[Objective] To investigate the effects of a metabolite cocktail composed of indole-3-propionic acid (IPA), sodium butyrate (SB), and valeric acid (VA) of gut microbiota on the proliferation of hepatocellular carcinoma cells. [Methods] The human hepatocellular carcinoma HepG2 cells were cultured in vitro and treated with the cocktail at different concentrations (1×, 2×, 3×, 4×, and 5×). The total cholesterol (TC) and triglyceride (TG) levels in the cells were determined by the total cholesterol and triglyceride assay kits. The Cell Counting Kit-8 (CCK-8) and colony formation assays were employed to examine the cell proliferation. Twelve BALB/c athymic nude mice were randomized into a control (Ctrl) group and a treatment (Treat) group and then subjected to subcutaneous injections of HepG2 cells. The tumor size was measured every three days, and the tumor volume and tumor inhibition rate were calculated. When the tumor volume reached 100 mm³, the mice in the Ctrl group were administered with sterile water by gavage daily, while those in the Treat group received the cocktail via gavage until euthanized under anesthesia. After 27 days of treatment, the body weights of mice in both groups were measured, and tumors were excised and weighed, with the tumor weight/body weight ratio calculated. The content of Ki-67 protein in the tumors was determined by immunohistochemical (IHC) staining, and the lipid accumulation within tumor cells was assessed by Oil Red O staining. [Results] The cocktail of IPA, SB, and VA lowered the levels of TC and TG in hepatocellular carcinoma HepG2 cells and exerted an inhibitory effect on the proliferation of HepG2 cells. Both CCK-8 and colony formation assays indicated that the cocktail inhibited the proliferation of HepG2 cells in a dose-dependent manner. The oral administration of the cocktail inhibited the growth of hepatocellular carcinoma cells, as evidenced by smaller and lighter tumors and lower tumor weight/body weight ratios in the Treat group than in the Ctrl group (Ctrl: 723 mm³, 0.47 g, 22.23%; Treat: 526 mm³, 0.32 g, 16.65%). IHC and Oil Red O staining further demonstrated reductions in Ki-67 expression and lipid accumulation in the mice administered with the cocktail via gavage. [Conclusion] The cocktail of IPA, SB, and VA can inhibit the proliferation and suppress the lipid synthesis of hepatocellular carcinoma cells.

Glucose-1-phosphate is a key precursor for starch biosynthesis of photoautotrophs. Phosphoglucomutases (PGMs) belonging to the phosphohexomutase family have a high conserved characteristic and perform the interconversion between glucose-6-phosphate and glucose-1-phosphate to regulate the starch biosynthesis. Compared with the higher plants, microalgae possess unique photosynthetic systems. Additionally, some microalgae strains can utilize organic carbon sources to produce valuable biomass by heterotrophic or mixotrophic cultivation, which might endow PGMs with specific structural features and biological functions in starch metabolism to regulate the levels of carbon fixation by photosynthesis, carbohydrate metabolism, and other pathways in microalgae. This article summarizes the molecular characteristics, functions, and activity regulation of PGMs for microalgae. Moreover, this article elucidates the potential mechanisms by which PGMs regulate microalgae starch synthesis to influence intracellular protein and lipid metabolic pathways. This review lays a theoretical foundation for microalgae carbon sequestration and the value-added utilization of microalgae resources, contributing to the achievement of China's "dual-carbon" goals.

