Structure-activity relationships of the intramolecular disulphide bonds in LEAP2, an antimicrobial peptide from Acrossocheilus fasciatus.

BACKGROUND: The liver-expressed antimicrobial peptide 2 (LEAP2) plays a pivotal role in the host's immune response against pathogenic microorganisms. Numerous such antimicrobial peptides have recently been shown to mitigate infection risk in fish, and studying those harboured by the economically important fish Acrossocheilus fasciatus is imperative for enhancing its immune responses against pathogenic microorganisms. In this study, we cloned and sequenced LEAP2 cDNA from A. fasciatus to examine its expression in immune tissues and investigate the structure-activity relationships of its intramolecular disulphide bonds. RESULTS: The predicted amino acid sequence of A. fasciatus LEAP2 was found to include a signal peptide, pro-domain, and mature peptide. Sequence analysis indicated that A. fasciatus LEAP2 is a member of the fish LEAP2A cluster and is closely related to Cyprinus carpio LEAP2A. A. fasciatus LEAP2 transcripts were expressed in various tissues, with the head kidney exhibiting the highest mRNA levels. Upon exposure to Aeromonas hydrophila infection, LEAP2 expression was significantly upregulated in the liver, head kidney, and spleen. A mature peptide of A. fasciatus LEAP2, consisting of two disulphide bonds (Af-LEAP2-cys), and a linear form of the LEAP2 mature peptide (Af-LEAP2) were chemically synthesised. The circular dichroism spectroscopy result shows differences between the secondary structures of Af-LEAP2 and Af-LEAP2-cys, with a lower proportion of alpha helix and a higher proportion of random coil in Af-LEAP2. Af-LEAP2 exhibited potent antimicrobial activity against most tested bacteria, including Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Staphylococcus warneri. In contrast, Af-LEAP2-cys demonstrated weak or no antibacterial activity against the tested bacteria. Af-LEAP2 had a disruptive effect on bacterial cell membrane integrity, whereas Af-LEAP2-cys did not exhibit this effect. Additionally, neither Af-LEAP2 nor Af-LEAP2-cys displayed any observable ability to hydrolyse the genomic DNA of P. aeruginosa. CONCLUSIONS: Our study provides clear evidence that linear LEAP2 exhibits better antibacterial activity than oxidised LEAP2, thereby confirming, for the first time, this phenomenon in fish.

Metabolomics and microbiome co-analysis reveals altered innate immune responses in Charybdis japonica following Aeromonas hydrophila infection.

A comprehensive bioinformatics analysis was conducted to elucidate the innate immune response of Charybdis japonica following exposure to Aeromonas hydrophila. This study integrated metabolomics, 16S rRNA sequencing, and enzymatic activity data to dissect the immune mechanisms activated in response to infection. Infection with A. hydrophila resulted in an increased abundance of beneficial intestinal genera such as Photobacterium spp., Rhodobacter spp., Polaribacter spp., Psychrilyobacter spp., and Mesoflavibacter spp. These probiotics appear to suppress A. hydrophila colonization by competitively dominating the intestinal microbiota. Key metabolic pathways affected included fatty acid biosynthesis, galactose metabolism, and nitrogen metabolism, highlighting their role in the crab's intestinal response. Enzymatic analysis revealed a decrease in activities of hexokinase, phosphofructokinase, and pyruvate kinase, which are essential for energy homeostasis and ATP production necessary for stress responses. Additionally, reductions were observed in the activities of acetyl-CoA carboxylase and fatty acid synthase. Gene expression analysis showed downregulation in Peroxiredoxin 1 (PRDX1), Peroxiredoxin 2 (PRDX2), glutathione-S-transferase (GST), catalase (CAT), and glutathione (GSH), with concurrent increases in malondialdehyde (MDA) levels, indicating severe oxidative stress. This study provides insights into the molecular strategies employed by marine crabs to counteract bacterial invasions in their natural habitat.

An esculentin-1 homolog from a dark-spotted frog (Pelophylax nigromaculatus) possesses antibacterial and immunoregulatory properties.

