Days Of Co-cultivation Research Articles

1,25-dihydroxyvitamin-D3 distinctly impacts the paracrine and cell-to-cell contact interactions between hPDL-MSCs and CD4+ T lymphocytes.

Human periodontal ligament-derived mesenchymal stromal cells (hPDL-MSCs) possess a strong ability to modulate the immune response, executed via cytokine-boosted paracrine and direct cell-to-cell contact mechanisms. This reciprocal interaction between immune cells and hPDL-MSCs is influenced by 1,25-dihydroxyvitamin-D3 (1,25(OH)2D3). In this study, the participation of different immunomodulatory mechanisms on the hPDL-MSCs-based effects of 1,25(OH)2D3 on CD4+ T lymphocytes will be elucidated using different co-culture models with various cytokine milieus. hPDL-MSCs and CD4+ T lymphocytes were co-cultured indirectly and directly with inserts (paracrine interaction only) or directly without inserts (paracrine and direct cell-to-cell contact interaction). They were stimulated with TNF-α or IL-1β in the absence/presence of 1,25(OH)2D3. After five days of co-cultivation, the CD4+ T lymphocyte proliferation, viability, and cytokine secretion were analyzed. Additionally, the gene expression of soluble and membrane-bound immunomediators was determined in hPDL-MSCs. In the indirect and direct co-culture model with inserts, 1,25(OH)2D3 decreased CD4+ T lymphocyte proliferation and viability. The direct co-culture model without inserts caused the opposite effect. 1,25(OH)2D3 mainly decreased the CD4+ T lymphocyte-associated secretion of cytokines via hPDL-MSCs. The degree of these inhibitions varied between the different co-culture setups. 1,25(OH)2D3 predominantly decreased the expression of the soluble and membrane-bound immunomediators in hPDL-MSCs to a different extent, depending on the co-culture models. The degree of all these effects depended on the absence and presence of exogenous TNF-α and IL-1β. These data assume that 1,25(OH)2D3 differently affects CD4+ T lymphocytes via the paracrine and direct cell-to-cell contact mechanisms of hPDL-MSCs, showing anti- or pro-inflammatory effects depending on the co-culture model type. The local cytokine microenvironment seems to be involved in fine-tuning these effects. Future studies should consider this double-edged observation by executing different co-culture models in parallel.

Characterization of the fungal genus Sphaerellopsis associated with rust fungi: species diversity, host-specificity, biogeography, and in-vitro mycoparasitic events of S. macroconidialis on the southern corn rust, Puccinia polysora

Sphaerellopsis species are putative hyperparasites of rust fungi and may be promising biological control agents (BCA) of rust diseases. However, few detailed studies limit potential BCA development in Sphaerellopsis. Here, we explored the biogeography, host-specificity, and species diversity of Sphaerellopsis and examined the early infection stage of one species, S. macroconidialis, to infer its trophic status. We randomly screened 5,621 rust specimens spanning 99 genera at the Arthur Fungarium for the presence of Sphaerellopsis. We identified 199 rust specimens infected with Sphaerellopsis species on which we conducted morphological and multi-locus phylogenetic analyses. Five Sphaerellopsis species were recovered, infecting a total of 122 rust species in 18 genera from 34 countries. Sphaerellopsis melampsorinearum sp. nov. is described as a new species based on molecular phylogenetic data and morphological features of the sexual and asexual morphs. Sphaerellopsis paraphysata was the most commonly encountered species, found on 77 rust specimens, followed by Sphaerellopsis macroconidialis on 56 and S. melampsorinearum on 55 examined specimens. The type species, Sphaerellopsis filum, was found on 12 rust specimens and Sphaerellopsis hakeae on a single specimen. We also recovered and documented for the first time, the sexual morph of S. macroconidialis, from a specimen collected in Brazil. Our data indicate that Sphaerellopsis species are not host specific and furthermore that most species are cosmopolitan in distribution. However, S. paraphysata is more abundant in the tropics, and S. hakeae may be restricted to Australia. Finally, we confirm the mycoparasitic strategy of S. macroconidialis through in-vitro interaction tests with the urediniospores of Puccinia polysora. Shortly after germination, hyphae of S. macroconidialis began growing along the germ tubes of P. polysora and coiling around them. After 12 days of co-cultivation, turgor loss was evident in the germ tubes of P. polysora, and appressorium-like structures had formed on urediniospores. The interaction studies indicate that Sphaerellopsis species may be more effective as a BCA during the initial stages of rust establishment.

