Treatment Of Supernatant Research Articles

Gut microbiota analysis and LC-MS-based metabolomics to investigate AMPK/NF-κB regulated by Clostridium butyricum in the treatment of acute pancreatitis

BackgroundAcute pancreatitis (AP) is an inflammatory condition with potentially life-threatening complications. This study investigates the therapeutic potential of Clostridium butyricum for modulating the inflammatory cascade through the AMPK/NF-κB signaling pathway, focusing on inflammation induced by AP. LC-MS analysis of serum samples from AP patients highlighted the regulation of lipid metabolism and inflammation, and found that metabolites involved in the inhibition of NF-κB phosphorylation and the AMPK activation pathway were downregulated. We hypothesized that pre-administration of Clostridium butyricum and its culture supernatant could mitigate AP-induced damage by modulating the AMPK/NF-κB pathway.MethodsLipopolysaccharide (LPS)-induced cell inflammation models. LPS combined with CAE induced acute pancreatitis in mice. We divided mice into four groups: Con, AP, AP + C.Buty (AP with Clostridium butyricum treatment), and AP + CFS (AP with culture supernatant treatment). Analyses were performed using WB, RT-qPCR, Elisa, flow cytometry, IHC, and HE, respectively.ResultsOur study shows that CFS can reduce the apoptosis of LPS-induced cellular inflammation and reduce the release of LPS-induced cytoinflammatory factors through the AMPK/NF-κB pathway in vitro. In vivo, Clostridium butyricum and its supernatant significantly reduced inflammatory markers, and corrected histopathological alterations in AP mice. Gut microbiota analysis further supported these results, showing that Clostridium butyricum and its supernatant could restore the balance of intestinal flora disrupted by AP.ConclusionsMechanistically, our results indicated that the therapeutic effects of Clostridium butyricum are mediated through the activation of AMPK, leading to the inhibition of the NF-κB pathway, thereby reducing the production of pro-inflammatory cytokines. Clostridium butyricum and its culture supernatant exert a protective effect against AP-induced damage by modulating the AMPK/NF-κB signaling pathway. Future studies will further elucidate the molecular mechanisms underlying the beneficial effects of Clostridium butyricum in AP and explore its clinical applicability in human subjects.

Protective effect of bone-marrow stromal cells on injured cortical neurons subjected to hemostimulation in vitro

Objective: Bone-marrow mesenchymal stem cells (BMSCs) are a subpopulation of cells found in the bone marrow stromal of mammals that have the potential to differentiate and form bone, cartilage, adipose, neural, and muscle cells, with strong proliferative ability, multidirectional differentiation potential, immunomodulatory function. Here, we reported the novel findings on the effect of BMSC in protecting injured cortical neurons induced by hemostimulation. Methods: Cortical neurons harvested from the neonatal rat and were isolated and incubated at 37°C in a 5% CO2 environment. During this process, part of cells was subjected to hemostimulation, and BMSC derived supernatant addition was performed to observe its effect on neuroprotection. Hoechst 33342/PI co-staining and CCK-8 cell viability assays were utilized to evaluate the effects of BMSC supernatant administration on primary cortical neurons. Results: BMSC derived supernatant administration effectively protects hemoglobin-induced neuronal damage, indicating by cell viability-detection. In addition, BMSCs exert optimal effect by inhibiting neuronal cell apoptosis. Conclusions: BMSC derived supernatant treatment effectively ameliorates hemoglobin-induced neuronal damage and attenuates neuronal apoptosis.

A new approach on the management of landfill leachate reverse osmosis concentrate: Solar distillation coupled with struvite recovery and biological treatment

