Mixed Culture System Research Articles

Bioleaching of Industrial Metallic Steel Waste by Mixed Cultures of Thermoacidophilic Archaea

Different mixed cultures of extremely thermoacidophilic microorganisms were used for bioleaching of metalliferous industrial dust waste derived from the basic oxygen furnace (BOF) steelmaking process. Such mixed cultures can extract various metals from multi-metallic BOF-dust waste, improving the metal dissolution and bioleaching performance in frames of metal recycling processes to assist circular economies and waste management. The results of the investigation showed that mixed cultures of thermoacidophilic archaea of the order Sulfolobales (Acidianus spp., Sulfolobus spp., and Metallosphaera sedula) during their growth in laboratory glass bioreactors provided a superior bioleaching system to Acidianus manzaensis alone. Depending on the composition of mixed thermoacidophilic cultures, extraction of various metals from BOF-dust could be achieved. Among the three different types of mixed cultures tested, the mixed culture system of A. manzaensis, A. brierleyi, and S. acidocaldarius was most effective for extraction of major elements (Fe, Ca, Zn, Mn, and Al). The mixed culture of A. manzaensis, A. brierleyi, and M. sedula showed high performance for bioleaching of most of the minor elements (Cu, Ni, Pb, Co, Mo, and Sr). The efficient ability of mixed cultures to colonise the mineral matrix of the metal waste product was observed via scanning electron microscopy, while their metal extraction capacities were analysed by inductively coupled plasma mass spectrometry. These investigations will promote the further design of microbial consortia in order to break down the solid matrix and efficiently extract metals from metalliferous waste materials.

Enhancing medium-chain fatty acids production via alkyl polyglucose modulation of electron acceptor generation, microbial community and metabolic traits in anaerobic fermentation of waste activated sludge

Production of medium chain fatty acids (MCFAs) through anaerobic fermentation is an essential pathway for efficient resource recovery and utilization of waste activated sludge (WAS). In this study, a novel strategy utilizing polyglucose (APG) to enhance MCFAs production from WAS by anaerobic fermentation was proposed. The results showed that the maximum MCFAs production reached 14745.0 mg COD/L with 0.3 g APG/g TSS, which was 20.2 times of that without APG treatment. The transformation of intermediates demonstrated that APG promoted the generation of electron acceptors (i.e., short-chain fatty acids) and conversion of substrate to MCFAs. Microbial community analysis indicated that APG selectively enriched microbe involved in hydrolysis, acidogenesis and chain elongation (CE). Also, the presence of APG had a negative impact on methanogens and potential excessive ethanol oxidation (EEO) microbe, which were competitors of CE bacteria in mixed-culture system. Metagenomic analysis showed an increase in relative abundance of genes encoding metabolic functions responsible for hydrolysis, acidogenesis, CE and energy generation, thus maintained high microbial activity in MCFAs biosynthesis. These enhanced metabolic functions were accompanied by a reduction in relative abundance of genes encoding methanogenesis and EEO metabolism, which together result in boosting the efficiency of substrate and energy flow to MCFAs production. This study presents a promising approach to improve the biotransformation of polysaccharides and proteins from WAS into MCFAs.

Study on the Mechanism of Algae Bacteria Symbiotic System in Treating Fracturing Flowback Fluid

Fracturing operations in oil and gas wells are one of the main measures to increase production. Fracturing flowback fluid has the characteristics of high viscosity, high COD value, and containing multiple chemical reagents, making it difficult to treat. It has become one of the main pollutants in oil fields. Directly discharging untreated fracturing fluid can cause serious pollution to the environment and soil. Therefore, in-depth research on the treatment methods of fracturing flowback fluid is of great significance for environmental protection and cost reduction in oil and gas fields. This article systematically analyzes the sources, components, pollution characteristics, and treatment status of fracturing flowback fluid. The efficient treatment of fracturing flowback fluid using a microalgae aerobic bacteria mixed culture system was conducted, and the mechanism of algae bacteria symbiotic treatment of oily wastewater and the mechanism of microalgae aerobic bacteria mixed culture purification of fracturing flowback fluid were analyzed in depth.