Antioxidative Response of Duckweed (Lemna minor L.) to Rhizosphere-Associated Pseudomonas Strains and Exogenous Indole-3-Acetic Acid

Aquatic plants, just like terrestrial plants, are exposed to oxidative stress. However, their responses are still under-researched. In this study, we examined the physiological and antioxidative responses of an aquatic plant, duckweed (Lemna minor L.), to four indole-3-acetic acid (IAA)-degrading and -producing Pseudomonas bacteria (Pseudomonas oryzihabitans D1-104/3, P. putida A3-104/5, P. gessardii C31-106/3 and P. yamanorum C44-104/1) and/or a supraphysiological level of IAA (10 mg L−1). Growth characteristics, total photosynthetic pigment content, histochemical localization of reactive oxygen species and antioxidant enzyme activity (SOD, CAT and POX) were evaluated at two time points, after 3 and 7 days of co-cultivation. Superoxide anion and hydrogen peroxide were produced and accumulated mainly in the roots, daughter fronds and veins of duckweeds. Duckweeds’ responses depended on the strain of Pseudomonas, time and exogenous IAA. Co-cultivation of duckweed with bacteria has positive or neutral effects. Exogenous application of IAA had a negative or neutral effect on enzyme activity and other parameters. Co-cultivation with P. gessardii C31-106/3 showed plant-growth-promoting effects on duckweed: increased biomass production, modulation of duckweeds’ antioxidant enzymatic activity and reduction in hydrogen peroxide content. This study widens our knowledge of aquatic plants and their response to oxidative stress, supports the hypothesis that plant growth-promoting bacteria (PGPB) induce tolerable levels of oxidative stress in plants and introduces a new PGPB strain, P. gessardii C31-106/3.

Bio-engineered As-Ag-TiO2 nanoparticles enhance the genetic transformation of Pisum sativum L. via proton-coupled electron transfer-dependent alternative protonation

Nanoparticle-assisted biomolecule delivery to improve plant gene transformation and expression is a, cutting-edge approach. In this study, we examined the effects of acetosyringone-doped BENPs on the enhanced genetic transformation of peas. The vir gene inducer acetosyringone was delivered into Agrobacterium via a nano-doping system with TiO2 and Ag NPs. Four BENPs, such as TiO2 (S1), Ag-TiO2 (S2), As-TiO2 (S3), and As-Ag-TiO2 (S4), were green-synthesized from the PLE using hydrothermal and calcination approaches. SEM, HR-TEM-EDAX, SAED, XRD, and XPS physicochemical characterizations ensured the BENPs structure and biomolecule doping. The potential bar transformants were chosen against the MIC of BASTA® using the EHA 105 Agrobacterium tumefaciens strain carrying the pCAMBIA 1304-plasmid. The delivery of nano-doped acetosyringone was studied with various applications, and the typical exogenous acetosyringone delivery in the absence of BENPs was also analyzed as a control experiment. The gus histochemical, PCR, and southern hybridization confirmed that, among the BENPs, S4 at a 4 ppm dose induced the highest gene transformation efficiency (36.33 %) in pea along with three days of co-cultivation, 30 sec of sonication, and 3 min of vacuum infiltration. In BENP-assisted pea gene transformation, S2 at 4 ppm (26.66 %), S3 at 3 ppm (23 %), and S1 at 4 ppm (17.66 %) showed the next three significant gene transformation efficiencies, respectively. The elevated virC and virG gene expression profiles of the S4 BENP-treated Agrobacterium were identified with gene-specific RT-PCR analysis. The PCET activity of S4 was critically confirmed with cyclic voltammetry, which revealed that BENPs might function as an alternative proton supplement system to improve the vir gene induction and transformation of T-DNA. Hence, this alternative PCET-dependent BENP-assisted acetosyringone delivery for Agrobacterium mediated gene transformation exhibits potential value in the development of genetically-modified peas and opens the door to crops for advanced genetic manipulation against various biotic and abiotic stresses.

Successful Isolation of Diverse Verrucomicrobiota Strains through the Duckweed-Microbes Co-cultivation Method.

