Melatonin alleviates cadmium toxicity by regulating root endophytic bacteria community structure and metabolite composition in apple.

The level of cadmium (Cd) accumulation in orchard soils is increasing, and excess Cd will cause serious damage to plants. Melatonin is a potent natural antioxidant and has a potential role in alleviating Cd stress. This study aimed to investigate the effects of exogenous melatonin on a root endophyte bacteria community and metabolite composition under Cd stress. The results showed that melatonin significantly scavenged the reactive oxygen species and restored the photosynthetic system (manifested by the improved photosynthetic parameters, total chlorophyll content and the chlorophyll fluorescence parameters (Fv/Fm)), increased the activity of antioxidant enzymes (the activities of catalase, superoxide dismutase, peroxidase and ascorbate oxidase) and reduced the concentration of Cd in the roots and leaves of apple plants. High-throughput sequencing showed that melatonin increased the endophytic bacterial community richness significantly and changed the community structure under Cd stress. The abundance of some potentially beneficial endophytic bacteria (Ohtaekwangia, Streptomyces, Tabrizicola and Azovibrio) increased significantly, indicating that the plants may absorb potentially beneficial microorganisms to resist Cd stress. The metabolomics results showed that melatonin significantly changed the composition of root metabolites, and the relative abundance of some metabolites decreased, suggesting that melatonin may resist Cd stress by depleting root metabolites. In addition, co-occurrence network analysis indicated that some potentially beneficial endophytes may be influenced by specific metabolites. These results provide a theoretical basis for studying the effects of melatonin on the endophytic bacterial community and metabolic composition in apple plants.

Dopamine alleviates cadmium stress in apple trees by recruiting beneficial microorganisms to enhance the physiological resilience revealed by high-throughput sequencing and soil metabolomics.

Dopamine has demonstrated promise as a stress-relief substance. However, the function of dopamine in Cd tolerance and its mechanism remains largely unknown. The current study was performed to investigate the mechanism of dopamine on alleviating apple Cd stress through regular application of CdCl2 and dopamine solution to potting soil. The results indicated that dopamine significantly reduced reactive oxygen species (ROS) and Cd accumulation and alleviated the inhibitory effect of Cd stress on the growth of apple plants through activation of the antioxidant system, enhancement of photosynthetic capacity, and regulation of gene expression related to Cd absorption and detoxification. The richness of the rhizosphere microbial community increased, and community composition and assembly were affected by dopamine treatment. Network analysis of microbial communities showed that the numbers of nodes and total links increased significantly after dopamine treatment, while the keystone species shifted. Linear discriminant analysis effect size indicated that some biomarkers were significantly enriched after dopamine treatment, suggesting that dopamine induced plants to recruit potentially beneficial microorganisms (Pseudoxanthomonas, Aeromicrobium, Bradyrhizobium, Frankia, Saccharimonadales, Novosphingobium, and Streptomyces) to resist Cd stress. The co-occurrence network showed several metabolites that were positively correlated with relative growth rate and negatively correlated with Cd accumulation, suggesting that potentially beneficial microorganisms may be attracted by several metabolites (L-threonic acid, profenamine, juniperic acid and (3ß,5ξ,9ξ)-3,6,19-trihydroxyurs-12-en-28-oic acid). Our results demonstrate that dopamine alleviates Cd stress in apple trees by recruiting beneficial microorganisms to enhance the physiological resilience revealed. This study provides an effective means to reduce the harm to agricultural production caused by heavy metals.

Comparative Metagenomic and Metatranscriptomic Analyses Reveal the Response of Black Soldier Fly (Hermetia illucens) Larvae Intestinal Microbes and Reduction Mechanisms to High Concentrations of Tetracycline.