BACKGROUND: Esculentin-1, initially discovered in the skin secretions of pool frogs (Pelophylax lessonae), has demonstrated broad-spectrum antimicrobial activity; however, its immunomodulatory properties have received little attention. RESULTS: In the present study, esculentin-1 cDNA was identified by analysing the skin transcriptome of the dark-spotted frog (Pelophylax nigromaculatus). Esculentin-1 from this species (esculentin-1PN) encompasses a signal peptide, an acidic spacer peptide, and a mature peptide. Sequence alignments with other amphibian esculentins-1 demonstrated conservation of the peptide, and phylogenetic tree analysis revealed its closest genetic affinity to esculentin-1P, derived from the Fukien gold-striped pond frog (Pelophylax fukienensis). Esculentin-1PN transcripts were observed in various tissues, with the skin exhibiting the highest mRNA levels. Synthetic esculentin-1PN demonstrated antibacterial activity against various pathogens, and esculentin-1PN exhibited bactericidal activity by disrupting cell membrane integrity and hydrolyzing genomic DNA. Esculentin-1PN did not stimulate chemotaxis in RAW264.7, a murine leukemic monocyte/macrophage cell line. However, it amplified the respiratory burst and augmented the pro-inflammatory cytokine gene (TNF-α and IL-1ß) expression in RAW264.7 cells. CONCLUSIONS: This novel finding highlights the immunomodulatory activity of esculentin-1PN on immune cells.

MAPK CcSakA of the HOG Pathway Is Involved in Stipe Elongation during Fruiting Body Development in Coprinopsis cinerea.

Mitogen-activated protein kinase (MAPK) pathways, such as the high-osmolarity glycerol mitogen-activated protein kinase (HOG) pathway, are evolutionarily conserved signaling modules responsible for transmitting environmental stress signals in eukaryotic organisms. Here, we identified the MAPK homologue in the HOG pathway of Coprinopsis cinerea, which was named CcSakA. Furthermore, during the development of the fruiting body, CcSakA was phosphorylated in the fast elongating apical part of the stipe, which meant that CcSakA was activated in the apical elongating stipe region of the fruiting body. The knockdown of CcSakA resulted in a shorter stipe of the fruiting body compared to the control strain, and the expression of phosphomimicking mutant CcSakA led to a longer stipe of the fruiting body compared to the control strain. The chitinase CcChiE1, which plays a key role during stipe elongation, was downregulated in the CcSakA knockdown strains and upregulated in the CcSakA phosphomimicking mutant strains. The results indicated that CcSakA participated in the elongation of stipes in the fruiting body development of C. cinerea by regulating the expression of CcChiE1. Analysis of the H2O2 concentration in different parts of the stipe showed that the oxidative stress in the elongating part of the stipe was higher than those in the non-elongating part. The results indicated that CcSakA of the HOG pathway may be activated by oxidative stress. Our results demonstrated that the HOG pathway transmits stress signals and regulates the expression of CcChiE1 during fruiting body development in C. cinerea.

Evaluation of the impact of transgenic maize BT799 on growth, development and reproductive function of Sprague-Dawley rats in three generations.

BT799 was Bacillus thuringiensis-genetic modified (GM) maize, and Sprague-Dawley (SD) rats were treated with different diet formulations containing BT799 maize grain (33% and 66%) or its non-transgenic Zhengdan 958 (ZD958, 33% and 66%). The feeding lasted for 10 (P)/14 (F1 and F2) weeks. The reproductive capacity and pathological responses were detected in each generation of rats fed with BT799 and ZD958. During the growth and development of parental rats, each group showed the same trend in body weight gain and food intake, with a few fluctuations at individual time points. No statistically significant difference was observed in reproductive data (copulation index, fertility index, and live birth rate) of rats fed with transgenic maize compared with non-transgenic maize. We observed some apparent changes in reproductive data (sperm numbers and motility) and pathological responses (organ relative weights, hematological parameters, serum chemistry parameters, and sex hormone levels) among rats fed with BT799 maize grain. However, these differences were within the laboratory's historical normal range of control SD rats and not maize grain dose-dependent. These changes were not considered to be adverse or toxic. No significant difference in macroscopic or histological adverse effects was observed between rats consuming transgenic BT799 diet and non-transgenic diet. In conclusion, the long-term intake of BT799 maize was as safe as the corresponding non-transgenic maize for three-generation SD rats.

Complete Genome Sequence of Tsuneonella flava SS-21NJ, a Potential Oil Sludge Bioremediation Agent.

We report here the complete genome sequence of Tsuneonella flava strain SS-21NJ, which was isolated from oil sludge from Shengli Oilfield in Dongying, Shandong Province, China. These results provide basic information for functional genomics and oil degradation research of Tsuneonella strains.

Chelatococcus composti sp. nov., isolated from penicillin fermentation fungi residue with pig manure co-compost.

A novel Gram-stain-negative bacterium, designated strain PC-2T, was isolated from penicillin fermentation fungi residue with pig manure co-compost in China. Phylogenetic analysis, based on 16S rRNA gene sequence comparisons, revealed that strain PC-2T should be assigned to the genus Chelatococcus and that it had 98.9 % similarity with Chelatococcus daeguensis, 98.8 % with Chelatococcus sambhunathii, 98.4 %, with Chelatococcus caeni and 96.0 % with Chelatococcus asaccharovorans. The G+C content of genomic DNA was 70.9 mol%. On the basis of the phylogenetic analysis, DNA-DNA relatedness values, phenotypic characteristics and chemotaxonomic data, strain PC-2 T represents a novel species of the genus Chelatococcus, for which the name Chelatococcus composti sp. nov. is proposed. The type strain is PC-2T (=DSM 101465T=CGMCC 1.15283T).