The management of reverse osmosis (RO) concentrate remains a challenging task for operators of Landfill Leachates Treatment Plants. In this article we suggest an integrated treatment scheme for RO concentrate that combines solar distillation, struvite precipitation to reduce ammonia content of the distillate and biological treatment of the supernatant either with mixed cultures of bacteria or with microalgae. Experiments in a pilot-scale solar still, equipped with underfloor heating system, showed that the production rate of the distillate ranged up to 3.17 L/d m2. The distillate was characterized by elevated average concentrations of ammonium nitrogen; 2028 mg/L and 1358 mg/L in the two experiments conducted, respectively. A decreasing trend on concentrations of NH4+-N was noticed during these experiments, while the opposite was observed for COD. Struvite recovery experiments showed that the optimum Mg:NH4:PO3 ratio was that of 2:1:5.8. Under these conditions, the NH4+-N removal reached 88%. Further treatment of the process supernatant into a 4-L hybrid sequencing batch reactor with biocarriers and activated sludge achieved NH4+-N removal higher than 98% in Phases C and D, where 450 and 600 mL of supernatant were added, respectively. Similar removal was also observed in a 2-L bioreactor with microalgae Chlorella sorokiniana when 150 mL of struvite supernatant were added (Phase B) while further increase of the amount of added supernatant to 200 mL resulted to a sharp stop of NH4+-N consumption (Phase C). Calculations for a landfill serving 20,000 inhabitants and a daily RO concentrate production of 6 m3/d showed that the required area for the construction of the solar still was 1893 m2 and the volumes of the hybrid and the microalgae reactor were 54 m3 and 60 m3, respectively. The recovered solid material of struvite process, after characterization for heavy metals and organic micropollutants, could be reused to the fertilizers industry.

The role of progranulin in macrophages of a glioblastoma model.

Glioblastoma (GBM), characterized by astrocytic tumorigenesis, remains one of the most prognostically challenging tumor types. Targeting entire GBM microenvironment using novel therapeutic factors is currently desired investigation approach. In this study, we focused on progranulin (PGRN), a regulator of diverse cellular functions. Recent studies implicated PGRN in the poor prognostics of GBM patients. However, the specific role of PGRN in the GBM microenvironment remains elusive. We utilized public databases of GBM patient and previous single-cell RNA sequence to examine association between PGRN expression and patient survival/grade, and expression levels of PGRN in each cell constituting the tumor microenvironment. To clarify the role of PGRN in Tumor-associated macrophage (TAM), we examined cell proliferation and expression of some proteins in murine GBM cells when cell supernatants derived from TAM of PGRN knockout (Grn-/-) or wild type mice were treated with murine GBM cells. Our results reveal significant PGRN expression in macrophages within the GBM environment, suggesting an association between increased PGRN expression in macrophages and tumor malignancy. TAM induction led to PGRN expression enhancement. Treatment with Grn-/- mouse -derived bone marrow-derived macrophage (BMDM) supernatant resulted in diminished GBM cell proliferation and cell cycle- and mesenchymal GBM subtype-associated reduced protein expression. Furthermore, the Grn-/- mouse-derived BMDM supernatant treatment reduced the phosphorylated STAT3 expression in GBM cells, while the expression of IL-6 and IL-10, known STAT3 pathway activators, diminished in Grn-/- mouse-derived BMDMs. Our results suggest that macrophage-derived PGRN is pivotal for fostering malignant transformations within the tumor microenvironment.

Synergistic Activity of Ingulados Bacteria with Antibiotics against Multidrug-Resistant Pathogens.

Antimicrobial resistance is a critical challenge due to the overuse of conventional antimicrobials, and alternative solutions are urgently needed. This study investigates the efficacy of compounds derived from lactic acid bacteria (LAB) fermentation combined with antibiotics against multidrug-resistant pathogens isolated from clinical cases in a hospital setting. Strains of Escherichia coli, Klebsiella pneumoniae, and Enterococcus faecium and faecalis were isolated and selected from blood, respiratory, and urine samples. They were tested against the fermentation products from the Ingulados LAB collection (BAL5, BAL6, BAL8, BAL13, and BAL16), recognized for their antimicrobial efficacy against veterinary pathogens. The activity against multidrug-resistant (MDR) pathogens was evaluated initially, followed by synergy tests using checkerboard assays and subsequent analysis. Bioinformatic assessments and supernatant treatments were performed to characterize the nature of the compounds responsible for the antimicrobial activity. Notably, BAL16 exhibited significant growth inhibition against multidrug-resistant E. faecium. Synergy tests highlighted its combined activity with tetracycline through FICI and surface analysis and bioinformatic analysis unveiled the protein fraction containing bacteriocins as the underlying mechanism. This study highlights BAL16 fermentation products potential as valuable antimicrobial agents against MDR E. faecium infections, attributed to bacteriocins. Further in-depth studies are necessary for complete bacteriocin characterization.