Black soldier fly (Hermetia illucens L) larvae (BSFL) possess remarkable antibiotic degradation abilities due to their robust intestinal microbiota. However, the response mechanism of BSFL intestinal microbes to the high concentration of antibiotic stress remains unclear. In this study, we investigated the shift in BSFL gut microbiome and the functional genes that respond to 1250 mg/kg of tetracycline via metagenomic and metatranscriptomic analysis, respectively. The bio-physiological phenotypes showed that the survival rate of BSFL was not affected by tetracycline, while the biomass and substrate consumption of BSFL was slightly reduced. Natural BSFL achieved a 20% higher tetracycline degradation rate than the germ-free BSFL after 8 days of rearing. Metagenomic and metatranscriptomic sequencing results revealed the differences between the entire and active microbiome. Metatranscriptomic analysis indicated that Enterococcus, Vagococcus, Providencia, and Paenalcaligenes were the active genera that responded to tetracycline. Furthermore, based on the active functional genes that responded to tetracycline pressure, the response mechanisms of BSFL intestinal microbes were speculated as follows: the Tet family that mediates the expression of efflux pumps expel tetracycline out of the microbes, while tetM and tetW release it from the ribosome. Eventually, tetracycline was degraded by deacetylases and novel enzymes. Overall, this study provides novel insights about the active intestinal microbes and their functional genes in insects responding to the high concentration of antibiotics.

Exogenous melatonin alleviates apple replant disease by regulating rhizosphere soil microbial community structure and nitrogen metabolism.

Apple replant disease (ARD) is a common soil-borne disease afflicting apple plants. Melatonin is a broad-spectrum oxygen scavenger that plays a key role in alleviating stress-induced damage in plants. In this study, we aimed to determine whether adding melatonin to replant soil can promote plant growth by improving the rhizosphere soil environment and nitrogen metabolism. In replant soil, chlorophyll synthesis was blocked, reactive oxygen species (ROS) accumulated in large quantities, and membrane lipid peroxidation was aggravated; this eventually resulted in slow plant growth. However, the application of 200 µM exogenous melatonin enhanced the tolerance of plants to ARD by up-regulating the expression of antioxidant enzyme-related genes and increasing ROS scavenging enzyme activity. Exogenous melatonin also increased the absorption and utilization of 15N by increasing the expression of nitrogen absorption genes and the activity of nitrogen metabolism enzymes. Exogenous melatonin enhanced the soil microbial environment by promoting soil enzyme activity and bacterial richness and decreasing the abundance of several harmful fungi in rhizosphere soil. Mantel test results showed that soil properties (except for AP) and growth indexes were positively correlated with the rate of 15N absorption and utilization. Spearman correlation analysis showed that the above factors were closely related to the richness and diversity of bacteria and fungi, indicating that the composition of microbial communities might play a key role in mediating change in the soil environment and thus affect nutrient absorption and growth. These findings provide new insights into how melatonin enhances ARD tolerance.

Biochar Can Improve Absorption of Nitrogen in Chicken Manure by Black Soldier Fly.

(1) Background: There is growing interest in using insects to treat nutrient-rich organic wastes, such as the black soldier fly (BSF), one of the most efficient organic waste recyclers for upcycling nutrients into the food system. Although biochar (BC) was shown to enhance nutrient retention and the final product quality during the composting of livestock and poultry manure in many previous studies, little information is available on the effect of BC on livestock manure bioconversion by black soldier fly larvae (BSFL). (2) Methods: This study investigated the effect of adding a small amount of BC to chicken manure (CM) on the bioconversion system of the black soldier fly (including N2O and NH3 emissions and the final distribution of nitrogen during the treatment process). (3) Results: The lowest N2O and NH3 emission and highest residual nitrogen in the substrate were observed in the 15% BC treatment. The highest bioconversion rate of CM (8.31%) and the peak of larval biomass was obtained in the 5% BC treatment. (4) Conclusions: The results demonstrate the feasibility of adding 5% BC to reduce pollution and achieve a satisfactory BSFL-based CM bioconversion efficiency.

Ionomic Combined with Transcriptomic and Metabolomic Analyses to Explore the Mechanism Underlying the Effect of Melatonin in Relieving Nutrient Stress in Apple.

Nutrient stress harms plant growth and yield. Melatonin is a biologically active, multifunctional hormone that relieves abiotic stress in plants. Although previous studies have shown that melatonin plays an important role in improving nutrient-use efficiency, the mechanism of its regulation of nutrient stress remains unclear. In this study, melatonin was applied to apple plants under nutrient stress, and morphological indices, physiological and biochemical indices, and stomatal morphology were evaluated. The response of apple plants to nutrient deficiency and the melatonin mechanism to alleviate nutrient stress were analyzed by combining ionome, transcriptome, and metabolome. The results showed that exogenous melatonin significantly alleviated the inhibitory effect of nutritional stress on the growth of apple plants by regulating stomatal morphology, improving antioxidant enzyme activity, promoting ion absorption, and utilizing and changing the absorption and distribution of minerals throughout the plant. The transcriptome results showed that melatonin alleviated nutrient stress and promoted nutrient absorption and utilization by regulating glutathione metabolism and upregulating some metal ion transport genes. The metabolome results indicated that levels of oxalic acid, L-ascorbic acid, anthocyanins (cyanidin-3-O-galactoside), lignans (lirioresinol A and syringaresinol), and melatonin significantly increased after exogenous melatonin was applied to plants under nutrient stress. These differentially expressed genes and the increase in beneficial metabolites may explain how melatonin alleviates nutrient stress in plants.