Investigating Pollen and Gene Flow of WYMV-Resistant Transgenic Wheat N12-1 Using a Dwarf Male-Sterile Line as the Pollen Receptor.

Pollen-mediated gene flow (PMGF) is the main mode of transgene flow in flowering plants. The study of pollen and gene flow of transgenic wheat can help to establish the corresponding strategy for preventing transgene escape and contamination between compatible genotypes in wheat. To investigate the pollen dispersal and gene flow frequency in various directions and distances around the pollen source and detect the association between frequency of transgene flow and pollen density from transgenic wheat, a concentric circle design was adopted to conduct a field experiment using transgenic wheat with resistance to wheat yellow mosaic virus (WYMV) as the pollen donor and dwarf male-sterile wheat as the pollen receptor. The results showed that the pollen and gene flow of transgenic wheat varied significantly among the different compass sectors. A higher pollen density and gene flow frequency was observed in the downwind SW and W sectors, with average frequencies of transgene flow of 26.37 and 23.69% respectively. The pollen and gene flow of transgenic wheat declined dramatically with increasing distance from its source. Most of the pollen grains concentrated within 5 m and only a few pollen grains were detected beyond 30 m. The percentage of transgene flow was the highest where adjacent to the pollen source, with an average of 48.24% for all eight compass directions at 0 m distance. Transgene flow was reduced to 50% and 95% between 1.61 to 3.15 m, and 10.71 to 20.93 m, respectively. Our results suggest that climate conditions, especially wind direction, may significantly affect pollen dispersal and gene flow of wheat. The isolation-by-distance model is one of the most effective methods for achieving stringent transgene confinement in wheat. The frequency of transgene flow is directly correlated with the relative density of GM pollen grains in air currents, and pollen competition may be a major factor influencing transgene flow.

Evaluation of aerobic co-composting of penicillin fermentation fungi residue with pig manure on penicillin degradation, microbial population dynamics and composting maturity.

Improper treatment of penicillin fermentation fungi residue (PFFR), one of the by-products of penicillin production process, may result in environmental pollution due to the high concentration of penicillin. Aerobic co-composting of PFFR with pig manure was determined to degrade penicillin in PFFR. Results showed that co-composting of PFFR with pig manure can significantly reduce the concentration of penicillin in PFFR, make the PFFR-compost safer as organic fertilizer for soil application. More than 99% of penicillin in PFFR were removed after 7-day composting. PFFR did not affect the composting process and even promote the activity of the microorganisms in the compost. Quantitative PCR (qPCR) indicated that the bacteria and actinomycetes number in the AC samples were 40-80% higher than that in the pig-manure compost (CK) samples in the same composting phases. This research indicated that the aerobic co-composting was a feasible PFFR treatment method.

Biotransformation of thianicotinyl neonicotinoid insecticides: diverse molecular substituents response to metabolism by bacterium Stenotrophomonas maltophilia CGMCC 1.1788.

The carbon atom that neighbors the tertiary amine attached to the 6-chloro-3-pyridinylmethyl moiety is the key active site in the hydroxylation of the neonicotinoids imidacloprid and thiacloprid as well as in the demethylation of acetamiprid by Stenotrophomonas maltophilia CGMCC 1.1788. In this study, thianicotinyl neonicotinoid insecticides having diverse molecular substituents were biotransformed by S. maltophilia CGMCC 1.1788. The results indicated that the substitution of 6-chloropyridyl in imidacloprid with 2-chlorothiazol in imidaclothiz did not affect the hydroxylation of imidaclothiz and its hydroxylated site, while the oxadiazinane ring in thiamethoxam was not hydroxylated or opened. Moreover, the N-methyl group in clothianidin and thiamethoxam was not demethylated by S. maltophilia CGMCC 1.1788. The biotransformation of imidaclothiz was inhibited by piperonyl butoxide, implying that both hydroxylation and dehydrogenation are mediated by a P450 monooxygenase. The bioassay results suggested that the activity of 5-hydroxy and olefin imidaclothiz was similar but less than that of imidaclothiz against the horsebean aphid Aphis craccivora and mosquito larva Culex pipiens, while 5-hydroxy IMT showed weak activity against the brown planthopper Nilaparvata lugens.

Permeabilization of Microbacterium oxylans shifts the conversion of puerarin from puerarin-7-O-glucoside to puerarin-7-O-fructoside.