Probiotic Properties and Anti-inflammatory Activity of Bacillus spp. Isolated from Ethnically Fermented Soybean

Bacillus spp. probiotics used as feed additives can form spores and tolerate the harsh conditions of the human digestive system and are beneficial for the treatment of inflammatory bowel syndrome. Since reports on probiotics and anti-inflammatory properties of Bacillus spp. isolated from the fermented food of Northeast India have not been explored much. The present study focused on Bacillus spp. BN5, AY5, and AN8, possessing these desired properties. In the probiotics study, the isolates were screened for their tolerance to acid and bile salt, auto-aggregation, hydrophobicity, cholesterol assimilation, antibiotic resistance, and antagonistic properties. It was found that these isolates possessed the desirable probiotic traits. The Bacillus spp. culture and their supernatant were also screened for their ability to reduce LPS-induced inflammation in murine macrophage (RAW 264.7) cells. All the Bacillus spp. culture and their supernatant treatments were found to reduced the Nitric oxide (NO) production by LPS-induced cell lines. The supernatant of LPS-induced cell lines were also analyzed to measure the level of inflammatory cytokine production. It was found that the levels of TNF-α, IL-6, and IL-1β were reduced after co-treatment with LPS and Bacillus spp. culture or LPS and Bacillus spp. supernatant. Results suggested that the Bacillus spp. are potential probiotic candidates with anti-inflammatory properties.

The Microbial Tryptophan Metabolite Contributes to the Remission of Salmonella typhimurium Infection in Mice.

Salmonella enterica serovar Typhimurium (S. Tm) causes severe foodborne diseases. Interestingly, gut microbial tryptophan (Trp) metabolism plays a pivotal role in such infections by a yet unknown mechanism. This study aimed to explore the impact of Trp metabolism on S. Tm infection and the possible mechanisms involved. S. Tm-infected C57BL6/J mice were used to demonstrate the therapeutic benefits of the Bacillus velezensis JT3-1 (B. velezensis/JT3-1) strain or its cell-free supernatant in enhancing Trp metabolism. Targeted Trp metabolomic analyses indicated the predominance of indole-3-lactic acid (ILA), an indole derivative and ligand for aryl hydrocarbon receptor (AHR). Based on the 16S amplicon sequencing and correlation analysis of metabolites, we found that B. velezensis supported the relative abundance of Lactobacillus and Ligilactobacillus in mouse gut and showed positive correlations with ILA levels. Moreover, AHR and its downstream genes (especially IL-22) significantly increased in mouse colons after B. velezensis or cell-free supernatant treatment, suggesting the importance of AHR pathway activation. In addition, ILA was found to stimulate primary mouse macrophages to secrete IL-22, which was antagonized by CH-223191. Furthermore, ILA could protect mice from S. Tm infection by increasing IL-22 in Ahr+/- mice, but not in Ahr-/- mice. Finally, Trp-rich feeding showed amelioration of S. Tm infection in mice, and the effect depended on gut microbiota. Taken together, these results suggest that B. velezensis-associated ILA contributes to protecting mice against S. Tm infection by activating the AHR/IL-22 pathway. This study provides insights into the involvement of microbiota-derived Trp catabolites in protecting against Salmonella infection.

