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In both underwater and semi-aquatic environments, the gut microbiota is of particular physiological importance for amphibious animals, given that the gut tract is among those organs in direct communication with the external environment. In this study, we examined the effects of these contrasting environments on the dominant bacteria in the guts of the amphibious mudskipper Boleophthalmus pectinirostris. Compared with the guts of normal mudskippers, in which the dominant bacteria were identified as Vibrio and Faecalibacterium, we found that Acinetobacter, Shigella, and Bacillus predominated in their guts after exposure to the semi-aquatic environment, whereas Escherichia, Bacteroides, and Bacillus were more prevalent in the guts in the underwater environment. The total number of cultured gut bacteria decreased significantly in the semi-aquatic environment. In semi-aquatic mudskippers, we also detected reductions and increases in the length and width of gut villi, respectively, whereas the width of gut villi declined and the number of goblet cells increased significantly in mudskippers maintained underwater. The mRNA expression of multiple gut transporters for glucose, long-chain fatty acids, and amino acids was found to increase markedly in both underwater and semi-aquatic environments, with the expression of most transporters being significantly higher in those mudskippers exposed to an underwater environment. Furthermore, we detected significant increases in the mRNA expression of pro-inflammatory cytokine transcripts in the guts of both underwater and semi-aquatic mudskippers on days 2, 4, and 6 of exposure, whereas the expression of IL-10 and TGFβ mRNA was more pronounced on days 4 and 8, respectively. Comparatively, we found that expression levels of cytokines in the guts of underwater mudskipper were substantially higher than those in the guts of semi-aquatic mudskippers. Collectively, our findings revealed notable differences in the gut microbiota and energy metabolism requirements of mudskippers exposed to underwater and semi-aquatic conditions, thereby providing a theoretical basis explaining the maintenance of a homeostatic state in mudskippers that constantly transition between these contrasting amphibious habitats.

Tetrodotoxin (TTX) is a kind of neurotoxin with great scientific research and medicinal value. Studies have confirmed that microorganism is one of the important sources of TTX. However, the specific biosynthetic and regulatory pathway of TTX production by microorganisms are still unclear. In this paper, out of twelve symbiotic bacteria screened from the wild puffer fish (Fugu rubripes) collected from coastal sea area of Ningbo (Zhejiang, China), a strain named He-1 isolated from the liver of puffer fish was found can produce TTX. It was identified as Bacillus cereus based on physiological, biochemical characteristics and 16S rDNA gene analysis. However, the strain He-2, which is the subculture strain of He-1, cannot produce TTX under the normal culture condition. Further study showed that by adding the metabolite of the He-1, the ability of TTX producing of He-2 could be restored. A peptide compound was obtained by separating and purifying of the metabolites from strain He-1, which could induce the TTX production of strain He-2. This work will provide a new way for further revealing the anabolic pathway of TTX.

Fasting alters the structure of the intestine and its bacterial diversity, affecting the metabolism of three nutrients (sugar, fat, and protein). However, very little information is available on the effects of fasting on fish intestinal structure, bacterial flora, nutrient metabolism, and inflammation. In this study, fasting modified the intestinal bacterial floral compositions and diversity in mudskippers (Boleophthalmus pectinirostris), and altered the dominant flora in both semiaquatic (SA) and underwater (UW) conditions. The length of the intestinal villi and the thickness of the intestinal mucosa/submucosa decreased significantly during fasting. The intestinal mucosa/submucosa thickness of the SA group was obviously thinner than that in the UW group. The constitutive expression of glucose, long-chain fatty acid, and amino acid transporter mRNA was detected by real-time quantitative PCR. Expression was dramatically increased in the intestine after fasting. Interestingly, the expression of most of the transporters mRNA associated with energy metabolism was higher in the SA group than that in the UW group. Fasting also induced a pro-inflammatory response in the early and middle stages and an anti-inflammatory response in the middle and late stages. The fish intestine is more likely to present pro-inflammatory symptoms in SA and anti-inflammatory symptoms in UW. The results demonstrated that fasting plays a crucial role in the modulation of structure and function in the intestine and bacterial flora. The function of the fish intestine recovers more quickly after fasting in SA than that in UW.