A dual-function mixed-culture biofilm for sulfadiazine removal and electricity production using bio-electrochemical system

Sulfadiazine (SDZ) is frequently detected in environmental samples, arousing much concern due to its toxicity and hard degradation. This study investigated the electricity generation capabilities, SDZ removal and microbial communities of a highly efficient mixed-culture system using repeated transfer enrichments in a bio-electrochemical system. The mixed-culture biofilm (S160-T2) produced a remarkable current density of 954.12 ± 15.08 μA cm−2 with 160 mg/L SDZ, which was 32.9 and 1.8 times higher than that of Geobacter sulfurreducens PCA with 40 mg/L SDZ and without additional SDZ, respectively. Especially, the impressive SDZ removal rate of 98.76 ± 0.79% was achieved within 96 h using the further acclimatized mixed-culture. The removal efficiency of this mixed-culture for SDZ through the bio-electrochemical system was 1.1 times higher than that using simple anaerobic biodegradation. Furthermore, the current density and removal efficiency in this system gradually decreased with increasing SDZ concentrations from 0 to 800 mg/L. In addition, community diversity data demonstrated that the dominant genera, Geobacter and Escherichia-Shigella, were enriched in mixed-culture biofilm, which might be responsible for the current production and SDZ removal. This work confirmed the important roles of acclimatized microbial consortia and co-substrates in the simultaneous removal of SDZ and electricity generation in an electrochemical system.

Evidence of autotrophic direct electron transfer denitrification (DETD) by Thiobacillus species enriched on biocathodes during deep polishing of effluent from a municipal wastewater treatment plant

Cathodes can play a pivotal role as electron donors for denitrification in wastewater treatment plants, facilitating the removal of nitrogen-containing compounds such as nitrate. Denitrifying bacteria can either accept electrons directly from the electrode or indirectly via electron shuttling intermediaries generated at the cathode. In this study, comparative analyses were conducted between two denitrifying electrochemical reactors with biocathodes poised at different potentials, one that generated H2 through water electrolysis and one that did not (−500 mV and −800 mV vs. SHE). Nitrogen removal efficiencies were much higher in the −500 mV (vs. SHE) system, >93 % compared to only 20 %. Notably, a biofilm formed exclusively on the −500 mV (vs. SHE) cathode, predominantly composed of Thiobacillus species, with the majority most similar to Thiobacillus strain 65-29. Metatranscriptomics showed that genes from Thiobacillus strain 65-29 encoding outer surface c-type cytochrome and porin proteins that could facilitate direct electron uptake, were markedly up-regulated in the −500 mV (vs. SHE) reactor. Reduced sulfur species generated by the cathode and sulfate reducing bacteria might have also provided a source of electrons for nitrate reduction. In fact, Thiobacillus in the −500 mV (vs. SHE) system exhibited elevated expression of genes involved in thiosulfate (soxABXYZ), sulfite (soeABC), and sulfide (fccAB) oxidation. These findings offer valuable insights into the mechanisms of autotrophic denitrification by Thiobacillus in mixed culture systems and provide practical guidance for engineering applications.

Effects of Weak Electric Fields on the Denitrification Performance of Pseudomonas stutzeri: Insights into Enzymes and Metabolic Pathways.

Enhanced denitrification has been reported under weak electric fields. However, it is difficult to investigate the mechanism of enhanced denitrification due to the complex interspecific interactions of mixed-culture systems. In this study, Pseudomonas stutzeri, capable of denitrification under anaerobic conditions, was selected for treating low COD/N (2.0, ratio between concentration of chemical oxygen demand and NO3--N) artificial wastewater under constant external voltages of 0.2, 0.4, and 0.6 V. The results revealed that P. stutzeri exhibited the highest efficiency in nitrate reduction at 0.2 V. Moreover, the maximum nitrate removal rate was 15.96 mg/(L·h) among the closed-circuit groups, 19.39% higher than that under the open-circuit group. Additionally, a notable reduction in nitrite accumulation was observed under weak electric fields. Enzyme activity analysis showed that the nitrate reductase activities were significantly increased among the closed-circuit groups, while nitrite reductase activities were inhibited. Transcriptomic analysis indicated that amino acid metabolism, carbohydrate metabolism, and energy metabolism were increased, enhancing the resistance of P. stutzeri to environmental stress and the efficiency of carbon source utilization for denitrification. The current study examined the impacts of weak electric fields on enzyme activities and microbial metabolic pathways and offers valuable insights into the mechanism by which denitrification is enhanced by weak electric fields.