The "duckweed-microbes co-cultivation method" is a microbial isolation technique that effectively recovers diverse microbes, including rarely cultivated bacterial phyla, from environmental samples. In this method, aseptic duckweed and microbes collected from an environmental sample are co-cultivated for several days, and duckweed-associated microbes are then isolated from its roots using a conventional agar plate-based cultivation method. We herein propose several improvements to the method in order to specifically obtain members of the rarely cultivated bacterial phylum, Verrucomicrobiota. In systems using river water as the inoculum, the marked enrichment of Verrucomicrobiota was observed after 10 days of co-cultivation, particularly in the roots and co-cultivated media. We also successfully isolated 44 strains belonging to subdivisions 1, 3, and 4 of the phylum Verrucomicrobiota from these systems. This was achieved by changing the concentration of nitrogen in the co-cultivation medium, which is known to affect duckweed growth and/or metabolism, and by subjecting the fronds and co-cultivated media as well as the roots after co-cultivation to microbial isolation.

Efficient Agrobacterium tumefaciens-mediated genetic transformation of Aloe vera

Genetic transformation of plants has emerged as a core research tool for the functional characterization of genes and cultivar improvement in the field of plant biology. Effectually, high transformation efficiency is a prerequisite for accomplishing the stated targets. Aloe vera, a tropical cactus plant in distinction to the family Liliaceae, has seized a long history of secular acceptance as a therapeutic agent and is reasonably the most popular herbal remedy these days. However, the genetic transformation efficiency of Aloe vera is relatively low and seldom reported formerly. This study aims to optimize Agrobacterium-mediated transformation in Aloe vera by refining several parameters like explant selection, the extent of explant injury during infection, Agrobacterium concentration, co-cultivation pH, and duration and desiccation of plant tissue to improve the infection efficiency. The results showed that nearly 83% transient transformation efficiency was attained by suspending the Agrobacterium tumefaciens (AGL1 strain) cells at a concentration of OD600: 0.4 in co-cultivation media at pH-5.6 to infect the stem of aloe by desiccation, followed by 3 days of co-cultivation. Desiccation during infection had proved to enhance T-DNA delivery, whereas a higher extent of explant injury was found to curtail the infection efficiency. The molecular analysis of the putative transformants was carried out by following GUS histochemical assay and PCR. Thereupon, Southern blotting was used to substantiate the authentication of positive transgenic plants and analyze the copy number of the hptII gene in the Aloe vera genome. A transformation efficiency of over 2% was obtained, which is higher than the previous report. This study bestows an improved Agrobacterium-mediated transformation protocol based on desiccation in Aloe vera, which might help in facilitating various functional genomic studies and gene regulation in the aloe plant, eventually allowing the genetic modification of aloe species in an effective manner. This research deliberates a meliorated Agrobacterium-mediated transformation methodology for Aloe vera, which may facilitate various gene functional investigations and bioengineering of aloe species in an effective salutary way.

Functional Characterization of a Novel Synthetic Herbicide Resistance Gene in a Model Plant

As crop losses can occur owing to the abrupt growth of uncontrollable weeds in the field, research is currently being conducted in Pakistan to eradicate herbs. To carry out our proposed research domain, we have designed a novel synthetically modified EPSPS gene that has a potent role in herbicide-resistance development. The novel codon-optimized synthesized mEPSPS sequence was inserted into the pXCSG-mYFP plant expression vector. Results of colony PCR (1400 bp) confirmed the integration of genes into bacteria. For functional validation of pXCSG-mYFP-EPSPS, transient expression in tobacco (Nicotiana benthamiana) in comparison with PBSF-16 was done. Benth infiltration results showed that transient expression was successfully confirmed through ELISA and western blot analysis via anti-YFP antibody in tobacco leaves. For further validation of transient expression, the stable transformation results of the pXCSG-mYFP-EPSPS vector showed that successful transformation was done via two days of co-cultivation followed by selection and regeneration of transformed tobacco plants. The regenerated tobacco plants were then confirmed through gene-specific-based PCR. After PCR-based confirmation, western blot further validates the 26 kDa anti-YFP antibodies expression in transformed tobacco plants. Another important finding of the study was the bar and PAT gene real-time expression elucidating that the bar gene was 4.9-fold more expressive under the 35S promoter than the PAT gene under the nos promoter.

Establishment of an Efficient Agrobacterium-Mediated Genetic Transformation System to Enhance the Tolerance of the Paraquat Stress in Engineering Goosegrass (Eleusine Indica L.)