Adipose-derived mesenchymal stem cell therapy for reverse bleomycin-induced experimental pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive respiratory disease. Arguably, the complex interplay between immune cell subsets, coupled with an incomplete understanding of disease pathophysiology, has hindered the development of successful therapies. Despite efforts to understand its pathophysiology and develop effective treatments, IPF remains a fatal disease, necessitating the exploration of new treatment options. Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of IPF, but further investigation is needed to understand its therapeutic effect. This study aimed to assess the therapeutic effect of adipose-derived mesenchymal stem cells in a bleomycin-induced pulmonary fibrosis model. First, MSC cells were obtained from mice and characterized using flow cytometry and cell differentiation culture methods. Then adult C57BL/6 mice were exposed to endotracheal instillation of bleomycin and concurrently treated with MSCs for reversal models on day 14. Experimental groups were evaluated on days 14, 21, or 28. Additionally, lung fibroblasts challenged with TGF-β1 were treated with MSCs supernatant or MSCs to explore the mechanisms underlying of pulmonary fibrosis reversal. Mesenchymal stem cells were successfully isolated from mouse adipose tissue and characterized based on their differentiation ability and cell phenotype. The presence of MSCs or their supernatant stimulated the proliferation and migration of lung fibrotic cells. MSCs supernatant reduced lung collagen deposition, improved the Ashcroft score and reduced the gene and protein expression of lung fibrosis-related substances. Bleomycin-challenged mice exhibited severe septal thickening and prominent fibrosis, which was effectively reversed by MSCs treatment. MSC supernatant could suppress the TGF-β1/Smad signaling pathway and supernatant promotes fibroblast autophagy. In summary, this study demonstrates that MSCs supernatant treatment is as effective as MSCs in revert the core features of bleomycin-induced pulmonary fibrosis. The current study has demonstrated that MSCs supernatant alleviates the BLM-induced pulmonary fibrosis in vivo. In vitro experiments further reveal that MSC supernatant could suppress the TGF-β1/Smad signaling pathway to inhibit the TGF-β1-induced fibroblast activation, and promotes fibroblast autophagy by Regulating p62 expression. These findings contribute to the growing body of evidence supporting the therapeutic application of MSCs in cell therapy medicine for IPF.

Potential of Bacillus amyloliquefaciens QY-1 as a biocontrol agent of Botrytis cinerea in postharvest blueberry

Botrytis cinerea is a prevalent disease of blueberry, causing significant postharvest losses. Bacillus amyloliquefaciens strain ‘QY-1’ is a strong antagonist of B. cinerea; therefore, it was isolated from an area severely impacted by blueberry gray mold for this study. In B. cinerea, QY-1 cell-free supernatant (CFS) treatment showed a high inhibition rate (86.93%), altered mycelial morphology, upregulated 1,3-β-glucosidase and chitinase, and downregulated ergosterol (ERG)-related gene expression, respectively. Moreover, QY-1 significantly reduced gray mold incidence in blueberries during storage without affecting fruit quality, thus highlighting its potential as an antibacterial and fresh-keeping agent for postharvest blueberries.

Environmental life cycle assessment of treatment and management strategies for food waste and sewage sludge

A consequential life cycle assessment (LCA) was utilized to compare the environmental impacts of food waste and sewage sludge management strategies. The strategies included a novel two-phase anaerobic digestion (AD) system and alternatives including landfill, waste-to-energy, composting, anaerobic membrane bioreactor, and conventional AD (wet continuous stirred-tank reactor [CSTR]). The co-management of food waste with sewage sludge was also considered for the two-phase AD system and for a conventional AD reactor. A multidimensional LCA approach was taken, considering the five-midpoint impact categories of global warming, smog, human health particulate, acidification, and eutrophication estimated using the U.S. EPA Tool for the Reduction and Assessment of Chemical and Other Environmental Impacts. Co-management of food waste and sewage sludge using the novel two-phase AD system was shown to maximize energy recovery and had a net global warming benefit while reducing other environmental impacts compared with the alternative management strategies. It had similar relative environmental advantages across all categories as conventional AD, with the advantage of a smaller physical footprint. However, both approaches featured net environmental burdens when the background electric grid intensity fell below 0.25 kg CO2-eq kWh−1, as could be expected in a decarbonized electric future. Upgrading the biogas produced from AD to renewable natural gas can displace the use of fossil natural gas for other non-electricity energy requirements that are difficult to decarbonize and may extend the time period of significant environmental benefits of utilizing AD for organic waste management. Treatment of the nutrient-rich supernatant generated by the novel two-phase AD system could be an obstacle for utilities with stringent nutrient discharge limits. Future research and full-scale implementation are needed to demonstrate the benefits of the two-phase AD system predicted through this analysis.