Resource recovery through bioremediation of fuel-synthesis wastewater in a biofilm photobioreactor using purple non-sulfur bacteria: A circular bioeconomy approach

In the current era of wastewater treatment, integrating reusable water production with resource recovery is a key goal. This study aims to treat fuel-synthesis wastewater (FSW), intending to recover various resources, including polyhydroxybutyrate (PHBs), single cell protein, bacteriochlorophylls, carotenoids, and coenzyme Q10 from suspended and biofilm growth to decrease the harvesting costs. The study considered the treatment process, biofilm growth, and resource recovery potential in a mixed-culture system enriched with purple non-sulfur bacteria for treating FSW. Specifically, the effects of four different FSW strengths (25–100 %) and nitrogen sufficiency (N+) or deficiency (N−) were evaluated in eight biofilm photobioreactors. This study observed a direct correlation between the concentration of FSW and PHB content; specifically, as the FSW content decreased from 100 % (undiluted) to 25 % the PHB content decreased. The undiluted condition achieved 17 % dry cell weight as PHB in the suspended growth and 22.6 % in the biofilm growth under N− condition. The protein content ranged between 33 and 44 %, and the presence of nitrogen had a slight positive effect on higher protein content. No trend was observed for carotenoids or bacteriochlorophylls in the N− condition. In contrast, for the N+ condition, the concentration of bacteriochlorophylls increased with decreasing wastewater concentration under suspended growth, while it decreased with decreasing wastewater concentration under biofilm growth. Coenzyme Q10 concentration was enhanced under the most growth-limited condition (25 %, N−). PHB and protein content of these resources seem most promising when using N− and N+ conditions, respectively.

Improving productivity and soil fertility in Medicago sativa and Hordeum marinum through intercropping under saline conditions

Background and aimsIntercropping is an agriculture system used to enhance the efficiency of resource utilization and maximize crop yield grown under environmental stress such as salinity. Nevertheless, the impact of intercropping forage legumes with annual cereals on soil salinity remains unexplored. This research aimed to propose an intercropping system with alfalfa (Medicago sativa)/sea barley (Hordeum marinum) to explore its potential effects on plant productivity, nutrient uptake, and soil salinity.MethodsThe experiment involved three harvests of alfalfa and Hordeum marinum conducted under three cropping systems (sole, mixed, parallel) and subjected to salinity treatments (0 and 150 mM NaCl). Agronomical traits, nutrient uptake, and soil properties were analyzed.Resultsrevealed that the variation in the measured traits in both species was influenced by the cultivation mode, treatment, and the interaction between cultivation mode and treatment. The cultivation had the most significant impact. Moreover, the mixed culture (MC) significantly enhanced the H. marinum and M. sativa productivity increasing biomass yield and development growth under salinity compared to other systems, especially at the second harvest. Furthermore, both intercropping systems alleviated the nutrient uptake under salt stress, as noted by the highest levels of K+/Na+ and Ca2+/Mg2+ ratios compared to monoculture. However, the intercropping mode reduced the pH and the electroconductivity (CEC) of the salt soil and increased the percentage of organic matter and the total carbon mostly with the MC system.ConclusionsIntercropped alfalfa and sea barely could mitigate the soil salinity, improve their yield productivity, and enhance nutrient uptake. Based on these findings, we suggest implementing the mixed-culture system for both target crops in arid and semi-arid regions, which further promotes sustainable agricultural practices.