Eleusine indica (goosegrass) is a problematic weed worldwide known for its multi-herbicide tolerance/resistance biotype. However, a genetic transformation method in goosegrass has not been successfully established, making a bottleneck for functional genomics studies in this species. Here, we report a successful Agrobacterium-mediated transformation method for goosegrass. Firstly, we optimized conditions for breaking seed dormancy and increasing seed germination rate. A higher callus induction rate from germinated seeds was obtained in N6 than in MS or B5 medium. Then the optimal transformation efficiency of the gus reporter gene was obtained by infection with Agrobacterium tumefaciens culture of OD600 = 0.5 for 30 min, followed by 3 days of co-cultivation with 300 μmol/L acetosyringone. Concentrations of 20 mg L-1 kanamycin and 100 mg L-1 timentin were used to select the transformed calli. The optimal rate of regeneration of the calli was generated by using 0.50 mg L-1 6-BA and 0.50 mg L-1 KT in the culture medium. Then, using this transformation method, we overexpressed the paraquat-resistant EiKCS gene into a paraquat-susceptible goosegrass biotype MZ04 and confirmed the stable inheritance of paraquat-resistance in the transgenic goosegrass lines. This approach may provide a potential mechanism for the evolution of paraquat-resistant goosegrass and a promising gene for the manipulation of paraquat-resistance plants. This study is novel and valuable in future research using similar methods for herbicide resistance.

Dormancy induced by oxidative damage during disinfection facilitates conjugation of ARGs through enhancing efflux and oxidative stress: A lagging response

Disinfection is known to greatly alter bacterial characteristics in water, and high horizontal gene transfer (HGT) frequency occurs in eutrophic conditions. Interestingly, these two seemingly irrelevant phenomena were closely linked by a lagging response of the increased conjugation frequency probably via daily water disinfection in this study. Three disinfection methods (UV, chlorine, and UV/chlorine) were selected to investigate the increased frequency of conjugation of ARGs during the stage of continuing culture after disinfection. The results showed that the conjugative transfer frequency was inhibited for all disinfection treatments after 24 h of co-incubation. Unexpectedly, after 3–7 days of co-cultivation, the HGT frequencies were increased by 2.71–5.61-fold and 5.46–13.96-fold in chlorine (30 min) and UV/chlorine (1 min) groups compared to the control, but not in UV-irradiated groups. A neglected lagging response was found for the first time, i.e., oxidative disinfection-induced dormancy promotes conjugative transfer of ARGs. Furthermore, mechanistic insights were gained from (1) membrane permeability, (2) conjugation-regulated system, (3) efflux pump system, and (4) oxidative stress system, suggesting the critical role of enhancing efflux and oxidative stress in the propagation of ARGs. Finally, the known instantaneous effect of oxidation disinfection was compared to address the controversial debate in this research field, proposing that the dormancy level of donor bacteria is the key to evaluating whether it can promote the HGT process. This study has important environmental implications for elucidating the transmission of ARGs after oxidation disinfection.

Agrobacterium rhizogenes-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA)

Agrobacterium rhizogenes-mediated genetic transformation of Musa acuminata cv. Vaibalhla (AAA) was successfully carried out using A. rhizogenes strain A4 harboring the binary vector pCAMBIA2301with VrNHX1gene. In the study, male flowers of M. acuminata cv. Vaibalhla were used as explants to obtain white bud-like structures by culturing on MS basal medium supplemented with 2 mg/l 6-Benzylaminopurine (BAP) and 0.5 mg/l 1-Naphthaleneacetic acid (NAA). The subsequent shoot induction and plantlet regeneration were carried out in MS medium supplemented with Kinetin (2 mg/l) and NAA (0.5 mg/l). For the genetic transformation, in vitro raised plantlets were inoculated with A. rhizogenes strain A4 harboring pCAMBIA2301VrNHX1 for 30 min followed by 2 days of co-cultivation in the dark in a semi solid MS basal medium. The treated plants were then transferred and maintained in MS basal medium supplemented with ascorbic acid (75 mg/l), kanamycin (150 mg/l) and cefotaxime (400 mg/l). Initiation of hairy roots were observed within 2 days of transfer which were evaluated for GUS activity and the subsequent GUS+ roots were assayed for transient transformation by PCR using nptII and NHX1 gene specific primers. The transfer of the plasmid as well as the gene was confirmed with positive bands observed at 540 bp and 1.6 kb respectively.

Overexpression of TcCHS Increases Pyrethrin Content When Using a Genotype-Independent Transformation System in Pyrethrum (Tanacetum cinerariifolium).