[Objective] A facultative anaerobic bacterium Klebsiella sp. CW-D3T utilizing sulfate as the terminal electron acceptor for anaerobic respiration was used for degradation of target pollutants in the system with phenanthrene (PHE)-Cd²⁺ co-contamination. The response mechanism of the strain to different Cd²⁺ concentrations in the sulfate reduction system and the anaerobic metabolic pathways of the strain for degrading PHE were studied. [Methods] A sulfate reduction system with an initial sulfate concentration of 20 mmol/L was developed to enhance the growth and metabolic activity of functional bacteria and improve the bacterial performance for remediating PHE-Cd²⁺ co-contamination. The changes in extracellular polymer secretion and the vibrational characteristics of characteristic peaks were analyzed to explore the cellular responses to different Cd²⁺ concentrations. Furthermore, qualitative and quantitative analyses of the metabolic products of PHE in the sulfate reduction system were conducted by GC-MS and HPLC. [Results] In the presence of 0.5–50.0 mg/L Cd²⁺, the sulfate reduction system of Klebsiella sp. CW-D3T enhanced the remediation efficiency of target compounds, with the PHE and Cd²⁺ removal rates above 70.00% when the initial Cd²⁺ concentration was below 10 mg/L. As the concentration of Cd²⁺ increased, the secretion of extracellular polysaccharides in extracellular polymeric substances (EPS) was more than that of extracellular proteins, and the intensity of characteristic peaks of polysaccharides and protein functional groups on the surface of bacterial cells was enhanced. The initial activation of PHE in the sulfate reduction system tended to favor carboxylation to produce 2-phenanthroic acid under Cd²⁺ stress. When the initial Cd²⁺ concentrations were 10 mg/L and 50 mg/L, the content of 2-phenanthroic acid peaked at 15.56 μg/L and 10.23 μg/L on day 5, respectively, which decreased by 27.56% and 52.37% compared with that of the control group without the addition of Cd²⁺. Cd²⁺ stress significantly affected the 2-phenanthroic acid content within the cycle and at the end of the cycle. [Conclusion] The biodegradation efficiency of PHE by Klebsiella sp. CW-D3T was significantly improved when sulfate was used as an electron acceptor in the presence of Cd²⁺. The extracellular polysaccharides and proteins played a positive role in enhancing the microbial tolerance to Cd²⁺ stress by regulating the detoxification process.

[Objective] The application of Bacillus subtilis in soil can promote plant growth, while few studies have reported the bacteriophages infecting B. subtilis. Therefore, the isolation, biological characterization, and genome sequencing of bacteriophages infecting B. subtilis from soil will contribute to the application of B. subtilis and enrich the biological information of bacteriophages infecting B. subtilis. [Methods] B. subtilis SM13 was used as the host to isolate a bacteriophage strain Bac-S from soil. Biological characterization, whole genome sequencing, gene function annotation, and phylogenetic analysis were performed for this bacteriophage strain. [Results] Transmission electron microscopy showed that Bac-S had a head with the diameter of about 43 nm and a tail too short to be measured. The optimal multiplicity of infection of Bac-S was 0.1. The one-step growth curve showed that Bac-S had an incubation period of about 10 min and a burst size of 30 PFU/cell. Bac-S had a wide host spectrum and can infect hosts of different genera. The sequencing results showed that the genome of Bac-S was 150 019 bp, with the G+C content of 42.6% and 237 open reading frames (ORFs). The BLASTn comparison with the sequences in the NCBI database showed that Bac-S was similar to other bacteriophages infecting B. subtilis. [Conclusion] A bacteriophage strain infecting B. subtilis with a short incubation period, a wide host spectrum, tolerance to high temperature, and intolerance to UV light was isolated from soil. The biological and genetic characterization of this bacteriophage enriches our knowledge about the bacteriophages infecting B. subtilis.

[Objective] To compare the metabolism and transcription between the probiotic Escherichia coli Nissle 1917 (EcN) and the model strains, thus providing a reference for the engineering and promoting the application of the food-safe strain EcN. [Methods] The genome and transcriptome were compared between EcN and model strains BL21(DE3) and W3110 by software, and plasmids were constructed to verify the differences. EcN-derived microcin was expressed in BL21(DE3) and the antibacterial effect of microcin was verified. [Results] A total of 904 differentially coding genes were identified. The differences in carbon source absorption and utilization of different strains were verified by experiments with different carbon sources as substrates. The expression of the promoter P_flic confirmed the differences in transcription among different strains. The recombinant strain of microcin showed an increase of 30.3% in the inhibition rate after 12 h of culture. [Conclusion] This study clarifies the metabolic characteristics of EcN and confirms the differences in transcription between EcN and model strains. Moreover, this study provides ideas for the development of microcin as a narrow-spectrum therapeutic drug to inhibit intestinal pathogens and reduce intestinal bacterial blooms.