Hypothermia promotes tunneling nanotube formation and the transfer of astrocytic mitochondria into oxygen-glucose deprivation/reoxygenation-injured neurons

Mitochondrial transfer occurs between cells, and it is important for damaged cells to receive healthy mitochondria to maintain their normal function and protect against cell death. Accumulating evidence suggests that the functional mitochondria of astrocytes are released and transferred to oxygen–glucose deprivation/reoxygenation (OGD/R)-injured neurons. Mild hypothermia (33 °C) is capable of promoting this process, which partially restores the function of damaged neurons. However, the pathways and mechanisms by which mild hypothermia facilitates mitochondrial transfer remain unclear. We are committed to studying the role of mild hypothermia in neuroprotection to provide reliable evidences and insights for the clinical application of mild hypothermia in brain protection. Tunneling nanotubes (TNTs) are considered to be one of the routes through which mitochondria are transferred between cells. In this study, an OGD/R-injured neuronal model was successfully established, and TNTs, mitochondria, neurons and astrocytes were double labeled using immunofluorescent probes. Our results showed that TNTs were present and involved in the transfer of mitochondria between cells in the mixed-culture system of neurons and astrocytes. When neurons were subjected to OGD/R exposure, TNT formation and mitochondrial transportation from astrocytes to injured neurons were facilitated. Further analysis revealed that mild hypothermia increased the quantity of astrocytic mitochondria transferred into damaged neurons through TNTs, raised the mitochondrial membrane potential (MMP), and decreased the neuronal damage and death during OGD/R. Altogether, our data indicate that TNTs play an important role in the endogenous neuroprotection of astrocytic mitochondrial transfer. Furthermore, mild hypothermia enhances astrocytic mitochondrial transfer into OGD/R-injured neurons via TNTs, thereby promoting neuroprotection and neuronal recovery.

Biodegradation of 17β‐estradiol by drug‐resistant Stenotrophomonas maltophilia MN08 and Pseudomonas aeruginosa KL10 isolated from the sediment sample

Abstract17 β‐Estradiol is produced by animals, and humans and excreted through excreta, and has endocrine‐disrupting activity. Bacterial degradation is one of the suitable methods used for the removal of 17 β‐estradiol (E2) from the natural environment. In this study, E2‐degrading bacteria were screened from the enriched sediment sample from the wastewater environment. Two bacterial strains of E2‐degrading bacteria (Stenotrophomonas maltophilia MN08 and Pseudomonas aeruginosa KL10) were determined and >70% of E2‐degradation was achieved. The strain MN08 could rapidly remove approximately 86.5% ± 1.3% of E2 within 7 days of treatment. In addition, the strain KL10 removed 79.5% ± 1% of E2 within the same period. Optimized culture temperature and pH of the medium could improve E2‐degradation. The results demonstrated that the strain MN08 exhibited maximum E2‐degradation activity at 35°C, and pH 8.0. The optimal pH for strain KL10 to degrade E2 ranged from 7.0 to 9.0. The E2‐degradation level for the mixture culture systems can exhibit synergistic E2‐degradation activity. The degradation of the MN08 and KL10 individual strains was 87.3% ± 1.2% and 81.4% ± 1.5%, respectively. The E2‐degradation potential for the mixed culture system was 91.3% ± 2.9% under optimized conditions in which 100 mg/L of E2 served as the sole source of carbon and energy. The present finding demonstrates that the co‐culture of S. maltophilia MN08 and P. aeruginosa KL10 may provide a promising bio‐treatment strategy for the removal of steroids from wastewater.

A mutant methionyl-tRNA synthetase-based toolkit to assess induced-mesenchymal stromal cell secretome in mixed-culture disease models

BackgroundMesenchymal stromal cells (MSCs) have a dynamic secretome that plays a critical role in tissue repair and regeneration. However, studying the MSC secretome in mixed-culture disease models remains challenging. This study aimed to develop a mutant methionyl-tRNA synthetase-based toolkit (MetRSL274G) to selectively profile secreted proteins from MSCs in mixed-culture systems and demonstrate its potential for investigating MSC responses to pathological stimulation.MethodsWe used CRISPR/Cas9 homology-directed repair to stably integrate MetRSL274G into cells, enabling the incorporation of the non-canonical amino acid, azidonorleucine (ANL), and facilitating selective protein isolation using click chemistry. MetRSL274G was integrated into both in H4 cells and induced pluripotent stem cells (iPSCs) for a series of proof-of-concept studies. Following iPSC differentiation into induced-MSCs, we validated their identity and co-cultured MetRSL274G-expressing iMSCs with naïve or lipopolysaccharide (LPS)-treated THP-1 cells. We then profiled the iMSC secretome using antibody arrays.ResultsOur results showed successful integration of MetRSL274G into targeted cells, allowing specific isolation of proteins from mixed-culture environments. We also demonstrated that the secretome of MetRSL274G-expressing iMSCs can be differentiated from that of THP-1 cells in co-culture and is altered when co-cultured with LPS-treated THP-1 cells compared to naïve THP-1 cells.ConclusionsThe MetRSL274G-based toolkit we have generated enables selective profiling of the MSC secretome in mixed-culture disease models. This approach has broad applications for examining not only MSC responses to models of pathological conditions, but any other cell type that can be differentiated from iPSCs. This can potentially reveal novel MSC-mediated repair mechanisms and advancing our understanding of tissue regeneration processes.

