Three novel C1q domain containing proteins from the disk abalone Haliotis discus discus: Genomic organization and analysis of the transcriptional changes in response to bacterial pathogens

Bathige, S.D.N.K.,Umasuthan, N.,Jayasinghe, J.D.H.E.,Godahewa, G.I.,Park, H.C.,Lee, J. Academic Press 2016 FISH AND SHELLFISH IMMUNOLOGY Vol.56 No.-

<P>The globular C1q (gC1q) domain containing proteins, commonly referred as C1q domain containing (C1qDC) proteins, are an essential family of proteins involved in various innate immune responses. In this study, three novel C1qDC proteins were identified from the disk abalone (Haliotis discus discus) transcriptome database and designated as AbC1qDC1, AbC1qDC2, and AbC1qDC3. The cDNA sequences of AbC1qDC1, AbC1qDC2, and AbC1qD0 consisted of 807, 1305, and 660 bp open reading frames (ORFs) encoding 269, 435, and 220 amino acids (aa), respectively. Putative signal peptides and the N-terminal gC1q domain were identified in all three AbC1qDC proteins. An additional predicted motif region, known as the coiled coil region (CCR), was identified next to the signal sequence of AbC1qDC2. The genomic organization of the AbC1qDCs was determined using a bacterial artificial chromosome (BAC) library. It was found that the CDS of AbC1qDC1 was distributed among three exons, while the CDSs of AbC1qDC2 and AbC1qDC3 were distributed between two exons. Sequence analysis indicated that the AbC1qDC proteins shared <40% identity with other counterparts from different species. According to the neighbor joining phylogenetic tree, the proteins were grouped within an invertebrate group with high evolutionary distances, which suggests that they are new members of the C1qDC family. Higher expression of AbC1qDC1 and AbC1qDC2 was detected in hepatopancreas, muscle, and mantle tissues compare to the other tissues analyzed, using reverse transcription, followed by quantitative real-time PCR (qPCR) using SYBR Green, whereas AbC1qDC3 was predominantly expressed in gill tissues, followed by muscles and the hepatopancreas. The temporal expression of AbC1qDC transcripts in gills after bacterial (Vibrio parahaemolyticus and Listeria monocytogenes) and lipopolysaccharide stimulation indicated that AbC1qDCs can be strongly induced by both Gram-negative and Gram-positive bacterial species with different response profiles. The results of this study suggest that AbC1qDCs are involved in immune responses against invading bacterial pathogens. (C) 2016 Published by Elsevier Ltd.</P>

A homolog of teleostean signal transducer and activator of transcription 3 (STAT3) from rock bream, <i>Oplegnathus fasciatus</i>: Structural insights, transcriptional modulation, and subcellular localization

Bathige, S.D.N.K.,Thulasitha, William Shanthakumar,Umasuthan, Navaneethaiyer,Jayasinghe, J.D.H.E.,Wan, Qiang,Nam, Bo-Hye,Lee, Jehee Elsevier 2017 Veterinary immunology and immunopathology Vol.186 No.-