The main product of the conversion of puerarin by unpermeabilized cells of bacterium Microbacterium oxydans CGMCC 1788 was puerarin-7-O-glucoside (241 +/- 31.9 microM). Permeabilization with 40% ethanol could not increase conversion yield, whereas it resulted in change of main product; a previous trace product became a main product (213 +/- 48.0 microM) which was identified as a novel puerarin-7-O-fructoside by electrospray ionization time-of-flight MS, (13)C NMR, (1)H NMR, and GC-MS analysis of sugar composition, and puerarin-7-O-glucoside became a trace product (14.8 +/- 5.4 microM). However, the extract from cells of M. oxydans CGMCC 1788 permeabilized with ethanol converted puerarin to form 113.9 +/- 27.7 microM puerarin-7-O-glucoside and 187.8 +/- 29.5 microM puerarin-7-O-fructoside under the same conditions. When unpermeabilized intact cells were recovered and used repeatedly for the conversion of puerarin, with increase of reuse times, the yield of puerarin-7-O-glucoside gradually decreased, whereas the yield of puerarin-7-O-fructoside increased gradually in the conversion mixture. The main product of the conversion of puerarin by the tenth recycled unpremerbilized cells was puerarin-7-O-fructoside (288.4 +/- 24.0 microM). Therefore, the change of permeability of cell membrane of bacterium M. oxydans CGMCC 1788 contributed to the change of conversion of the product's composition.

Preparation of a super-long two column chromatography system and its application in separating glycosylated puerarin.

Separation of Puerarin-7-O-glucoside from its precursor, puerarin, using a common chromatography column packed with AB-8 macroporous resin was unsuccessful. Therefore, in this study a 8 m super-long flexible reinforced PVC column was externally added to the common column in order to improve the chromatography efficiency by increasing the number of theoretical plates. Both the PVC and common columns were separately packed with AB-8 macroporous resin slurry. The packed PVC column was coiled after washing and stored until use. The microbial transformation mixture with puerarin-7-O-glucoside and puerarin (250 mL) was loaded onto the common column, followed by washing with 2000 mL H(2)O. After attaching the coiled external PVC column to the common column, a linear gradient of 10-30% ethanol was applied to elute the target compound. Two peaks appeared: peak I contained puerarin-7-O-glucoside at 97.9% purity and 88.1% recovery rate, and peak II was puerarin at 98.7% purity and 87.0% recovery rate. The use of the coiled external flexible reinforced PVC column avoided spatial restriction for long columns, which made it much more convenient for column packing and chromatography operations. Furthermore, this method eliminated the resin blockage problem caused by stationary water pressure in a rigid vertical long column. Using an external super-long column, the PVC tube was connected with the common column only during elution, which avoided delay in time period during sample loading and column washes associated with the use of long external columns.

Hydroxylation of thiacloprid by bacterium Stenotrophomonas maltophilia CGMCC1.1788.

Chloropyridinyl neonicotinoid insecticides play a major role in crop protection and flea control on cats and dogs. Imidacloprid, thiacloprid and acetamiprid have in common the 6-chloro-3-pyridinylmethyl group but differ in the nitroguanidine or cyanoamidine substituent on an acyclic or cyclic moiety. Our previous study found that Stenotrophomonas maltophilia CGMCC 1.1788 could hydroxylate imidacloprid to 5-hydroxy imidacloprid, and 5-hydroxy imidacloprid was easily converted to 10-19 times higher insecticidal olefin imidacloprid against aphid or whitefly. Acetamiprid could be transformed by S. maltophilia to form N-demethylation product(IM 2-1). In this paper, we examined S. maltophilia CGMCC 1.1788's ability of transformation of thiacloprid. S. maltophilia CGMCC 1.1788 can hydroxylate thiacloprid to 4-hydroxy thiacloprid characterized by HPLC-MS/MS and NMR analysis, however 4-hydroxy thiacloprid could not be converted to olefin thiacloprid under acid conditions like imidacloprid, whereas oxidized and decyonated simultaneously to form 4-ketone thiacloprid imine in alkaline solution. Bioassays indicated that 4-hydroxy thiacloprid had 156 times lower insecticidal activity than thiacloprid, and the ketone-imine derivative almost had no toxicity towards aphid. Though both imidacloprid and thiacloprid are hydroxylated by S. maltophilia CGMCC 1.1788 at the same carbon atom position, however the structural difference between in imidacloprid and thiacloprid, originate the entire discrepancy in bioefficacy of metabolite and its further degrading pathway. These results explain that why thiacloprid is classified as not relevant grade for soil and seed applications, whereas imidacloprid is recommended and acetamiprid is limited.