Antibiofilm activity of cell-free supernatants of vaginal isolates of Corynebacterium amycolatum against Pseudomonas aeruginosa and Klebsiella pneumoniae

Biofilm formation is an important factor in the development of antibiotic resistance and chronic infection. In this study, we demonstrated that the cell-free supernatant of vaginal isolates of C. amycolatum caused a reduction in biofilm formation, destroyed the preformed biofilms, altered the cell surface properties and reduced the production of exopolysaccharides in clinical isolates of P. aeruginosa и Kl. pneumoniae. Microscopic observations showed that P. aeruginosa and Kl. pneumoniae biofilm formed small clusters scattered over the surface after treatment with cell-free supernatant of C. amycolatum ICIS 99, in contrast to the dense aggregates observed in controls, as well as the flat, scattered, and unstructured biofilm architecture after treatment of preformed biofilms cell-free supernatant. The cells were flat and relatively unstructured. Based on these results, we hypothesize that C. amycolatum likely produces secondary metabolites with antimicrobial activity and utilizes a similar mechanism of action to bacteriocins and/or biosurfactants. The data obtained open the prospect of studying the metabolic profile of the cell-free supernatant of C. amycolatum to understand the nature and mechanism of the detected antibacterial action and provide further support for the probiotic potential of C. amycolatum vaginal isolates.

Commensal Bacillus siamensis LF4 induces antimicrobial peptides expression via TLRs and NLRs signaling pathways in intestinal epithelial cells of Lateolabrax maculatus

Antimicrobial peptides (AMPs) play an important role in modulating intestinal microbiota, and our previous study showed that autochthonous Baccilus siamensis LF4 could shape the intestinal microbiota of spotted seabass (Lateolabrax maculatus). In the present study, a spotted seabass intestinal epithelial cells (IECs) model was used to investigate whether autochthonous B. siamensis LF4 could modulate the expression of AMPs in IECs. And then, the IECs were treated with active, heat-inactivated LF4 and its supernatant to illustrate their AMPs inducing effects and the possible signal transduction mechanisms. The results showed that after 3 h of incubation with 108 CFU/mL B. siamensis LF4, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic propylic transaminase (GPT) activities in supernatant decreased significantly and obtained minimum values, while supernatant alkaline phosphatase (AKP) activity, β-defensin protein level and IECs Na+/K+-ATPase activity, AMPs (β-defensin, hepcidin-1, NK-lysin, piscidin-5) genes expression increased significantly and obtained maximum values (P < 0.05). Further study demonstrated that the active, heat-inactivated LF4 and its supernatant treatments could effectively decrease the LDH, GOT, and GPT activities in IECs supernatant, increase AKP activity and β-defensin (except LF4 supernatant treatment) protein level in IECs supernatant and Na+/K+-ATPase and AMPs genes expression in IECs. Treatment with active and heat-inactivated B. siamensis LF4 resulted in significantly up-regulated the expressions of TLR1, TLR2, TLR3, TLR5, NOD1, NOD2, TIRAP, MyD88, IRAK1, IRAK4, TRAF6, TAB1, TAB2, ERK, JNK, p38, AP-1, IKKα, IKKβ and NF-κB genes. Treatment with B. siamensis LF4 supernatant also resulted in up-regulated these genes, but not the genes (ERK, JNK, p38, and AP-1) in MAPKs pathway. In summary, active, heat-inactivated and supernatant of B. siamensis LF4 can efficiently induce AMPs expression through activating the TLRs/NLRs-MyD88-dependent signaling, active and heat-inactivated LF4 activated both the downstream MAPKs and NF-κB pathways, while LF4 supernatant only activated NF-κB pathway.

Genomic, metabolomic, and functional characterisation of beneficial properties of Pediococcus pentosaceus ST58, isolated from human oral cavity.

Bacteriocins produced by lactic acid bacteria are proteinaceous antibacterial metabolites that normally exhibit bactericidal or bacteriostatic activity against genetically closely related bacteria. In this work, the bacteriocinogenic potential of Pediococcus pentosaceus strain ST58, isolated from oral cavity of a healthy volunteer was evaluated. To better understand the biological role of this strain, its technological and safety traits were deeply investigated through a combined approach considering physiological, metabolomic and genomic properties. Three out of 14 colonies generating inhibition zones were confirmed to be bacteriocin producers and, according to repPCR and RAPD-PCR, differentiation assays, and 16S rRNA sequencing it was confirmed to be replicates of the same strain, identified as P. pentosaceus, named ST58. Based on multiple isolation of the same strain (P. pentosaceus ST58) over the 26 weeks in screening process for the potential bacteriocinogenic strains from the oral cavity of the same volunteer, strain ST58 can be considered a persistent component of oral cavity microbiota. Genomic analysis of P. pentosaceus ST58 revealed the presence of operons encoding for bacteriocins pediocin PA-1 and penocin A. The produced bacteriocin(s) inhibited the growth of Listeria monocytogenes, Enterococcus spp. and some Lactobacillus spp. used to determine the activity spectrum. The highest levels of production (6400 AU/ml) were recorded against L. monocytogenes strains after 24 h of incubation and the antimicrobial activity was inhibited after treatment of the cell-free supernatants with proteolytic enzymes. Noteworthy, P. pentosaceus ST58 also presented antifungal activity and key metabolites potentially involved in these properties were identified. Overall, this strain can be of great biotechnological interest towards the development of effective bio-preservation cultures as well as potential health promoting microbes.

