The Inhibitory Effect of Magnolol on the Human TWIK1 Channel Is Related to G229 and T225 Sites.

TWIK1 (K2P1.1/KCNK1) belongs to the potassium channels of the two-pore domain. Its current is very small and difficult to measure. In this work, we used a 100 mM NH4+ extracellular solution to increase TWIK1 current in its stable cell line expressed in HEK293. Then, the inhibition of magnolol on TWIK1 was observed via a whole-cell patch clamp experiment, and it was found that magnolol had a significant inhibitory effect on TWIK1 (IC50 = 6.21 ± 0.13 µM). By molecular docking and alanine scanning mutagenesis, the IC50 of TWIK1 mutants G229A, T225A, I140A, L223A, and S224A was 20.77 ± 3.20, 21.81 ± 7.93, 10.22 ± 1.07, 9.55 ± 1.62, and 7.43 ± 3.20 µM, respectively. Thus, we conclude that the inhibition of the TWIK1 channel by magnolol is related to G229 and T225 on the P2- pore helix.

Immune-related genes expression analysis of Western mosquitofish (Gambusia affinis) challenged with Aeromonas hydrophila.

The great Gambusia affinis (G. affinis) is considered as an important animal model to study the endocrine disruption, ecological behavior, and environmental pollutant. The present study aims to build a new promising infection model with Aeromonas hydrophila (A. hydrophila) in aquaculture. The mRNA expression of Rac1, MyD88, IRAK4, TAK1, IKKß, and IL-1ß in G. affinis were significance higher (P < 0.05) in the liver of G. affinis than that of brain and intestine. And the PI3K mRNA expression level was significant lower (P < 0.05) in the intestine than that of brain and liver. The mRNA levels of AKT3 were significant higher (P < 0.05) in the brain than that of liver and intestine. And then the brain, liver, and intestine were collected at different time points (0 h, 3 h, 9 h, 18 h, 24 h, 48 h) after post injection of LD50 of A. hydrophila. The 0.85% NaCl was used as a negative control for the LD50 of A. hydrophila. The RT-PCR results showed that mRNA expressions of TLR2/4 pathway downstream genes MyD88, IRAK4, TAK1, Rac1, IKKß, and IL-1ß were firstly significantly up-regulated (P < 0.05) and were then backed to the 0 h group levels in three tissues. In contrast, mRNA expressions of TLR2/4 pathway downstream genes PI3K and AKT3 were firstly significantly decreased (P < 0.05) and were then increased to the 0 h group levels in brain and intestine. In summary, the results indicated that A. hydrophila could cause inflammatory reaction in intestinal and brain. In addition, the liver showed a provocative reaction when infected with A. hydrophila.

Expression of HSP70 family mRNAs in albino northern snakehead, Channa argus: Response to extreme temperature stress and bacterial infection.

The wild albino northern snakehead, Channa argus, has only been found in Jialing Rivers System of China so far. It is easy to be affected by the environmental factors such as temperature changes and bacterial infection, thus causing a huge economic loss. Therefore, this study cloned a 2,213 bp cDNA that encodes a protein of heat shock protein 70 (CaHSP70), which has an open reading frame (ORF) that encodes 639 amino acids and the corresponding polypeptides of 70.50 kDa. And the oretical isoelectric point (pI) of CaHSP70 is 5.79. Additionally, we also cloned a cDNA for heat shock cognate protein 70 (CaHSC70) with a total length of 2,300 bp. And the ORF of CaHSC70 encodes 648 amino acids and 71.18 kDa peptides. The pI of CaHSC70 is 5.22. Moreover, the cDNA length of stress-70 protein mitochondrial (CaHSPA9) is 2,944 bp with an ORF that encodes 679 amino acids, polypeptides of 73.74 kDa, and a pI of 6.68. The quantitative real-time PCR (qRT-PCR) analysis showed that the mRNA expression levels of CaHSP70, CaHSC70, and CaHSPA9 genes were tissue-specific in the control groups. After the heat shock at 37 °C, the mRNA expression levels of CaHSP70 were extremely significantly upregulated in the kidney, liver, spleen, and brain tissues, while fewer mRNA expression levels of CaHSC70 and CaHSPA9 showed a relatively induction in these tissues. In contrast, after the cold shock at 8.5 °C, fewer mRNA expression levels of CaHSP70, CaHSC70, and CaHSPA9 showed the changes of expression in all the tissues, compared to heat shock. In addition, CaHSP70, CaHSC70, and CaHSPA9 mRNA expression levels showed an overall trend of first upregulating and then falling after Edwardsiella tarda (strain No. DL1,476) challenge. In conclusion, this study demonstrated that temperature had a great effect on the mRNA expression levels of CaHSP70, CaHSC70, and CaHSPA9, and the mRNA expression levels of these three genes were also sensitive to pathogen infection, especially CaHSP70 in the albino C. argus.