The blood clam, Tegillarca granosa, is a benthic filter feeder with a strong capacity to accumulate and tolerate cadmium (Cd). In our previous study, DPEP1 was shown to be significantly up-regulated under Cd stress based on proteomic analysis. To investigate whether DPEP1 is involved in Cd-induced response, the function of DPEP1 in T. granosa was investigated by integrated molecular and protein approaches. Rapid amplification cDNA end (RACE) assay was established to achieve the complete cDNA sequence of DPEP1 from T. granosa. The full-length cDNA of DPEP1 was 1811 bp, and it contained a 1359-bp open reading frame (ORF), including a 22-amino acid signal peptide. qRT-PCR analysis revealed that DPEP1 was expressed in all examined tissues with the highest expression in gills. At the same time, we investigated DPEP1 gene expression changes after Cd stress at different time points over 96 h. We found that the expression of DPEP1 increased upon initial Cd stress, then it was inhibited, and finally, it was maintained at a low level. Moreover, recombinant DPEP1 showed that higher glutathione (GSH) hydrolysis activity in the temperature range of 30–40°C, and its maximum activity was at pH = 6. Additionally, the results of immunohistochemistry also confirmed that DPEP1 protein was expressed in all test tissues with the highest expression in gills. In addition, there was a positive correlation between QRT-PCR and immunohistochemistry. These results suggested that DPEP1 is probably involved in Cd-induced response by balancing GSH.

Recent studies have shown that activation of liver X receptors (LXRs) attenuates the development of atherosclerosis, not only by regulating lipid metabolism but also by suppressing inflammatory signaling. Sphingosine 1-phosphate receptor 2 (S1PR2), an important inflammatory gene product, plays a role in the development of various inflammatory diseases. It was proposed that S1PR2 might be regulated by LXR-α. In the present study, the effect of LXR-α on tumor necrosis factor-α (TNF-α)-induced S1PR2 expression in human umbilical vein endothelial cells (HUVECs) was investigated and the underlying mechanism was explored. The results demonstrated that TNF-α led to an increase in S1PR2 expression and triggered a downregulation of LXR-α expression in HUVECs as well. Downregulation of LXR-α with specific small interfering RNA (siRNA) remarkably enhanced the primary as well as TNF-α-induced expression of S1PR2 in HUVECs. Activation of LXR-α by agonist GW3965 inhibited both primary and TNF-α-induced S1PR2 expression. GW3965 also attenuated S1PR2-induced endothelial barrier dysfunction. The data further showed that TNF-α induced a significant decrease in miR-130a-3p expression. Overexpression of miR-130a-3p with mimic product reduced S1PR2 protein expression, and inhibition of miR-130a-3p by specific inhibitor resulted in an increase in S1PR2 protein expression. Furthermore, activation of LXRs with agonist enhanced the expression of miR-130a-3p, and knockdown of LXR-α by siRNA suppressed miR-130a-3p expression. These results suggest that LXR-α might downregulate S1PR2 expression via miR-130a-3p in quiescent HUVECs. Stimulation of TNF-α attenuates the activity of LXR-α and results in enhanced S1PR2 expression.

It was previously reported that low‑dose sphingosine‑1‑phosphate (S1P) enhanced endothelial barrier integrity, whereas high‑dose S1P induced endothelial monolayer hyperpermeability responses. A number of studies have revealed the underlying molecular mechanisms of the physiological‑dose of S1P on barrier‑protective effect. However, little work has been performed to determine the effect of S1P‑induced endothelial barrier responses. In the present study, the role of moesin phosphorylation in excessive S1P‑induced endothelial hyperpermeability was investigated by western blotting, fluorescence staining and transendothelial electrical resistance detection. It was revealed that S1P induced moesin phosphorylation in a time‑ and concentration‑dependent manner. In addition, it was confirmed that high‑dose S1P‑induced moesin phosphorylation occurred via S1P receptor 2 (S1PR2). Moesin phosphorylation was required for S1P‑induced F‑actin rearrangement and endothelial barrier disruption. The results suggested that the S1PR2‑moesin axis is involved in high‑dose S1P‑induced endothelial barrier responses. The results of the present study may provide novel therapeutic targets for endothelial injury‑associated vascular disorders.