<P><B>Abstract</B></P> <P>Signal transducer and activator of transcription 3 (STAT3) is one of the crucial transcription factors in the Janus kinase (JAK)/STAT signaling pathway, and it was previously considered as acute phase response factor. A number of interleukins (ILs) such as IL-5, IL-6, IL-9, IL-10, IL-12, and IL-22 are known to be involved in activation of STAT3. In addition, various growth factors and pathogenic or oxidative stresses mediate the activation of a wide range of functions via STAT3. In this study, a STAT3 homolog was identified and functionally characterized from rock bream (RbSTAT3), <I>Oplegnathus fasciatus</I>. <I>In silico</I> characterization revealed that the RbSTAT3 amino acid sequence shares highly conserved common domain architectural features including N-terminal domain, coiled coil domain, DNA binding domain, linker domain, and Src homology 2 (SH2) domains. In addition, a fairly conserved transcriptional activation domain (TAD) was located at the C-terminus. Comparison of RbSTAT3 with other counterparts revealed higher identities (>90%) with fish orthologs. The genomic sequence of <I>RbSTAT3</I> was obtained from a bacterial artificial chromosome (BAC) library, and was identified as a multi-exonic gene (24 exons), as found in other vertebrates. Genomic structural comparison and phylogenetic studies have showed that the evolutionary routes of teleostean and non-teleostean vertebrates were distinct. Quantitative real time PCR (qPCR) analysis revealed that the spatial distribution of <I>RbSTAT3</I> mRNA expression was ubiquitous and highly detectable in blood, heart, and liver tissues. Transcriptional modulation of <I>RbSTAT3</I> was examined in blood and liver tissues after challenges with bacteria (<I>Edwardsiella tarda</I> and <I>Streptococcus iniae</I>), rock bream irido virus (RBIV), and immune stimulants (LPS and poly (I:C)). Significant changes in <I>RbSTAT3</I> transcription were also observed in response to tissue injury. In addition, the transcriptional up-regulation of <I>RbSTAT3</I> was detected in rock bream heart cells upon recombinant rock bream IL-10 (rRbIL-10) treatment. Subcellular localization and nuclear translocation of rock bream STAT3 following poly (I:C) treatment were also demonstrated. Taken together, the results of the current study provide important evidence for potential roles of rock bream STAT3 in the immune system and wound healing processes.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Identification of STAT3 (RbSTAT3) ortholog from rock bream. </LI> <LI> Genomic structure and putative promoter analysis of <I>RbSTAT3</I>. </LI> <LI> Expression profile (<I>In vivo</I>) of <I>RbSTAT3</I> upon pathogenic and PAMP stress, and tissue injury. </LI> <LI> Expression profile (<I>In vitro</I>) of <I>RbSTAT3</I> in response to rock bream IL-10. </LI> <LI> Subcellular localization of RbSTAT3. </LI> </UL> </P>

A TELEOST SPECIFIC GLUTATHIONE S-TRANSFERASE FAMILY PROTEIN FROM BLACK ROCKFISH

J.D.H.E. Jayasinghe,S.D.N.K. Bathige,Jehee Lee 한국수산과학회 양식분과 2015 한국수산과학회 양식분과 학술대회 Vol.2015 No.5

A cytosolic JAK/STAT member from rock bream, Oplegnathus fasciatus

S.D.N.K Bathige,William Shanthakumar Thulasith,Navaneethaiyer Umasuthan,G.I Godahewa,J.D.H.E. Jayasinghe,Jehee Lee 한국수산과학회 양식분과 2015 한국수산과학회 양식분과 학술대회 Vol.2015 No.10

MOLECULAR INSIGHTS AND POTENT IMMUNE RESPONSES OF THREE NOVEL C1q DOMAIN CONTAINING (C1qDC) PROTEINS FROM DISK ABALONE Haliotis discus discus

S.D.N.K. Bathige,Navaneethaiyer Umasuthan,J.D.H.E. Jayasinghe,G.I. Godahewa,Jehee Lee 한국수산과학회 양식분과 2015 한국수산과학회 양식분과 학술대회 Vol.2015 No.5