Cytochrome P450 1A mRNA in the Gambusia affinis and Response to Several PAHs.

Cytochrome P4501A (CYP1A) has been used as a specific biomarker for monitoring water contamination such as PAHs, PCBs and dioxins. In the present study, the cyp1a gene of Gambusia affinis was cloned and sequenced and their expressions under PAHs exposure were characterized. The newly identified cyp1a encodes a protein with 521 amino acids that shared 96-80% identity with other Cyprinodontiformes fishes. RT-PCR analysis revealed that the basal mRNA level of cyp1a was highly expressed in liver and intestine. The expression level of cyp1a was significantly induced by exposure to 100 µg/L 3, 4-Benzopyrene (BaP) for 5 days in the muscle, testis, brain, liver and intestine of adult male fish. Except in the testis, the induced mRNA level of cyp1a ultimately decreased after prolonging the exposure time to 25 days. As for testis, the induced mRNA level of cyp1a was maintained at a high level during the entire exposure time under 100 µg/L BaP exposure. Furthermore, the expression of cyp1a increased with exposure time under a relatively low exposure concentrations 1 µg/L. Regarding the effects of other PAHs, D(a,h)A, BbF, and BaA showed a statistically significant effect of induction on mRNA level of cyp1a in the muscle, testis, brain, liver and intestine.

Insights Into the Albinism Mechanism for Two Distinct Color Morphs of Northern Snakehead, Channa argus Through Histological and Transcriptome Analyses.

The great northern snakehead (Channa argus) is one of the most important economic and conservational fish in China. In this study, the melanocytes in the skin of two distinct color morphs C. argus were investigated and compared through employment of the microscopic analysis, hematoxylin and eosin (H&E) and Masson Fontana staining. Our results demonstrated the uneven distribution of melanocytes with extremely low density and most of them were in the state of aging or death. Meanwhile, there was no obvious pigment layer and melanocytes distribution pattern found in the albino-type (AT), while the melanocytes were evenly distributed with abundance in the bicolor-type (BT). The transcriptome analysis through Illumina HiSeq sequencing showed that a total of 34.93 Gb Clean Data was obtained, and Q30 base percentage reached 92.66%. The BT and AT northern snakeheads transcriptome data included a total of 56,039,701 and 60,410,063 clean reads (n = 3), respectively. In gene expression analyses, the sample correlation coefficients (r) were ranged between 0.92 and 1.00; the contribution of PC1 and PC2 were 50.25 and 13.73% by using PCA cluster analysis, the total number of DEGs were 1024 (559 up-regulated and 465 down-regulated), and the number of annotated DEGs was 767 (COG 172, KEGG 262, GO 288, SwissProt 548, Pfam 579 and NR 765). Additionally, 46,363 ± 873 and 44,947 ± 392 single nucleotide polymorphisms (SNPs) were compiled via genetic structure analysis, respectively. Ten key pigment-related genes were screened using qRT-PCR. And all of them revealed extremely higher expression levels in the skin of BT than those of AT. This is the first study to analyze the mechanism of albino characteristics of Channa via histology and transcriptomics, and also provide the oretical and practical support for the protection and development of germplasm resources for C. argus.