Agar is one of the important components in the cell wall of red algae. The biodegradation of agar affects marine ecological processes, such as nutrient recycling, succession of large seaweed communities, heavy metal pollution, and carbon sequestration. In addition, the degradation products of agar demonstrate great application potential in aquaculture, agriculture, medicine, health products, bioenergy, etc. Therefore, the biodegradation of agar and its ecological and application values have become research hotspots in recent years. This article reviews the research progress in the significance of agar degradation, microbial agarases, and agar metabolic pathways, providing theoretical support for the research on the ecological effects and comprehensive utilization of the agar from red algae.

[Objective] Keratinases, a class of serine proteases capable of degrading keratin, have important application potential and research value in the utilization of keratin resources. The efficient industrial production of keratinase is helpful to promoting its application in leather, textiles, feed, chemical fertilizers, daily chemicals, and medicine. In this study, we optimized the fermentation conditions of Bacillus subtilis WB600-pMA5-KerBv, a recombinant keratinase-producing strain constructed in our laboratory, to improve the enzyme production. Furthermore, we explored the potential application of keratinase in the degradation of fibrin. [Methods] First, the composition of the fermentation medium was determined by single factor experiments. Then, response surface methodology was employed to optimize the medium formula for producing keratinase, and the factors significantly affecting the growth and enzyme production of bacteria and the optimum concentrations were determined. Subsequently, the DoseResp model was adopted to predict the optimal growth point of the strain and thus guide the expansion of enzyme production in a 5 L fermenter. Finally, the blood clot and fibrinogen degradation experiments were carried out to evaluate the degradation performance of the keratinase. [Results] The formula of the fermentation medium for producing keratinase by the recombinant strain was optimized as follows (g/L): glucose 25.0, yeast powder 25.0, soybean meal 15.0, dipotassium phosphate 14.04, potassium dihydrogen phosphate 2.58, and magnesium chloride 0.3. The optimal growth point of the strain was predicted based on the DoseResp model to guide the expansion of production in a 5 L fermenter. Under the optimized conditions, the OD₆₀₀ (bacterial biomass) increased from 2.45 in a shake flask to 77.80, and the enzyme activity increased by about 4.76 times from 4 471 U/mL in a shake flask to 21 301.67 U/mL. In addition, the keratinase showcased remarkable degradation ability on fibrinogen and blood clots. [Conclusion] The systematic fermentation optimization and model-based prediction of enzyme production in fermenters improved the production of keratinase in Bacillus subtilis. The findings provided a research basis for the application of keratinase in thrombolysis.

[Objective] To provide candidate strains and effective strategies for the control of mulberry fruit sclerotiniose, we screened out the endophytic bacteria with biocontrol potential for mulberry fruit sclerotiniose from a resistant mulberry cultivar. [Methods] The endophytic bacteria antagonistic to mulberry fruit sclerotiniose were isolated from mulberry plants by the tissue culture and confrontation culture methods. The antagonistic strain was identified based on morphological features, physiological and biochemical characteristics, and the phylogenetic relationship based on 16S rRNA gene sequences. The antimicrobial spectrum and control efficiency to detached mulberry fruits were determined to evaluate the application potential of the antagonistic strain. Furthermore, we observed the inhibitory effect of the fermentation supernatant of the strain on the mycelial growth of the pathogen, measured the variations in glycogen and reactive oxygen species accumulation of the pathogen treated with the antagonistic strain, and determined the expression of pathogen-related genes after treatment with the antagonistic strain to decipher the antagonistic mechanism of this strain. [Results] An endophytic bacterial strain C1R32 with strong and stable antagonistic activity on Sclerotinia sclerotiorum PZ-2 (the pathogen of mulberry fruit sclerotiniose) was isolated from a healthy mulberry branch. C1R32 showed similar morphological features and physiological and biochemical characteristics with Bacillus. The phylogenetic analysis based on 16S rRNA gene sequences revealed that C1R32 was located in the same clade with B. subtilis. Therefore, strain C1R32 was identified as B. subtilis. B. subtilis C1R32 had antagonistic activities against a variety of phytopathogens including S. sclerotiorum. The suspension and fermentation supernatant of B. subtilis C1R32 showed the control effects of 52.94% and 46.43%, respectively, on sclerotiniose of detached mulberry fruits. The cell-free fermentation supernatant of B. subtilis C1R32 caused the hypha swelling and distorting, cell wall breaking, and cytoplasm leakage of S. sclerotiorum PZ-2. Moreover, B. subtilis C1R32 inhibited S. sclerotiorum PZ-2 by reducing glycogen accumulation, promoting reactive oxygen species burst, and influencing the expression of genes associated with antioxidant activity. [Conclusion] We isolated an endophytic B. subtilis strain capable of controlling mulberry fruit sclerotiniose from a resistant mulberry cultivar and preliminarily explored its antagonistic mechanism, providing potential strain resources for the biocontrol of mulberry fruit sclerotiniose.