Genome-centric metagenomics provides new insights into metabolic pathways of polyhydroxyalkanoates biosynthesis and functional microorganisms subsisting on municipal organic wastes

The microbial community of a sequencing batch reactor operated under feast and famine conditions for production of polyhydroxyalkanoates (PHAs) was characterized through high-throughput sequencing and metagenomic analysis. The fermented food waste and chemically-enhanced primary sludge was fed in this bioreactor. After acclimation, the PHA yield achieved as high as 0.60–0.69 g CODPHA/g CODS. The complete changes of microbial community structure were found during shifts of feedstock. A synthesis of SCL/MCL-PHAs pathway was established for PHA-producing bioreactor in this mixed-culture system. The structure-performance relationship of PHA-producing microbial community and feedstock composition was investigated. The results showed that microbial community tends to be decentralized and prefer team work for PHA synthesis to consume the multiple substrates and digest inevitable non-VFA contents in fermented liquor. This study also discovered unreported potential PHA producers (e.g., genera Tabrizicola, Nannocystis, Ga0077539, Ga0077559, JOSHI-001, SNC69–320 and UBA2334) subsisting on municipal organic wastes and expands the current knowledge about mixed-culture system that the PHA synthesis pathway is widely existed in activated sludge.

Identification of a novel combination treatment strategy in clear cell renal cell carcinoma stem cells with shikonin and ipilimumab.

Management of clear cell renal cell carcinoma (ccRCC) has changed rapidly in recent years with the advent of immune checkpoint inhibitors (ICIs). However, only a limited number of patients can sustainably respond to immune checkpoint inhibitors and many patients develop resistance to therapy, creating an additional need for therapeutic strategies to improve the efficacy of systemic therapies. Binding probability and target genes prediction using online databases, invasion, migration, and apoptosis assays as well as the inhibition of cancer stem cells (CSCs) markers in ccRCC cell lines were used to select the most promising phytochemicals (PTCs). Mixed lymphocyte tumor cell culture (MLTC) system and flow cytometry were performed to confirm the potential combination strategy. The potential immunotherapeutic targets and novel CSC markers were identified via the NanoString analysis. The mRNA and protein expression, immune signatures as well as survival characteristics of the marker in ccRCC were analyzed via bioinformation analysis. Shikonin was selected as the most promising beneficial combination partner among 11 PTCs for ipilimumab for the treatment of ccRCC patients due to its strong inhibitory effect on CSCs, the significant reduction of FoxP3+ Treg cells in peripheral blood mononuclear cells (PBMCs) of patients and activation of the endogenous effector CD3+CD8+ and CD3+CD4+ T cells in response to the recognition of tumor specific antigens. Based on NanoString analysis VCAM1, CXCL1 and IL8 were explored as potential immunotherapeutic targets and novel CSC markers in ccRCC. The expression of VCAM1 was higher in the tumor tissue both at mRNA and protein levels in ccRCC compared with normal tissue, and was significantly positively correlated with immune signatures and survival characteristics in ccRCC patients. We propose that a combination of shikonin and ipilimumab could be a promising treatment strategy and VCAM1 a novel immunotherapeutic target for the treatment of ccRCC.