Inhibition of STAT3 signaling as critical molecular event in HUC-MSCs suppressed Glioblastoma Cells.

Objective: We investigated the effect of human umbilical cord mesenchymal stem cells (HUC-MSCs) supernatants on proliferation, migration, invasion, and apoptosis in glioblastoma (GBM) cell lines RG-2, U251, U87-MG, and LN-428, as well as their apoptosis and autophagy-mediated through IL-6/JAK2/STAT3 signaling pathway to explore the molecular mechanisms. Methods: In this study, RG-2, U251, U87-MG, and LN-428 cells were treated with 9 mg/ml HUC-MSCs supernatants. Their responses to HUC-MSCs supernatants treatment and the status of STAT3 signaling were analyzed by multiple experimental approaches to elucidate the importance of HUC-MSCs supernatants for GBM. Results: The results demonstrated that after treatment with HUC-MSCs supernatants, in vitro proliferation of RG-2, U251, U87-MG, and LN-428 cells were inhibited, and their sustained growth was also blocked. RG-2, U251, and U87-MG cells showed significant S phase accumulation, while LN-428 cells were blocked in G0/G1 phase. Their migratory invasive capacities were inhibited, and their apoptosis and autophagy ratios were increased. These effects were mediated through the IL-6/JAK2/STAT3 and its downstream signaling pathway. Conclusion: Our data showed that HUC-MSCs supernatants had anti-tumor effects on GBM cells. It inhibited the proliferation, migration, and invasion of GBM cells and promoted their apoptosis. Negative regulation of the IL-6/JAK2/STAT3 signaling pathway enhanced apoptosis and autophagy in tumor cells, thereby improving the therapeutic effect on GBM.

Comparison of the Efficacy of Enrofloxacin and Lactobacillus Plantarum Cell-Free Supernatant Treatments on Vaginitis in Ewes

The aim of the study was to investigate the effect of different intravaginal treatment strategies on the vaginal discharge score, vaginal microbiota, bacterial and Enterobacteriaceae counts in nulliparous Merino ewes. All ewes (n=45) received intravaginal sponges containing 60 mg medroxyprogesterone acetate for 7 days and allocated into three equal groups (n=15). Sponges were injected Lactobacillus plantarum cell-free supernatant (SUPER), enrofloxacin (ENRO), or physiologic saline (CON) prior to sponge insertion. At sponge removal, 500 IU equine chorionic gonadotropin (eCG) were administered in all ewes. For the detection of vaginal microbiota, bacterial and Enterobactericeae counts, samples were collected prior to sponge insertion, at sponge withdrawal, and 48 h later after sponge withdrawal. Vaginal discharge score was not different in ENRO (2.26±0.18) and SUPER (2.20±0.14) compared to CON (2.46±0.16). The time-dependent alteration was significant for the mean bacterial and Enterobacteriaceae count in all groups (P

Iron-containing carriers stimulate nitrogen conversions in one-stage reactors treating N-rich digester supernatant