[Objective] We characterized the uterine microbiota in healthy felines and felines with pyometra, aiming to reveal the effect of pyometra on the uterine microbiota of felines and explore the potential pathogens causing pyometra. [Methods] High-throughput sequencing of the full-length 16S rRNA gene was employed to determine and compare the uterine microbiota in healthy felines and felines with pyometra. The key strains were isolated and identified by the culture method. [Results] The dominant bacterial genera in the uterus of healthy felines were Acinetobacter, Pseudomonas, Sphingomonas, and Weissella. The dominant bacterial genus and species in the uterus of felines with pyometra were Escherichia-Shigella and Escherichia coli, respectively. Functional prediction showed that pathways such as protein export, amino acid-related enzymes, protein processing in endoplasmic reticulum, and aminoacyl tRNA biosynthesis in the pyometra group were significantly reduced. The results of isolation and identification showed that the prevalent bacterial species in the uterus of felines with pyometra was E. coli. The isolates all belonged to the phylogroup B2 and were mostly tested positive for hylA, fimH, iroN, cnf1, papC, kpsMTII, and iutA. [Conclusion] We compared the uterine microbiota in healthy felines and felines with pyometra. The dominant bacteria in the uterus of healthy felines were mostly non-pathogenic, while those in the uterus of felines with pyometra changed significantly, with E. coli being dominant and carrying multiple virulence genes. The findings provide a theoretical basis for treating pyometra in felines.

Beneficial bacteria in the gut affect human health, and it is generally believed that the assemblage of healthy gut flora is achieved through vertical transmission of by breastfeeding in early infancy. There is limited evidence for the difference in the composition of beneficial bacteria across different mother-infant cohorts and the presence of population-specific microbial taxa. [Objective] To investigate Lactobacillus spp. and the vertical transmission and genetic differences of the dominant species Lacticaseibacillus paracasei among mother-infant cohorts of different ethnic groups, providing a theoretical basis for developing personalized probiotic regimens. [Methods] Lactobacillus strains were isolated from 39 mother-infant pairs of three ethnic groups without mixed marriage in China and identified by repetitive extragenic palindromic PCR (rep-PCR) and groEL sequences. The genetic differences of 83 strains of L. paracasei, a representative species, were analyzed by multilocus sequence typing (MLST). [Results] The species and abundance of Lactobacillus varied among the mother-infant pairs of different ethnic groups. A total of 945 Lactobacillus strains were isolated, belonging to 15 species of 4 genera. L. rhamnosus (20.07%), L. paracasei (16.54%), and L. casei (11.90%) were dominant species in the Han ethnic group, while L. casei (13.55%), L. paracasei (12.69%), and Ligilactobacillus salivarius (11.47%) were dominant bacteria in Uighur ethnic group in Hotan. The dominant species in the Li ethnic group in Hainan were Limosilactobacillus oris (24.55%), L. paracasei (15.85%), and Lactobacillus gasseri (10.87%). The 83 strains of L. paracasei were classified into 11 phylogenetic groups by rep-PCR and 31 sequence types (STs) by MLST, demonstrating ethnic specificity. L. paracasei isolates from the same mother-infant pair had the same STs, and isolates from the mother-infant pairs of the same ethnic group had higher genetic similarity. [Conclusion] Lactobacillus species varied in the mother-infant pairs of different ethnic groups, and L. paracasei strains from the same origin displayed higher genetic similarity, which supported vertical transmission at strain level and ethnic specificity.