Anticancer polypyrrole-polyethylenimine drug-free nanozyme for precise B-cell lymphoma therapy

As an alternative strategy for cancer treatment, the combination of cancer nanomedicine and immunotherapy is promising with regard to efficacy and safety; however, precise modulation of the activation of antitumor immunity remains challenging. Therefore, the aim of the present study was to describe an intelligent nanocomposite polymer immunomodulator, drug-free polypyrrole-polyethyleneimine nanozyme (PPY-PEI NZ), which responds to the B-cell lymphoma tumor microenvironment, for precision cancer immunotherapy. Earlier engulfment of PPY-PEI NZs in an endocytosis-dependent manner resulted in rapid binding in four different types of B-cell lymphoma cells. The PPY-PEI NZ effectively suppressed B cell colony-like growth in vitro accompanied by cytotoxicity via apoptosis induction. During PPY-PEI NZ-induced cell death, mitochondrial swelling, loss of mitochondrial transmembrane potential (MTP), downregulation of antiapoptotic proteins, and caspase-dependent apoptosis were observed. Deregulated AKT and ERK signaling contributed to glycogen synthase kinase-3-regulated cell apoptosis following deregulation of Mcl-1 and MTP loss. Additionally, PPY-PEI NZs induced lysosomal membrane permeabilization while inhibiting endosomal acidification, partly protecting cells from lysosomal apoptosis. PPY-PEI NZs selectively bound and eliminated exogenous malignant B cells in a mixed culture system with healthy leukocytes ex vivo. While PPY-PEI NZs showed no cytotoxicity in wild-type mice, they provided long-term and efficient inhibition of the growth of B-cell lymphoma-driven nodules in a subcutaneous xenograft model. This study explores a potential PPY-PEI NZ-based anticancer agent against B-cell lymphoma.

Mixed culture biocatalytic production of the high-value biochemical 7-methylxanthine

Background7-Methylxanthine, a derivative of caffeine noted for its lack of toxicity and ability to treat and even prevent myopia progression, is a high-value biochemical with limited natural availability. Attempts to produce 7-methylxanthine through purely chemical methods of synthesis are faced with complicated chemical processes and/or the requirement of a variety of hazardous chemicals, resulting in low yields and racemic mixtures of products. In recent years, we have developed engineered microbial cells to produce several methylxanthines, including 3-methylxanthine, theobromine, and paraxanthine. The purpose of this study is to establish a more efficient biosynthetic process for the production of 7-methylxanthine from caffeine.ResultsHere, we describe the use of a mixed-culture system composed of Escherichia coli strains engineered as caffeine and theobromine “specialist” cells. Optimal reaction conditions for the maximal conversion of caffeine to 7-methylxanthine were determined to be equal concentrations of caffeine and theobromine specialist cells at an optical density (600 nm) of 50 reacted with 2.5 mM caffeine for 5 h. When scaled-up to 560 mL, the simple biocatalytic reaction produced 183.81 mg 7-methylxanthine from 238.38 mg caffeine under ambient conditions, an 85.6% molar conversion. Following HPLC purification and solvent evaporation, 153.3 mg of dried 7-methylxanthine powder was collected, resulting in an 83.4% product recovery.ConclusionWe present the first report of a biocatalytic process designed specifically for the production and purification of the high-value biochemical 7-methylxanthine from caffeine using a mixed culture of E. coli strains. This process constitutes the most efficient method for the production of 7-methylxanthine from caffeine to date.

Co-culturing Immune Cells and Mouse-Derived Mixed Cortical Cultures with Oxygen-Glucose Deprivation to In Vitro Simulate Neuroinflammatory Interactions After Stroke.

Studying interactions between neural cells and glial cells in vitro remains an essential tool for scientists worldwide, and with the addition of oxygen-glucose deprivation (OGD) can be particularly useful for identifying mechanisms related to ischemic stroke-related injury and repair. In developing these protocols in the lab, however, we discovered the limitation of co-culturing immune cells with pure neuronal cultures as the standard media for immune cells impair neuronal growth and vice versa. Thus, we optimized a mixed cortical cell culture system that does not require the use of glial-conditioned media to support the viability and growth of neurons but can nonetheless be used to quantify neuronal survival and dendritic arborization. The following methods provide a guide as to how to culture mixed cortical cells from mouse pups (postnatal day 0-2). Additionally, we demonstrate how to co-culture mixed cortical cells with immune cells (e.g., B cells) to study neuro-immune interactions in vitro.