Bacillus velezensis EEAM 10B Strengthens Nutrient Metabolic Process in Black Soldier Fly Larvae (Hermetia illucens) via Changing Gut Microbiome and Metabolic Pathways.

Insects are a potential alternative protein source to solve the food shortage crisis. Previous studies have illustrated that probiotics can improve the substrate conversion efficiency of insects and increase insect protein content. However, the effects of probiotics on insect physiology and nutrient metabolism are still not well understood. Here, the black soldier fly larvae (BSFL), Hermetia illucens (Diptera: Stratiomyidae), was used as a study subject to deeply investigate the specific interaction among a novel probiotic, Bacillus velezensis EEAM 10B (10B), intestinal microbiota, and the host. In this study, the effects of 10B on the survival and physiology of BSFL were first analyzed. It shows that 10B significantly elevated the substrate conversion rate, average dry weight, and protein content of BSFL by 5%, 0.13 g/pc, and 8%, respectively. Then, we assessed the effect of 10B on the microbial community composition in the gut and frass of BSFL using Illumina Miseq sequencing. It shows that 10B significantly altered the microbial composition of the gut, but not that of the frass. Pearson's correlation analysis further showed that the Bacillus, unclassified_of_Caloramatoraceae, and Gracilibacillus were positively correlated with the survival rate, crude protein content, and substrate conversion rate of BSFL. To further investigate the effect of 10B on host metabolism, metabolic analyses on germ-free BSFL, monobacterial intestinal BSFL, and natural BSFL were also performed. The results proved that 10B (i) played a vital role in the survival of BSFL; and (ii) regulated the amino acid synthetic and metabolic process of BSFL, thus leading to the rise of the protein content of BSFL. In addition, vitamin backfill assays verified that the BSFL survival rate was significantly improved by supplying the germ-free BSFL with riboflavin, which further suggests that 10B determines the survival of BSFL via delivering riboflavin. Overall, this study provides a reference for understanding the comprehensive contribution of a specific probiotic to its host.

Integrated Analysis of MicroRNA and mRNA Expression Profiles in the Fat Bodies of MbMNPV-Infected Helicoverpa armigera.

MicroRNAs (miRNAs), are a novel class of gene expression regulators, that have been found to participate in regulating host-virus interactions. However, the function of insect-derived miRNAs in response to virus infection is poorly understood. We analyzed miRNA expression profiles in the fat bodies of Helicoverpa armigera (H. armigera) infected with Mamestra brassicae multiple nucleopolyhedroviruses (MbMNPV). A total of 52 differentially expressed miRNAs (DEmiRNAs) were filtered out through RNA-seq analysis. The targets of 52 DEmiRNAs were predicted and 100 miRNA-mRNA interaction pairs were obtained. The predicted targets of DEmiRNAs were mainly enriched in the Wnt signaling pathway, phagosome, and mTOR signaling pathway, which are related to the virus infection. Real-time PCR was used to verify the RNA sequencing results. ame-miR-317-3p, mse-miR-34, novel1-star, and sfr-miR-6094-5p were shown to be involved in the host response to MbMNPV infection. Results suggest that sfr-miR-6094-5p can negatively regulate the expression of four host genes eIF3-S7, CG7583, CG16901, and btf314, and inhibited MbMNPV infection significantly. Further studies showed that RNAi-mediated knockdown of eIF3-S7 inhibited the MbMNPV infection. These findings suggest that sfr-miR-6094-5p inhibits MbMNPV infection by negatively regulating the expression of eIF3-S7. This study provides new insights into MbMNPV and H. armigera interaction mechanisms.

A meta-analysis of early oral refeeding and quickly increased diet for patients with mild acute pancreatitis.