Iron incorporation in biomass carriers (0%, 1%, 5%, and 10% of carrier weight) at nitrogen loading rates (NLR) from 0.08 to 0.30 g/(g MLVSS·d) was tested during the treatment of nitrogen-rich digester supernatant in one-stage reactors, in which hybrid biomass (suspended/attached) was inhabited by ammonia-oxidizing bacteria, Anammox bacteria, and denitrifiers. The Anammox process dominated at the highest NLR and increased the volumetric nitrogen removal rate to about 500 g/(m3·d). Total microbial composition was most strongly affected by NLR, however, Anammox bacteria abundance was also affected by the iron content. These bacteria predominated in reactors with 5%-iron carriers, which ensured the highest nitrogen removal efficiency (up to 75%) and kinetics: the average ammonium removal rate was at least 40% higher than in the reactor without iron. The presence of 10%-iron carriers in the reactor inhibited the ammonium oxidation rate; an increase in ammonium removal rate with increasing initial ammonium concentration was the lowest. The contribution of Anammox to nitrogen removal was demonstrated by the simultaneous increase in biomass heme c content to about 0.1 µmol/mg protein along with increasing iron content to 5% and NLR. The incorporation of iron into the carrier structure allowed for a long-term release of mostly ferrous iron to the supernatant, thus accelerating its treatment.

Biocontrol potential of endosymbiotic bacteria of entomopathogenic nematodes against the tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae)

BackgroundThe tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) is a major pest of tomato plants threatening global tomato production. The control of the pest is becoming increasingly difficult due to the rapid development of resistance to insecticides. Entomopathogenic nematodes (EPNs) of families Steinernematidae and Heterorhabditidae are successful biocontrol agents for many insect pests. Recently, their bacteria, Xenorhabdus spp. and Photorhabdus spp. have attracted great attention due to their major role in the pathogenicity of EPNs. In the present study, the pathogenicity of eleven EPNs isolates belonging to Steinernema feltiae and Heterorhabditis bacteriophora species was screened against the 1st/2nd and 3rd/4th instar larvae of T. absoluta. The cell-free supernatants and cell suspensions of the symbiotic bacteria from the most efficient isolates were further evaluated for their biocontrol potential in the oral and contact treatments on the larvae of T. absoluta.ResultsKBC-4 and MCB-8 isolates of S. feltiae showed superior virulence relative to other EPNs species/isolates and induced 90% larval mortality against the 3rd/4th instar of T. absoluta larvae, whereas there were no clear differences in the efficacy of EPNs species/isolates against the 1st/2nd instar of T. absoluta. The 1st/2nd instar of T. absoluta larvae was more susceptible to cell-free supernatants and cell suspension of selected EPNs. The highest mortality (80%) was obtained from X. bovienii MCB-8 strain in the contact treatment of supernatants. In contact treatment of cell suspension, higher mortalities were obtained compared to oral treatments. Mortality rates ranged between 30 and 57.5% in the contact treatments of cell suspensions while the highest mortality did not exceed 20% in oral treatments. The antifeedant activity was observed in oral treatments of cell suspension and most of the larvae avoided feeding on treated leaves.ConclusionThe results indicated that symbiotic bacteria of EPNs had a great potential against T. absoluta larvae and contact treatment of cell-free supernatants against early instars of T. absoluta can be an ideal application. However, further studies are needed to investigate the field effectiveness of symbiotic bacteria.

Identification and Biocontrol Potential of Entomopathogenic Nematodes and Their Endosymbiotic Bacteria in Apple Orchards against the Codling Moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae).

The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is one of the major pests in pome fruit production worldwide. Heavy treatment of the larvae of C. pomonella with insecticides triggered the development of resistance to many groups of insecticides. In addition, the increasing concern about the adverse effects of synthetic insecticides on human health and the environment has led to the development of sustainable and eco-friendly control practices for C. pomonella. The entomopathogenic nematodes (EPNs) (Steinernema and Heterorhabditis spp.) and their endosymbionts (Xenorhabdus and Photorhabdus spp.) represent a newly emerging approach to controlling a wide range of insect pests. In the present study, field surveys were conducted in apple orchards to isolate and identify EPNs and their endosymbionts and evaluate their insecticidal efficacy on the larvae of C. pomonella. EPNs were isolated from 12 of 100 soil samples (12%). Seven samples were identified as Steinernema feltiae (Filipjev, 1934) (Rhabditida: Steinernematidae), whereas five samples were assigned to Heterorhabditis bacteriophora (Poinar, 1976) (Rhabditida: Heterorhabditidae). The pathogenicity of the EPN species/isolates was screened on the last instar larvae of G. mellonella. The two most pathogenic isolates from each EPN species were tested against fifth instar larvae of C. pomonella under controlled conditions. The maximum mortality (100%) was achieved by all EPN species/isolates at a concentration of 100 IJs/larva 96 h after treatment. The endosymbionts of selected H. bacteriophora and S. feltiae species were identified as Photorhabdus luminescens subsp. kayaii and Xenorhabdus bovienii, respectively. The mortality rates ranged between 25 and 62% when the fifth larval instar larvae of C. pomonella were exposed to the treatment of cell-free supernatants of symbiotic bacteria. In essence, the present survey indicated that EPNs and their symbiotic bacteria have good potential for biological control of C. pomonella.