Implications of plant N/P stoichiometry influenced by arbuscular mycorrhizal fungi for stability of plant species and community in response to nutrient limitation

Arbuscular mycorrhizal fungi (AMF) influence plant nitrogen/phosphorus (N/P) by modifying plant N and P uptake, which further affects plant stoichiometric N/P homeostasis. Plant species and community stoichiometric N/P homeostasis can impact plant species and community stability, respectively, in response to variations of soil N and P availabilities. We investigated interspecific plant interactions via AMF in regard to plant and soil microbial N/P stoichiometry across different soil N and P availabilities induced by N and P addition (0 mg N kg−1, 25 mg N kg−1, 50 mg N kg−1, 30 mg P kg−1 and 100 mg P kg−1). We selected one dominant (Bothriochloa ischaemum; C4 grass) and one subordinate (Lespedeza davurica; legume) species in a natural grassland climax community. We examined how AMF influences stoichiometric N/P homeostasis in monoculture and mixed culture systems, and the resulting consequences for temporal stability of plant species and community in response to variations in soil N and P availability.The AMF mitigated the P limitation of soil microbial communities and decreased the degree of stoichiometric N/P homeostasis of host plants in monoculture. Through their resource‐scavenging and soil organic matter mineralisation functions, AMF enhances plant ‘luxury consumption', promoting species stability in monoculture in response to soil N and P availability variations. Compared with plants in monoculture, the interaction between B. ischaemum and L. davurica via AMF increased shoot N/P under soil N‐poor conditions, leading to an enhanced degree of stoichiometric N/P homeostasis in both plant species, especially the legume.Our results suggest that interspecific plant interaction between C4 grass and legume mediated by AMF confers an advantage in complementarity in plant N acquisition under N‐poor conditions, leading to increased stability of plant communities and better maintenance of subordinate species (legume) in response to soil N deficiency.

Spherical Nucleic Acid Probe Based on 2′-Fluorinated DNA Functionalization for High-Fidelity Intracellular Sensing

Traditional spherical nucleic acids (SNAs) based on gold nanoparticles (AuNPs) assembled through Au-S covalent bonds are widely used in DNA-programmable assembly, biosensing, imaging, and therapeutics. However, biological thiols and other chemical substances can break the Au-S bonds and cause response distortion during the application process, specifically in cell environments. Herein, we report a new type of SNAs based on 2'-fluorinated DNA-functionalized AuNPs with excellent colloidal stability under high salt conditions (up to 1 M NaCl) and over a broad pH range (1-14), as well as resistance to biothiols. The fluorinated spherical nucleic acid probe (Au/FDNA probe) could detect targeted cancer cells with high fidelity. Compared to the traditional thiolated DNA-functionalized AuNP probe (Au-SDNA probe), the Au/FDNA probe exhibited a higher sensitivity to the target and a lower signal-to-background ratio. Furthermore, the Au/FDNA probe could discriminate target cancer cells in a mixed culture system. Using the proposed FDNA functionalization method, previously developed SNAs based on AuNPs could be directly adapted, which might open a new avenue for the design and application of SNAs.

Establishing an Easy-to-Use 3D BM Niche Model for the Co-Culture of Primary Bone Marrow Mesenchymal Stromal Cells (BM-MSC) with Hematopoietic Stem and Progenitor Cells (HSPC)