BACKGROUND/AIM: The objective of the study is to clarify whether early oral refeeding (EORF) and quickly increasing diet (QID) are of benefit to patients with mild acute pancreatitis compared with a traditional oral refeeding strategy. MATERIALS AND METHODS: Studies were searched in PubMed, Cochrane library, ScienceDirect, SpringerLink, China Biology Medicine disc and Embase. A meta-analysis was then performed, using relapse of abdominal pain, nausea/vomiting, and length of hospital stay (LOHS) as the evaluation indices. RESULTS: Eight trials met the inclusion criteria. For the oral refeeding time group, EORF could significantly decrease the LOHS (mean deviation [MD] -1.97; 95% confidence interval (CI) -3.32 to -0.62;P = 0.004), and there was no significant difference for relapse of abdominal pain (relative risk [RR] 1.17; 95% CI 0.69-2.00;P = 0.56) or nausea/vomiting (RR 1.30; 95% CI 0.19-8.82;P = 0.79) when compared with conventional oral refeeding. For the oral refeeding material group, there was no significant difference for relapse of abdominal pain (RR 0.86; 95% CI 0.53-1.40;P = 0.54), nausea/vomiting (risk difference -0.01; 95% CI -0.19-0.18;P = 0.94), or LOHS (MD -0.88; 95% CI -2.24-0.48;P = 0.20) between the QID and stepwise increasing diet groups. CONCLUSION: Pure EORF or QID caused no damage to patients with mild acute pancreatitis, and EORF could significantly decrease the LOHS.

Accumulation of Citrulline by Microbial Arginine Metabolism during Alcoholic Fermentation of Soy Sauce.

Citrulline, the major precursor of ethyl carbamate in soy sauce, is an intermediate catabolite of arginine produced by bacteria present in soy sauce moromi mash. Pediococcus acidilactici is responsible for the formation of citrulline during the lactic acid fermentation process of soy sauce. However, citrulline accumulation during the alcoholic fermentation process and the corresponding bacteria involved have not been identified. Salt-tolerant, arginine-utilizing bacteria were isolated from moromi mash during the alcoholic fermentation process. Under normal cultivation conditions, arginine utilization by these strains did not contribute to citrulline accumulation. However, the conversion of arginine to citrulline by these bacteria increased when cultivated during the alcoholic fermentation process. Additionally, the ethanol-enhanced solubility of free fatty acids in moromi mash stimulated the accumulation of citrulline. Staphylococcus exhibited the highest capability in the conversion of arginine to citrulline.

Characterization of a Bacillus amyloliquefaciens strain for reduction of citrulline accumulation during soy sauce fermentation.

OBJECTIVES: To reduce the amount of citrulline produced by arginine-consuming bacteria in the moromi mash during soy sauce production. RESULTS: Bacillus amyloliquefaciens JY06, a salt-tolerant strain with high arginine consumption ability and low citrulline accumulation capacity, was isolated from moromi mash. The concentration of citrulline was decreased from 26.8 to 5.1 mM and ethyl carbamate in soy sauce, after sterilization, decreased from 97 to 17 µg kg(-1) when B. amyloliquefaciens JY06 was added during fermentation. The aroma of the sauce was improved by increasing the ester content. CONCLUSIONS: B. amyloliquefaciens JY06 is a beneficial bacterium that can be used in soy sauce fermentation to eliminate ethyl carbonate and enhance the flavor of the sauce.

[Metabolism of ethyl carbamate precursors in soy sauce by Zygosaccharomyces rouxii ZQ02Metabolism of ethyl carbamate precursors in soy sauce by Zygosaccharomyces rouxii ZQ02].

Objective: To study nitrogen metabolism of Zygosaccharomyces rouxii and its relationship with the formation of soy sauce ethyl carbamate precursors. Methods: Z. rouxii ZQ02 was cultivated with single source of nitrogen, preferred nitrogen sources or under salt stress, to investigate its ability of using arginine, citrulline and urea. Results: Alanine, glycine and asparaginate were confirmed to be the preferred nitrogen sources of Z. rouxii ZQ02. Addition of preferred nitrogen sources did not inhibit the use of urea and citrulline, on the contrary, the consumption of urea and citrulline by Z. rouxii ZQ02 was stimulated with the addition of alanine and glycine. Z. rouxii ZQ02 did not accumulate any citrulline and urea from degradation of arginine. Urea and citrulline were used by Z. rouxii ZQ02 in the medium with single source of nitrogen. However, use of citrulline and urea by Z. rouxii ZQ02 was strongly inhibited under saline stress, resulting in the incomplete use of ethyl carbamate precursors. Conclusion: Use of citrulline and urea by Z. rouxii ZQ02 was strongly inhibited under high salt stress, resulting in the accumulation of ethyl carbamate precursors produced by other microorganisms during soy sauce fermentation.