CONTROLLED ELECTRIC FIELD-ASSISTED JETTING FROM VISCOUS AND NANOSUSPENSION MEDIA DROPLETS

S. N. JAYASINGHE 성균관대학교(자연과학캠퍼스) 성균나노과학기술원 2006 NANO Vol.1 No.3

This paper reports a study into forming a jet-on-demand to continuous microthreads by subjecting electric fields on high viscosity and low conducting media (concentrated nanosuspensions and dielectric mediums) droplets, placed on a conducting copper plate, which has a similar plate above at a distance of ~ 10 mm. The media used in this investigation has a viscosity ≫ 1000 mPa s and an electrical conductivity ≪ 10-6 Sm-1 and in the case of nanomaterial loading in suspension is 003E; 15 wt.%. The investigation illustrates both the ability to form jets in this configuration and the importance of the volume of media placed as a droplet which has a direct result on the formation of a jet subsequently fragmentating to droplets. At a droplet volume of < Q0, the resting droplet when under the influence of an applied electric field deforms forming a cone, much like those referred to as the "Taylor Cones". On increasing the volume of the droplet to Q0 and applying a voltage of ~ 4.6 kV across the plates, the apex of the cone was observed to pulsate. On further increasing the applied voltage, giving rise to an electric field strength of ~ 0.55 kV/mm, the pulsating apex stabilizes to evolve a stable jet which undergoes instabilities promoting the generation of droplets. Consequently, a fine jet-on-demand is obtained. On increasing the droplet volume to > Q0, forms jets on both plates. The study elucidates the importance of this jetting approach for forming droplet relics containing self-assembled nanoparticles to continuous microthreads from concentrated nanosuspensions and dielectric media for forming structures by deposition that are most useful and have widespread applications in materials science and engineering. Hence, the physical behavior of this droplet deformation — jetting — forming droplets under an imposed field, outlines the discussion presented in this paper.

A cytosolic JAK/STAT member from rock bream, Oplegnathus fasciatus: molecular characteristics and transcription profiles upon pathogenic stress

S.D.N.K Bathige,William Shanthakumar Thulasitha,Navaneethaiyer Umasuthan,G.I Godahewa,J.D.H.E. Jayasinghe,Jehee Lee 한국수산해양기술학회(구 한국어업기술학회) 2015 한국수산해양기술학회 학술발표대회 Vol.2015 No.10

Molecular cloning, transcriptional profiling, and subcellular localization of signal transducer and activator of transcription 2 (STAT2) ortholog from rock bream, Oplegnathus fasciatus

Bathige, S.D.N.K.,Umasuthan, N.,Priyathilaka, T.T.,Thulasitha, W.S.,Jayasinghe, J.D.H.E.,Wan, Q.,Nam, B.H.,Lee, J. Elsevier/North-Holland 2017 Gene Vol.626 No.-

Signal transducer and activator of transcription 2 (STAT2) is a key element that transduces signals from the cell membrane to the nucleus via the type I interferon-signaling pathway. Although the structural and functional aspects of STAT proteins are well studied in mammals, information on teleostean STATs is very limited. In this study, a STAT paralog, which is highly homologous to the STAT2 members, was identified from a commercially important fish species called rock bream and designated as RbSTAT2. The RbSTAT2 gene was characterized at complementary DNA (cDNA) and genomic sequence levels, and was found to possess structural features common with its mammalian counterparts. The complete cDNA sequence was distributed into 24 exons in the genomic sequence. The promoter proximal region was analyzed and found to contain potential transcription factor binding sites to regulate the transcription of RbSTAT2. Phylogenetic studies and comparative genomic structure organization revealed the distinguishable evolution for fish and other vertebrate STAT2 orthologs. Transcriptional quantification was performed by SYBR Green quantitative real-time PCR (qPCR) and the ubiquitous expression of RbSTAT2 transcripts was observed in all tissues analyzed from healthy fish, with a remarkably high expression in blood cells. Significantly (P<0.05) altered transcription of RbSTAT2 was detected after immune challenge experiments with viral (rock bream irido virus; RBIV), bacterial (Edwardsiella tarda and Streptococcus iniae), and immune stimulants (poly I:C and LPS). Antiviral potential was further confirmed by WST-1 assay, by measuring the viability of rock bream heart cells treated with RBIV. In addition, results of an in vitro challenge experiment signified the influence of rock bream interleukin-10 (RbIL-10) on transcription of RbSTAT2. Subcellular localization studies by transfection of pEGFP-N1/RbSTAT2 into rock bream heart cells revealed that the RbSTAT2 was usually located in the cytoplasm and translocated near to the nucleus upon poly I:C administration. Altogether, these findings suggest that RbSTAT2 is involved in various biologically crucial mechanisms, and provides immune protection to the rock bream.