Background BM-MSC are a collection of primitive cell populations, a major stromal component of the BM niche and are essential to maintain tissue homeostasis. 2D culture protocols are popular, though not standardized, and effective for BM-MSC expansion, but display limited niche function in co-culture systems. 3D co-culture leverages the importance of cell signaling networks activated by cell-cell and cell-matrix interactions in native tissues. Here, we establish a superior and easy-to-use 3D culture protocol and compare to conventional 2D approaches. Methods, Results and Discussion First, we compared commercially available expansion media to the most widely cited investigator-defined media for optimal 2D expansion and 3D culture of ex vivo murine BM-MSC. Based on this, we developed a five-marker flow cytometry panel to accurately quantify BM-MSC culture purity. To test BM-MSC niche function in 2D vs 3D cultures, we generated 3D spheroid hanging drop (HD) cultures from 2D expanded BM-MSC. In HD spheroids, cells are in direct contact with each other as well as extracellular matrix components and can be used to co-culture multiple different cell types. To assess the niche-like function of 3D HD cultures compared to 2D adherent cultures, we assessed Nestin protein expression since Nestin is a marker of HSC niches within mouse BM in vivo. BM-MSC were cultured in 2D or HD and Nestin protein expression assessed by immunofluorescence imaging, showing a significant increase in percent of Nestin-positive cells in HD-cultured BM-MSC, compared to matched 2D controls, in agreement with previous reports in human BM-MSC 3D culture. To assess 3D in vitro BM-MSC niche (IVBMN) function, we assayed stemness markers and survival of FACS isolated primary HSPC (Lineage-Sca1+cKit+, LSK) from mouse BM. LSK cells were cultured as standard (alone) or seeded as co-cultures with BM-MSC within HD with specific cytokine support, in low oxygen (6.5%) conditions (IVBMN) (Figure 1). LSK cells cultured within IVBMN exhibited superior survival as well as retention of stemness markers, demonstrating the benefit of BM-MSC support. In vivo, the BM niche also includes BM niche resident macrophages (BM-Mɸ), which were recently shown to be a limiting factor for engraftment and HSC reconstitution in irradiated mice, and are part of long term and short term HSC niches. To further extend our IVBMN, BM-Mɸ [F4/80+ Ly6G- VCAM1+ CD169+] were collected by FACS and added to IVBMN cultures (IVBMN-Mɸ). Notably, the IVBMN-Mɸ co-culture model further enhanced survival and stemness of primary LSK cells, when compared to LSK cultured alone or within IVBMN. Based on these preliminary data, we demonstrate that our IVBMN-Mɸ culture system is a unique tool which mimics niche properties in vitro and is easy-to-use for HSPC manipulation. Conclusions Here we describe a standardized flow panel for reliable murine BM-MSC immunophenotyping, and characterize a simple but effective 3D BM-MSC co-culture system which allows niche-like function in vitro. Fig. 1. In vitro BM niche (IVBMN) experimental workflow: A) Ex vivo expanded primary BM-MSC (20x) (i) -/+ FACS isolated BM niche resident Mɸ (ii) in co-culture with primary BM HPSC (LSK) (iii). B) Cells were cultured in a 3D mixed culture system with specific cytokine support, in 6.5% low oxygen. C) IVBMN-Mɸ [MSC+ BM-Mɸ +LSK] co-culture promotes maintenance of primary viable LSK compared to IVBMN [MSC+LSK] or LSK standard culture conditions alone, after 5 days culture. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ALL-MALE AND MIXED SEX PRAWN (MACROBRACHIUM ROSENBERGII) PRODUCTION UNDER POLYCULTURE WITH CARPS

An experiment was conducted at Dumurpota of Satkhira district for six months to compare the production performance of prawn (Macrobrachium rosenbergii) under all-male and mixed sex culture systems with carps (Labeo rohita and Catla catla). A total of six experimental ghers were used for two treatments in triplicates: carps with all-male prawn (T1) and carps with mixed sex prawn (T2). In both treatments, prawns were stocked at a rate of 10,000 PL/ha while C. catla and L. rohita were stocked each at a rate of 650 fingerlings ha-1. In T1, the initial average body weight of M. rosenbergii, L. rohita and C. catla was 7.33, 54.83 and 54.5 g respectively, whereas after 180 days of culture period the final average weights became 79.33, 256.5 and 253.47 g, respectively. In T2, the initial average body weight of M. rosenbergii, L. rohita and C. catla were 7.33, 55.07 and 54.17 g respectively, which resulted in the final average weights of 58.67, 258.87 and 252.57 g, respectively. The average production of prawn, L. rohita and C. catla in T1 was 745.97, 160.89 and 170.17 kg ha-1, respectively, while that in T2 was 540.81, 159.34 and 162.98 kg ha-1, respectively. The survival rates of M. rosenbergii, L. rohita and C. catla in T1 were found to be 91.28%, 89.93% and 87.93% respectively, whereas those in T2 were 87.56%, 90.3% and 88.3%, respectively. It was found that the production of carps was similar in both the treatments, but the production of prawn in T1 was substantially higher than that of T2 resulting in a net income difference of 77429.40 Tk ha-1. These results indicate that in polyculture with carps, the sex composition of prawn has no effect on the production of carp species but stocking of all male prawn can significantly increase the production of prawn and the economic return in such type of farming.