HUMSCs Transplantation Regulates AMPK/NR4A1 Signaling Axis to Inhibit Ovarian Fibrosis in POI Rats.

The mechanism of human Umbilical Cord Mesenchymal Stem Cells (hUMSCs) transplantation to improve ovarian function in the rats with Premature Ovarian Insufficiency (POI) is still unclear. The aim of this study is to investigate the signal axis mechanism that is involved in the ovarian function recovery of POI rats following hUMSCs transplantation. The rat model with POI was established by intraperitoneal injection of cisplatin. The hUMSCs were transplanted by caudal vein injection into POI rats.Hematoxylin-eosin (H&E) staining was performed to examine the morphology of rat ovarian tissue.Masson staining, Sirus red staining and immunofluorescence were used to observe the fibrosis extent of ovarian tissue. The levels of serum sex hormones and the expression of fibrosis related markers in ovarian tissueswere measured by enzyme-linked immunosorbent assay (ELISA).The expression of NR4A1, Phospho-NR4A1 and AMP-activated protein kinase (AMPK) signaling in rat ovarian tissues was measured by immunohistochemistry and immunofluorescence. The role of AMPK/NR4A1 signaling axis in the regulation of ovarian function recovery in POI rats following hUMSCs transplantation was further investigated by adenovirus and siRNA intervention in isolated stromal cells. The results showed that the hUMSCs transplantation significantly inhibited ovarian tissue fibrosis and restored the ovarian function in POI rats.The level of NR4A1 and AMPK expression in ovarian tissue of POI rats after hUMSCs transplantation was significantly increased compared with the control group.In the cultured ovarian stromal cells, the similar results were obtained on the expression of NR4A1 and its regulation on fibrosis related molecular markers in Cisplatin (CDDP) damaged stromal cells following hUMSCs supernatant treatment. Both hUMSCs supernatant treatment and the addition of AMPK inhibitors increased NR4A1 expression in stromal cells. And after NR4A1 molecular intervention, fibrosis-related indicators in stromal cells changed. The data suggests that the AMPK/NR4A1 signaling axis is involved in the ovarian function changes in POI rats following hUMSCs transplantation. The data from this study indicate that the inhibition of tissue fibrosis and recovery of ovarian function is regulated by AMPK/NR4A1 signaling axis in POI rats following hUMSCs transplantation.

A New Perspective: Revealing the Algicidal Properties of Bacillus subtilis to Alexandrium pacificum from Bacterial Communities and Toxins.

Algicidal bacteria are important in the control of toxic dinoflagellate blooms, but studies on the environmental behavior of related algal toxins are still lacking. In this study, Bacillus subtilis S3 (S3) showed the highest algicidal activity against Alexandrium pacificum (Group IV) out of six Bacillus strains. When treated with 0.5% (v/v) S3 bacterial culture and sterile supernatant, the algicidal rates were 69.74% and 70.22% at 12 h, respectively, and algicidal substances secreted by S3 were considered the mechanism of algicidal effect. During the algicidal process, the rapid proliferation of Alteromonas sp. in the phycosphere of A. pacificum may have accelerated the algal death. Moreover, the algicidal development of S3 released large amounts of intracellular paralytic shellfish toxins (PSTs) into the water, as the extracellular PSTs increased by 187.88% and 231.47% at 12 h, compared with the treatment of bacterial culture and sterile supernatant at 0 h, respectively. Although the total amount of PSTs increased slightly, the total toxicity of the algal sample decreased as GTX1/4 was transformed by S3 into GTX2/3 and GTX5. These results more comprehensively reveal the complex relationship between algicidal bacteria and microalgae, providing a potential source of biological control for harmful algal blooms and toxins.