A homolog of Kunitz-type serine protease inhibitor from rock bream, Oplegnathus fasciatus: Molecular insights and transcriptional modulation in response to microbial and PAMP stimulation, and tissue injury

Bathige, S.D.N.K.,Umasuthan, N.,Godahewa, G.I.,Jayasinghe, J.D.H.E.,Whang, I.,Noh, J.K.,Lee, J. Academic Press 2015 FISH AND SHELLFISH IMMUNOLOGY Vol.46 No.2

Serine proteases and their inhibitors play vital roles in diverse biological processes. In this study, we identified and characterized cDNA coding for a Kunitz-type serine protease inhibitor (SPI), which we designated as RbKSPI, in a commercially important species, rock bream. The full-length cDNA sequence of RbKSPI consisted of 2452 bp with an open reading frame (ORF) of 1521 bp encoding a polypeptide of 507 amino acid (aa) residues. In the RbKSPI protein, MANEC, PKD, LDLa, and two Kunitz domains responsible for various functions were identified as characteristic features. Homology analysis revealed that RbKSPI shared the highest identity with the Kunitz homolog in Takifugu rubripes (77.6%). Phylogenetic analysis indicated that RbKSPI clusters with other teleostean KSPIs. In tissue-specific expression analysis, RbKSPI transcripts were detected in all the tested tissues, with the highest expression in gill tissue, followed by kidney and intestine. The mRNA expression of RbKSPI significantly increased in blood cells upon stimulation with two strains of bacteria (Edwardsiella tarda and Streptococcus iniae) and two pathogen-associated molecular patterns (PAMPs; LPS and poly I:C). Meanwhile, down-regulated expression of RbKSPI was observed in response to tissue injury. Collectively, these results suggest that the RbKSPI may be involved in essential immune defense against microbial pathogens and in the wound-healing process.

Molecular insights of two STAT1 variants from rock bream (Oplegnathus fasciatus) and their transcriptional regulation in response to pathogenic stress, interleukin-10, and tissue injury

Bathige, S.D.N.K.,Umasuthan, N.,Godahewa, G.I.,Thulasitha, W.S.,Jayasinghe, J.D.H.E.,Wan, Q.,Lee, J. Academic Press 2017 Fish & shellfish immunology Vol.69 No.-

Signal transducers and activators of transcription 1 (STAT1) is critically involved in mediating cytokine-driven signaling, and triggers the transcription of target genes to activate cellular functions. Although the structural and functional aspects of STAT members have been well described in mammals, only limited information is available for the STAT genes in teleost fishes. In the present study, two variants of STAT1 genes (RbSTAT1 and RbSTAT1L) were identified from rock bream and characterized at the cDNA and genomic sequence levels. RbSTAT1 and RbSTAT1L were found to share a common domain architecture with mammalian STAT1. Phylogenetic analysis revealed that RbSTAT1 shows a common evolutionary trajectory with other STAT1 counterparts, whereas RbSTAT1L showed a separate path, implying that it could be a novel member of the STAT family. The genomic organizations of RbSTAT1 and RbSTAT1L illustrated a similar exon-intron pattern with 23 exons in the coding sequence. Transcription factor-binding sites, which are mostly involved in the regulation of immune responses, were predicted at the putative promoter regions of the RbSTAT1 and RbSTAT1L genes. SYBR Green qPCR analysis revealed the ubiquitous expression of RbSTAT1 and RbSTAT1L transcripts in different fish tissues with the highest level observed in peripheral blood cells. Significantly modulated transcripts were noted upon viral (rock bream iridovirus [RBIV]), bacterial (Edwardsiella tarda and Streptococcus iniae), and pathogen-associated molecular pattern (lipopolysaccharide and poly I:C) stimulations. The WST-1 cell viability assay affirmed the potential antiviral capacity of RbSTAT1 and RbSTAT1L against RBIV. A possible role of RbSTAT1 and RbSTAT1L in the wound healing process was revealed according to their modulated expression in injured fish. In addition, the transcriptional regulation of RbSTAT1 and RbSTAT1L was analyzed by qPCR following stimulation with rock bream interleukin-10. Taken together, these findings suggest that the STAT1-mediated Janus kinase/STAT pathway might at least in part be involved in the regulatory mechanisms underlying the immune defensive roles against microbial pathogens and the wound healing process.