donors in promoting the malignant process of adjacent epithelial cells. This finding hints at a new potential strategy for the prevention of PCa.