Pyrethrum (Tanacetum cinerariifolium) is one of the most important industrial crops for the extraction of pyrethrins, which are natural insecticidal compounds. Progress in pyrethrum molecular breeding with the objective of increasing pyrethrin content has been slow for lack of a suitable gene transfer system. Regeneration recalcitrance is a crucial barrier to establishing a genetic transformation system in pyrethrum. Therefore, in this study, an Agrobacterium-mediated transformation system in pyrethrum was developed using shoot apical meristems from germinated seedlings. Factors affecting transformation efficiency were optimized. Optimal conditions included explants at the “no true leaf” stage with a half apical meristem, an Agrobacterium tumefaciens cell density of OD600 = 0.5, two days of cocultivation, and the incorporation of 1.5 mg L−1 6-BA and 30 mg L−1 kanamycin into the selection medium. Under the optimized conditions, two expression cassettes (proTcCHS-GUS and proRbcS-TcCHS) were successfully transformed into pyrethrum. Polymerase chain reaction (PCR), Southern blotting, reverse-transcription quantitative PCR (RT-qPCR), and histochemical staining confirmed the identity of proTcCHS-GUS transgenic plants. PCR and RT-qPCR analyses confirmed the identity of proRbcS-TcCHS transgenic plants. The transformation efficiency was 0.83% (5 transgenic lines/600 infected explants). The relative concentration of pyrethrins in proRbcS-TcCHS transformants (OX T0-1: 1.50% or OX T0-2: 1.24%) was higher than that in nontransformed plants (WT: 0.76%). Thus, the genetic transformation system overcame the low regeneration efficiency and integrated a foreign gene into the pyrethrum genome. The new system is a suitable and effective tool for creating high-yielding cultivars of pyrethrum.

An Efficient Hairy Root System for Validation of Plant Transformation Vector and CRISPR/Cas Construct Activities in Cucumber (Cucumis sativus L.).

Hairy root induction system has been applied in various plant species as an effective method to study gene expression and function due to its fast-growing and high genetic stability. Recently, these systems have shown to be an effective tool to evaluate activities of CRISPR/Cas9 systems for genome editing. In this study, Rhizobium rhizogenes mediated hairy root induction was optimized to provide an effective tool for validation of plant transformation vector, CRISPR/Cas9 construct activities as well as selection of targeted gRNAs for gene editing in cucumber (Cucumis sativus L.). Under the optimized conditions including OD650 at 0.4 for infection and 5 days of co-cultivation, the highest hairy root induction frequency reached 100% for the cucumber variety Choka F1. This procedure was successfully utilized to overexpress a reporter gene (gus) and induce mutations in two Lotus japonicus ROOTHAIRLESS1 homolog genes CsbHLH66 and CsbHLH82 using CRISPR/Cas9 system. For induced mutation, about 78% of transgenic hairy roots exhibited mutant phenotypes including sparse root hair and root hair-less. The targeted mutations were obtained in individual CsbHLH66, CsbHLH82, or both CsbHLH66 and CsbHLH82 genes by heteroduplex analysis and sequencing. The hairy root transformation system established in this study is sufficient and potential for further research in genome editing of cucumber as well as other cucumis plants.

An improved Agrobacterium mediated transformation and regeneration protocol for successful genetic engineering and genome editing in eggplant

Successful genetic manipulation largely depends on effective transformation and regeneration methods. The Agrobacterium mediated transformation has always been the method of choice for crop improvement through genetic engineering and the recently developed CRISPR/Cas9 based genome editing approaches. In this study, we have developed an improved regeneration and Agrobacterium mediated transformation protocols for three high yielding eggplant varieties BARI begun 2, 4 and 6. Several key parameters were intensively studied which include culture media composition, concentration of growth regulators, compatible antibiotic selection, hyperhydricity and rooting process. For the three different explants used in the study, it was found that MS+2.5 mg/l BAP alone is optimal as a hormone supplement for maximum shoot regeneration. Significant reduction (16.67 ± 0.11%) in hyperhydricity was observed when casein hydrolysate along with sorbitol was used at a concentration of 0.2 mg/l and 0.1 mg/l respectively in MS+BAP 2.5 mg/l supplemented medium. Transformation efficiency was found to be highest with an Agrobacterium concentration of 0.6 at OD 600 nm, 10 min of infection and two days of co-cultivation. We found 100 mg/l concentration of kanamycin to be suitable as a selection pressure for screening eggplant transformation events. Rooting media composition standardized in this protocol provides a higher rate of root formation (85%) in both in vitro and ex vitro conditions. Using this protocol, existing problems in eggplant genetic engineering can easily be overcome with higher transformation efficiency.

