Methods: We have employed high-throughput RNA-Seq technology to uncover the transcriptome changes of P. monodon hepatopancreas when challenged with VpAHPND. The shrimps were challenged with VpAHPND through immersion method with dissected hepatopancreas samples for the control group (APm-CTL) and treatment group at 3 (APm-T3), 6 (APm-T6), and 24 (APm-T24) hours post-AHPND infection sent for RNA-Seq. The transcriptome de novo assembly and Unigene expression determination were conducted using Trinity, Tgicl, Bowtie2, and RSEM software. The differentially expressed transcripts were functionally annotated mainly through COG, GO, and KEGG databases.
Results: The sequencing reads generated were filtered to obtain 312.77 Mb clean reads and assembled into 48662 Unigenes. Based on the DEGs pattern identified, it is inferred that the PAMPs carried by VpAHPND or associated toxins are capable of activating PRRs, which leads to subsequent pathway activation, transcriptional modification, and antibacterial responses (Phagocytosis, AMPs, proPO system). DAMPs are released in response to cell stress or damage to further activate the sequential immune responses. The comprehensive interactions between VpAHPND, chitin, GbpA, mucin, chitinase, and chitin deacetylase were postulated to be involved in bacterial colonization or antibacterial response.
Conclusions: The outcomes of this research correlate the different stages of P. monodon immune response to different time points of AHPND infection. This finding supports the development of biomarkers for the detection of early stages of VpAHPND colonization in P. monodon through host immune expression changes. The potential genes to be utilized as biomarkers include but not limited to C-type lectin, HMGB1, IMD, ALF, serine proteinase, and DSCAM.
Methods: In this study, the dystrophin gene from M. rosenbergii (MrDys) was first characterised followed by the characterization of dystrophin gene from a closely related shrimp species, Penaeus monodon (PmDys). Dystrophin sequences from different phyla were then used for evolutionary comparison through BLAST analysis, conserved domain analysis and phylogenetic analysis. The changes in mRNA expression levels of dystrophin and the alteration of intracellular calcium concentrations in WSSV infected muscle cells were then studied.
Results: A 1246 base pair long dystrophin sequence was identified in the giant freshwater prawn, Macrobrachium rosenbergii (MrDys) followed by 1082 base pair long dystrophin sequence in P. monodon (PmDys). Four conserved domains were identified from the thirteen dystrophin sequences compared which were classified into 5 different phyla. From the phylogenetic analysis, aside from PmDys, the characterised MrDys was shown to be most similar to the invertebrate phylum of Nematoda. In addition, an initial down-regulation of dystrophin gene expression followed by eventual up-regulation, together with an increase in intracellular calcium concentration [Ca2+]
i
were shown upon WSSV experimental infection.
Discussion: Both the functionality of the dystrophin protein and the intracellular calcium concentration were affected by WSSV infection which resulted in progressive muscle degeneration. An increased understanding of the role of dystrophin-calcium in MrDys and the interactions between these two components is necessary to prevent or reduce occurrences of muscle degeneration caused by WSSV infection, thereby reducing economic losses in the prawn farming industry from such disease.
METHODOLOGY/PRINCIPAL FINDINGS: Four ligands (1-4) and their respective nickel-containing complexes (5-8) were synthesized and characterized. The compounds synthesized were tested for their effects on NF-κB nuclear translocation, pro-inflammatory cytokines secretion and NF-κB transactivation activity. The active compound was further evaluated on its ability to suppress carrageenan-induced acute inflammation in vivo. A potential binding target of the active compound was also predicted by molecular docking analysis.
CONCLUSIONS/SIGNIFICANCE: Among all synthesized compounds tested, we found that complex [Ni(H2L1)(PPh3)]Cl (5) (complex 5), potently inhibited IκBα degradation and NF-κB p65 nuclear translocation in LPS-stimulated RAW264.7 cells as well as TNFα-stimulated HeLa S3 cells. In addition, complex 5 significantly down-regulated LPS- or TNFα-induced transcription of NF-κB target genes, including genes that encode the pro-inflammatory cytokines TNFα, IFNβ and IL6. Luciferase reporter assays confirmed that complex 5 inhibited the transactivation activity of NF-κB. Furthermore, the anti-inflammatory effect of complex 5 was also supported by its suppressive effect on carrageenan-induced paw edema formation in wild type C57BL/6 mice. Interestingly, molecular docking study showed that complex 5 potentially interact with the active site of IKKβ. Taken together, we suggest complex 5 as a novel NF-κB inhibitor with potent anti-inflammatory effects.