The arginine deiminase pathway of koji bacteria is involved in ethyl carbamate precursor production in soy sauce.

Ethyl carbamate (EC) is a group 2A carcinogen generated from a few precursors in many fermented foods and alcoholic beverages. Citrulline, urea, carbamoyl phosphate, and ethanol are common precursors detected in fermented foods. In this study, citrulline was proved to be the main EC precursor in soy sauce, which was found to be accumulated in moromi mash period and correlated with the utilization of arginine by koji bacteria. Six koji isolates belonging to three genera were identified to be able to accumulate citrulline via the arginine deiminase (ADI) pathway. Among these strains, only Pediococcus acidilactici retained high activities in synthesis and accumulation of citrulline in the presence of high concentration of sodium chloride. These results suggested that P. acidilactici is responsible for the accumulation of citrulline, one of the EC precursors, in the process of soy sauce fermentation.

Lethal effect of a hyperthermic CO2 pneumoperitoneum on gastric cancer cells.

OBJECTIVES: We investigated the lethal effect of a hyperthermic CO2 pneumoperitoneum on gastric cancer cells. This could form the theoretical basis for further studies of the feasibility and safety of inflating hyperthermic CO2 in the abdominal cavity of gastric cancer patients during laparoscopy. METHODS: An in vitro hyperthermic CO2 pneumoperitoneum experimental model was built, where gastric cancer cell line SGC-7901 cells were grouped according to temperature. Cytotoxicity was detected using a cell counting kit; apoptosis was detected by Annexin V-FITC/PI flow cytometry and Hoechst 33342/PI fluorescent microscopy. Morphological alterations were observed by transmission electron microscopy. Invasion and migration were detected by a scratch test and by transwell migration, respectively. RESULTS: Cytotoxicity assays showed that a hyperthermic CO2 pneumoperitoneum significantly inhibited the proliferation of SGC-7901 cells (P<0.05); it also significantly induced apoptosis of SGC-7901 cells (P<0.05). Morphological observations showed that the cell membrane and nucleus had an apoptotic phenotype. The invasiveness and migration ability of the gastric cancer cells subjected to hyperthermic CO2 were significantly reduced. CONCLUSIONS: A hyperthermic CO2 pneumoperitoneum had a lethal effect on gastric cancer SGC-7901 cells by inhibiting their invasion and migration, and by inducing apoptosis.

High expressions of galectin-1 and VEGF are associated with poor prognosis in gastric cancer patients.

High expressions of galectin-1 and vascular endothelial growth factor (VEGF) are correlated with biological behavior in some cancers. The aim of this study is to evaluate the expressions of galectin-1 and VEGF in gastric cancer and investigate their relationships with clinicopathological factors and prognostic significance. Immunohistochemical analyses for galectin-1 and VEGF expression were performed on 108 cases of gastric cancer. The relationship between the expression and staining intensity of galectin-1 and VEGF, clinicopathological variables, and survival rates was analyzed. Immunohistochemical staining demonstrated that 68 of 108 gastric cancer samples (63.0%) were positive for galectin-1 and 62 out of 108 gastric cancer samples (57.4%) were positive for VEGF. Galectin-1 expression was associated with tumor size, differentiation grade, TNM stage, lymph node metastases, and VEGF expression. VEGF expression was related to tumor size, TNM stage, and lymph node metastases. Kaplan-Meier survival analysis showed that high galectin-1 and VEGF expressions exhibited significant correlations with poor prognosis for gastric cancer patients. Multivariate analysis revealed that galectin-1 and VEGF expressions were independent prognostic parameters for the overall survival rate of gastric cancer patients. The results of the present study suggest that galectin-1 expression is positively associated with VEGF expression. Both galectin-1 and VEGF can serve as independent prognostic indicators of poor survival for gastric cancer.