Different Early Responses of Laminariales to an Endophytic Infection Provide Insights About Kelp Host Specificity

The filamentous algal endophyte Laminarionema elsbetiae is highly prevalent in European populations of the brown alga Saccharina latissima, but has also been found occasionally in the other kelp species Laminaria digitata. The presence of L. elsbetiae coincides with morphological changes in the hosts such as twisted stipes and deformed blades, however, little is known about the molecular bases of these algal host-endophyte interactions. Using a co-cultivation experiment, we showed that physiological and gene regulation responses, and later endophyte prevalences are different between the main and the occasional host. The contact with the endophyte L. elsbetiae induced a stronger and faster transcriptomic regulation in the occasional host L. digitata after 24 h, from which growth rate was later affected. During the first two days of co-cultivation, only 21 differentially expressed genes (DEGs) were common in both kelps, indicating a crucial difference between the molecular responses of the two hosts. By functional annotation, we identified DEGs related to host-endophyte recognition, defense response and cell wall modification. Our results suggest that expression pattern differences between the two kelps related to the recognition of the endophyte and later defense reactions could explain the variability of observed physiological responses and host-endophyte specificity in kelp natural populations.

Antagonistic effect of the promising fungal producer strain of microbiopreparation T-1 Trichoderma sp. on oil flax Fusarium blight

Fusarium blight is the most harmful disease of oil flax, which affects crops in all cultivation regions during the growing season. Considering the value of flaxseed oil, which is also used for medical purposes, it is advisable to use biological preparations to obtain environmentally safe products, which allow not only to ensure the protection of plants from diseases but also to improve the ecological situation. In recent years (2018-2020), at the biomethod laboratory of V.S. Pustovoit All-Russian Research Institute of Oil Crops has been carried out the search of the promising producer strains for developing microbiopreparations against the pathogen of oil flax Fusarium blight with a multifunctional type of action and a prolonged shelf life. These studies involve the study of the antagonistic mechanisms of promising producer strains of microbiopreparations on the disease pathogen. The article presents the effect of the promising antagonist strain T-1 Trichoderma sp. on the pathogen of oil flax Fusarium blight Fusarium oxysporum Schlecht. emend. Shyd. et Hans. var. orthoceras (App. et Wr.) Bilai, identified as a result of a gradual screening. High competition of the antagonist strain for the feeding area, its hyperparasitic and antibiotic activity was established during the co-cultivation of the antagonist and the pathogen by the method of double cultures. During the study of the mechanism of the antagonistic action of the promising producer strain on the Fusarium blight pathogen using a light microscope, it was found that even before coalescence with the antagonist colony on the second day of coincubation, the shrinking of certain areas in the pathogen mycelium was noted, which proves the antibiotic activity of the fungal antagonist T-1 Trichoderma sp. On the 4th day of co-cultivation, after coalescence of the pathogen and antagonist colonies, aggregation of the pathogen mycelium into the cords and the effect of the antagonist mycelium coiling around the pathogen hyphae were observed . On the 6th day, the effect of the antagonist mycelium coiling around the pathogen hyphae increased. By the 8th-10th days of interaction, there was observed degradation and lysis of the aerial and substrate mycelium of the pathogen of oil flax Fusarium blight. Over the entire observation period, there was noted the normal development of the oil flax Fusarium blight pathogen Fusarium oxysporum var. orthoceras in the control.

Effective and Ineffective Samples of Mesenchymal Multipotent Stromal Cells Used for Acute Graft-Versus-Host Disease Prophylaxis after Allogeneic Bone Marrow Transplantation Differ in the Expression Level of HLA-DR When Co-Cultured with Activated Lymphocytes