New Findings What is the central question of this study? Why do different doses of sphingosine-1-phosphate (S1P) induce distinct biological effects in endothelial cells? What is the main finding and its importance? S1P at physiological concentrations preserved endothelial barrier function by binding to S1P receptor 1, then triggering Ca2+ release from endoplasmic reticulum through phosphoinositide phospholipase C and inositol triphosphate, and consequently strengthening tight junction and F-actin assembly through Rac1 activation. Excessive S1P induced endothelial malfunction by activating S1P receptor 2 and RhoA/ROCK pathway, causing F-actin and tight junction disorganisation. Extracellular Ca2+ influx was involved in this process. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid in plasma, and its plasma concentration can be adjusted through a complex metabolic process. The alterations in S1P levels and the activation of receptors collaboratively regulate distinct biological effects. This study was performed to investigate comparatively the effect of different concentrations of S1P on endothelial barrier function and to explore the roles of S1P receptors (S1PRs), Rho GTPases and calcium in S1P-induced endothelial responses. Endothelial barrier function was studied using transendothelial electric resistance and a resistance meter in human umbilical vein endothelial cells. Specific agonists or antagonists were applied to control the activation of S1P receptors and the release of calcium from different cellular compartments. The results indicated that at physiological concentrations, S1P preserved endothelial barrier function by binding with S1PR1. The activation of S1PR1 triggered the release of intracellular Ca2+ from the endoplasmic reticulum through the PI–phospholipase C and inositol trisphosphate pathways. Consequently, the Rho GTPase Rac1 was activated, strengthening the assembly of tight junction proteins and F-actin. However, excessive S1P induced endothelial barrier dysfunction by activating S1PR2 followed by the RhoA/RhoA kinase pathway, causing the disorganization of F-actin and the disassembly of the tight junction protein ZO-1. An influx of extracellular Ca2+ was involved in this process. These data suggest that physiological and excessive amounts of S1P induce different responses in human umbilical vein endothelial cells; the activation of the 1PR1–PLC–IP3R–Ca2+–Rac1 pathway governs the low-dose S1P-enhanced endothelial barrier integrity, and the activation of S1PR2–calcium influx–RhoA/ROCK dominates the high-dose S1P-induced endothelial monolayer hyperpermeability response.

The potential enhancement of Pichia membranifaciens by ammonium molybdate (NH(4)Mo) to control blue mould caused by Penicillium expansum on peach fruit was investigated. Combining P. membranifaciens at 1x10(8) cell/ml with 1 mM NH(4)Mo provided a more effective control of blue mould rot than applying the yeast or NH(4)Mo alone. Addition of 1 mM NH(4)Mo significantly increased the growth of P. membranifaciens in peach wounds, but did not affect the population in nutrient yeast dextrose broth medium. The in vitro experiment showed that the combined treatment inhibited spore germination and germ tube elongation of P. expansum in comparison with the treatment of P. membranifaciens or NH(4)Mo alone. Moreover, P. membranifaciens, NH(4)Mo, and the combination of them did not impair the quality parameters including fruit firmness and content of total soluble solids, titratable acidity and vitamin C of peach fruit after 6 days of storage at 20 degrees C. These results suggested that the use of NH(4)Mo is a useful approach to improve the efficacy of P. membranifaciens for postharvest disease control in peach fruit.

Disorders of angiogenesis are related to microangiopathies during the development of diabetic vascular complications, but the effect of advanced glycation end products (AGEs) on angiogenesis and the mechanism has not been completely unveiled. We previous demonstrated that moesin belonging to the ezrin-radixin-moesin (ERM) protein family protein played a critical role in AGE-induced hyper-permeability in human umbilical vein endothelial cells (HUVECs). Here, we investigated the impact of moesin on AGE-induced HUVEC proliferation, migration, and tubulogenesis. Silencing of moesin decreased cell motility and tube formation but not cell proliferation. It also attenuated cellular F-actin reassembly. Further, phosphorylation of threonine at the 558 amino acid residue (Thr 558) in moesin suppressed AGE-induced HUVEC proliferation, migration, and tube formation, while the activating mutation of moesin at Thr 558 enhanced HUVEC angiogenesis. Further, the inhibition of either RhoA activity by adenovirus or ROCK activation with inhibitor Y27632 decreased AGE-induced moesin phosphorylation and subsequently suppressed HUVEC angiogenesis. These results indicate that the Thr 558 phosphorylation in moesin mediates endothelial angiogenesis. AGEs promoted HUVEC angiogenesis by inducing moesin phosphorylation via RhoA/ROCK pathway.

A bacterium, identified as Ochrobactrum intermedium on the basis of phenotypic characterization and 16S rRNA gene analysis, capable of degrading nicotine was isolated and characterized. The strain, DN2, degraded all nicotine in basal medium containing 500–4000 mg nicotine L−1. Analysis of nicotine degradation by cell extracts provided evidence that the nicotine-degrading enzymes were localized in the cell-wall membrane.

Ochrobactrum intermedium DN2 was used to degrade nicotine in tobacco waste extracts. The optimal temperature and pH of nicotine degradation by strain DN2 was 30-37 degrees C and 7.0, respectively. Under these optimal conditions, the average degradation rate of nicotine in a 30L fed-batch culture was 140.5 mg 1(-1) h(-1). The results of this study indicate that strain DN2 may be useful for reducing the nicotine content of reconstituted tobacco.

Response surface methodology (RSM) was used to optimize a protective medium for enhancing the cell viability of Lactobacillus delbrueckii subsp. bulgaricus LB14 during freeze-drying. Using a previous Plackett-Burman design, it was found that sucrose, glycerol, sorbitol and skim milk were the most effective freeze-drying protective agents for L. bulgaricus LB14. A full factorial central composite design was applied to determine the optimum levels of these four protective agents. The experimental data allowed the development of an empirical model (P