IntroductionMultipotent mesenchymal stromal cells (MSCs) are widely used for the prevention and treatment of acute graft versus host disease (aGVHD) after allogeneic bone marrow transplantation (allo-BMT). In some cases prophylaxis of aGVHD failed. The reasons of the failure could be either the result of particular qualities of donor-recipient interaction, patient status or differences in MSC samples. In order to discriminate effective and ineffective for aGVHD prophylaxis MSCs the growth and differentiation characteristics, relative expression levels of different genes and changes in the expression of surface MSC molecules during co-cultivation with activated lymphocytes were investigated in all MSC samples.MethodsMSCs from the bone marrow of corresponding hematopoietic stem cells donor were injected into 59 patients after allo-BMT at the moment of blood count recovery. MSCs from the bone marrow of those donors were analyzed by cultivation in standard conditions (aMEM, 10% fetal calf serum) for 3 passages. Relative expression level (REL) of 30 genes involved in proliferation, differentiation and immunomodulation was estimated by RT-qPCR in all MSC samples. MSCs were seeded 105 cells per T25 flask, and then 106 allogeneic lymphocytes from single donor were added to all MSCs cultures. For lymphocytes activation 5 mg/ml phytohemagglutinin (PHA) was added to half of these cultures. Lymphocytes were removed from MSCs on 1st and 4th days of co-cultivation. Than trypsinized MSCs were collected from the bottom of the flask and expression of HLA-DR, HLA-ABC, CD73, PD-L1, CD54, CD80, CD86, CD105, CD146 and FAS-L on their surface was measured by flow cytometry. p<0.05 was considered statistically significant, all data is present as medium ± SEM.ResultsThe injection of MSCs reduced the incidence of aGVHD 2 times and increased the 5 years overall survival of patients (p=0.047). Eight of 51 MSC samples had been ineffective for preventing aGVHD. Analysis of individual donor characteristics (gender, age, body mass index) found no significant differences between the MSCs, effective and ineffective for preventing aGVHD. REL analysis of 30 genes involved in proliferation, differentiation and immunomodulation in MSCs reviled only 4 genes differs significantly between effective and ineffective MSC samples (table). During 4 days of cultivation the MFI of HLA-DR did not change on MSCs. When MSCs co-cultivated with non-activated lymphocytes for 4 days MFI of HLA-DR increased only in 1.3 fold (p=0.03). Co-cultivation of MSCs with activated lymphocytes led to 7 fold increases in HLA-DR expression between 1st and 4th days (p≤0,001). On 4th day of co-cultivation MSCs with activated lymphocytes the MFI of HLA-DR was 3 fold lower in ineffective MSC samples than in effective (table). These data indicate that the effectiveness of MSCs used depends on the degree of their immunogenicity and, accordingly, on the immunomodulating potential. Among studied molecules only Fas L was significantly lower in effective MSC samples than in ineffective (table). It is possible that such MSC samples survived in the organism better than ineffective and their immunomodulating function lasts longer in the organism.ConclusionsIn summary, this study showed that MSC injections resulted in a significant 2-fold decrease in aGvHD development compared with the standard prophylaxis treatment. When compared with ineffective MSC, effective MSC samples are characterized by higher total cell production and REL of CFH, FGFR1, PDGFRa, and ICAM1. ExpressionofHLA-DR increased significantly in MSCs co-cultivated with lymphocytes, so these cells became immunogenic in organism. It is evident that the level of HLA-DR expression on MSCs important for MSCs efficiency in aGVHD prophylaxis. It is possible to predict efficiency of MSCs injection by simple test of co-cultivation of these cells with activated lymphocytes and measuring the expression level of HLA-DR on them.The materials are supported by grant from the Russian Science Foundation, Project № 16-15-00102 [Display omitted] DisclosuresShipounova:Russian Foundation for Basic Research, Project № 15-04-02514: Research Funding.

The common gamma-chain cytokines IL-2, IL-7 and IL-15 impact on proliferation of lymphocytes in vitro in breast cancer patients.

e13060 Background: Monotherapy with low doses of cytokines does not provide significant therapeutic results, while treatment with high doses leads to a number of side effects, for example, cytokine release syndrome, etc. Therefore, it is necessary to study the effect of cytokines on immune cells which are involved in the regulation of pro- and antitumor immune response. Thus, the aim of this study was to analyze the dynamics in the proliferation of the total fraction of lymphocytes after expansion and exposure to γc-cytokines: IL-2, IL-7 and IL-15 and their combinations in vitro in patients with diagnosed breast cancer. Methods: The study included 10 patients with locally advanced stage I-III breast cancer. Peripheral blood mononuclear cells (PBMC, ficoll-hypaque) were used as material. After density separation the cells were cultured at a dose of 500 thousand cells / ml in 6-well plates (Biofil, China) in RPMI 1640 (Gibco, USA) supplemented with 10% FBS (HyClone, USA) at 37° C, 5% CO2. Expansion of lymphocytes was performed by kit with anti-biotin magnetic particles MACSiBead to CD2, CD3 and CD28 (Miltenyi Biotec, Germany), according to the manufacturer's protocol. Stimulation of lymphocyte proliferation was performed on days 4, 8 and 12 by introducing recombinant cytokines - IL-2, IL-7, IL-15 (Miltenyi Biotec, Germany) and their combinations. Cytokines were introduced at a concentration of 40 ng / ml each in the following variants: IL-2; IL-2 / IL-15; IL-2 / IL-15 / IL-7; IL-15; IL-15 / IL-7. Plates with cell suspension were cultured at 37 ° C, 5% CO2 for 15 days. Results: The data obtained on the 11th day of incubation demonstrated statistically significant differences in cell viability in samples with the addition of interleukin IL-7 / IL-15 combination (778%) compared to control samples by 1.5 times (p &lt; 0.05). The proportion of lymphocytes in samples with the addition of only IL-15 (702%) and IL-7 / IL-15 (756%) combination was 1.47 and 1.6 times higher, respectively, compared with the control (p &lt; 0.05) at the 12th day of co-cultivation. Conclusions: Stimulation of lymphocyte proliferation by the IL-7 / IL-15 combination results in a high frequency of viable cell production. Despite the only culture stage, the results of the study may be important for optimizing the use of γc-cytokines in the treatment of patients with breast cancer.

Optimization of parameters to improve transformation efficiency of elephant foot yam (Amorphophallus paeoniifolius (Dennst.) Nicolson.

The online version contains supplementary material available at 10.1007/s13205-021-02824-6.

Phytoremediation of CYN, MC-LR and ANTX-a from Water by the Submerged Macrophyte Lemna trisulca.

Cyanotoxins are harmful to aquatic and water-related organisms. In this study, Lemna trisulca was tested as a phytoremediation agent for three common cyanotoxins produced by bloom-forming cyanobacteria. Cocultivation of L. trisulca with Dolichospermum flos-aquae in BG11 medium caused a release of the intracellular pool of anatoxin-a into the medium and the adsorption of 92% of the toxin by the plant—after 14 days, the total amount of toxin decreased 3.17 times. Cocultivation with Raphidopsis raciborskii caused a 2.77-time reduction in the concentration of cylindrospermopsin (CYN) in comparison to the control (62% of the total pool of CYN was associated with the plant). The greatest toxin limitation was noted for cocultivation with Microcystis aeruginosa. After two weeks, the microcystin-LR (MC-LR) concentration decreased more than 310 times. The macrophyte also influenced the growth and development of cyanobacteria cells. Overall, 14 days of cocultivation reduced the biomass of D. flos-aquae, M. aeruginosa, and R. raciborskii by 8, 12, and 3 times, and chlorophyll a concentration in comparison to the control decreased by 17.5, 4.3, and 32.6 times, respectively. Additionally, the macrophyte stabilized the electrical conductivity (EC) and pH values of the water and affected the even uptake of cations and anions from the medium. The obtained results indicate the biotechnological potential of L. trisulca for limiting the development of harmful cyanobacterial blooms and their toxicity.

Synthesis and release of fatty acids under the interaction of Ulva pertusa and Heterosigma akashiwo by stable isotope analysis

Symbiosis of marine algae is inevitable in the marine environment, and species may occur interaction on the growth. In this study, the macroalgae Ulva pertusa and marine microalgae Heterosigma akashiwo were selected as target species to study the interaction mechanism between them. After the 8 days of co-cultivation, the inhibition on growth was observed for both of U. pertusa and H. akashiwo. Eight fatty acids in U. pertusa was detected, with the significant decrease in contents of polyunsaturated fatty acids (PUFAs) especially for C18:2, C18:3n-3 and C18:3n-6. Twelve fatty acids in H. akashiwo was detected, with the significant change for PUFAs. PUFA concentrations in the co-culture group were less than those in the mono-culture. Meanwhile the principal component analysis was conducted to insight into the interaction between U. pertusa and H. akashiwo by fatty acids content and carbon stable isotope ratio of fatty acids (δ13CFAs). Fatty acid content could not distinguish mono and co-culture. However, δ13CFAs could distinguish not only the culture time of algae, but also the living environment of algae. In addition, this study combined fatty acids content and δ13CFAs to explore the release of fatty acids by algae into the seawater. The C18:3n-3 was identified as the allelochemical released by U. pertusa to inhibit the growth of H. akashiwo. The ratio of δ13CFAs in seawater decreased. This study provides a theoretical basis for the symbiosis of marine algae, and a new method of compound-specific stable carbon isotopes was used to better explore the metabolism of fatty acids in algae.