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Peroxiredoxin 5 (Prdx5) is the last recognized member of Prdx family. It is a unique, atypical, 2-Cys antioxidant enzyme, protecting cells from death caused by reactive oxygen species (ROS). In this study, the Prdx5 ortholog of Amphiprion clarkii (AcPrdx5) was identified and characterized to explore its specific structural features and functional properties. The open reading frame of AcPrdx5 is 573 bp long and encodes 190 amino acids containing a mitochondrial targeting sequence, thioredoxin domain, and two conserved cysteine residues responsible for antioxidant function. The predicted molecular weight and theoretical isoelectric point of AcPrdx5 are 20.3 kDa and 9.01, respectively. AcPrdx5 sequences were found to be highly conserved across the other orthologs from various organisms and it distinctively clustered within the fish Prdx5 subclade of the phylogenetic tree. The expression of AcPrdx5 was ubiquitously detected among twelve tested tissues, with the highest level in the brain. Furthermore, the mRNA levels of AcPrdx5 in the blood and head-kidney tissues were significantly (p < 0.05) upregulated following polyinosinic-polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi immune challenge. A concentration-dependent antioxidant potential of recombinant AcPrdx5 was observed in insulin disulfide bond reduction, heavy metal detoxification, free radical and hydrogen peroxide (H 2 O 2 ) scavenging assays. Additionally, AcPrdx5 overexpression in fathead minnow (FHM) cells upregulated the antioxidant-associated gene (Rrm1, MAPK, SOD2, and PRDX1) expression after H 2 O 2 treatment, and promoted cell viability upon arsenic (As) exposure. In macrophages, AcPrdx5 overexpression effectively suppressed substantial nitric oxide production under lipopolysaccharide treatment. Collectively, our results suggest that AcPrdx5 may play roles in both antioxidant defense system and innate immune response against pathogenic invasions in A. clarkii., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Thioredoxin domain-containing protein 12 (TXNDC12) is a member of the thioredoxin-like superfamily that contributes to various thiol-dependent metabolic activities in all living organisms. In this research, the TXNDC12 gene from yellowtail clownfish (Amphiprion clarkii) was structurally characterized using in silico tools, assessed for immunological expression, and evaluated for biological activity using recombinant protein and cellular overexpression. The deduced coding sequence of AcTXNDC12 comprised a 522-bp nucleotide, encoding 173 amino acids with a predicted molecular mass of 19.198 kDa. The AcTXNDC12 protein consists of a 66 WCGAC 70 active motif and a 170 GDEL 173 signature. The highest tissue-specific expression of AcTXNDC12 was observed in the brain tissue, with significant modulation observed in the blood and gill tissues following stimulation of polyinosinic: polycytidylic acid, lipopolysaccharides (LPS), and Vibrio harveyi. In functional assays, recombinant AcTXNDC12 protein (rAcTXNDC12) showed insulin disulfide reduction activity, 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) decolorization antioxidant capacity, and ferric (Fe 3+ ) reducing antioxidant potential. Additionally, a significant reduction in nitric oxide production was observed in AcTXNDC12-overexpressed RAW 264.7 cells upon LPS stimulation. Furthermore, genes associated with the regulation of oxidative stress, including nuclear factor erythroid 2-related factor 2 (Nrf2), catalase (Cat), peroxiredoxin 1 (Prx1), and ribonucleotide reductase catalytic subunit M1 (Rrm1) were significantly upregulated in fathead minnow cells overexpressing AcTXNDC12 in response to H 2 O 2 treatment. The scratch wound healing assay demonstrated tissue regeneration and cell proliferation ability upon AcTXNDC12 overexpression. Altogether, the current study elucidated the antioxidant activity, immunological importance, and wound-healing effect of the AcTXNDC12 gene in yellowtail clownfish, providing valuable insights for advancing the aquaculture of A. clarkii fish., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Membrane-associated Ring-CH 5 (MARCH5) is a mitochondrial E3 ubiquitin ligase playing a key role in the regulation of mitochondrial dynamics. In mammals, MARCH5 negatively regulates mitochondrial antiviral signaling (MAVS) protein aggregation during viral infection and hampers downstream type I interferon signaling to prevent excessive immune activation. However, its precise functional role in the teleost immune system remains unclear. This study investigated the molecular characteristics and immune response of the MARCH5 ortholog in Amphiprion clarkii (A. clarkii; AcMARCH5). The predicted AcMARCH5 protein sequence consists of 287 amino acids with a molecular weight of 32.02 kDa and a theoretical isoelectric point of 9.11. It contains four C-terminal transmembrane (TM) domains and an N-terminal RING cysteine-histidine (CH) domain, which directly regulates ubiquitin transfer. Multiple sequence alignment revealed a high level of conservation between AcMARCH5 and its orthologs in other vertebrate species. Under normal physiological conditions, AcMARCH5 showed the highest mRNA expression in the muscle, brain, and kidney tissues of A. clarkii. Upon stimulation with polyinosinic:polycytidylic acid (Poly I:C), lipopolysaccharide (LPS), and Vibrio harveyi, AcMARCH5 expression was drastically modulated. Functional assays showed that overexpression of AcMARCH5 in fathead minnow (FHM) cells downregulated antiviral gene expression, accompanied by enhanced viral hemorrhagic septicemia virus (VHSV) replication. In murine macrophages, AcMARCH5 overexpression markedly reduced the production of pro-inflammatory cytokines in response to poly I:C treatment. Additionally, AcMARCH5 exhibited an anti-apoptotic effect in H 2 O 2 -treated FHM cells. Collectively, these results suggest that AcMARCH5 may play a role in maintaining cellular homeostasis under disease and stress conditions in A. clarkii., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Interferon-related developmental regulator 1 (IFRD1) is a viral responsive gene associated with interferon-gamma. Herein, we identified the IFRD1 gene (EaIFRD1) from red-spotted grouper (Epinephelus akaara), evaluated its transcriptional responses, and investigated its functional features using various biological assays. EaIFRD1 encodes a protein comprising 428 amino acids with a molecular mass of 48.22 kDa. It features a substantial domain belonging to the interferon-related developmental regulator superfamily. Spatial mRNA expression of EaIFRD1 demonstrated the highest expression levels in the brain and the lowest in the skin. Furthermore, EaIFRD1 mRNA expression in grouper tissues exhibited significant modulation in response to immune stimulants, including poly (I:C), LPS, and nervous necrosis virus (NNV) infection. We analyzed downstream gene regulation by examining type Ⅰ interferon pathway genes following EaIFRD1 overexpression. The results demonstrated a significant upregulation in cells overexpressing EaIFRD1 compared to the control after infection with viral hemorrhagic septicemia virus (VHSV). A subcellular localization assay confirmed the nuclear location of the EaIFRD1 protein, consistent with its role as a transcriptional coactivator. Cells overexpressing EaIFRD1 exhibited increased migratory activity, enhancing wound-healing capabilities compared to the control. Additionally, under H 2 O 2 exposure, EaIFRD1 overexpression protected cells against oxidative stress. Overexpression of EaIFRD1 also reduced poly (I:C)-mediated NO production in RAW267.4 macrophage cells. In FHM cells, EaIFRD1 overexpression significantly reduced VHSV virion replication. Collectively, these findings suggest that EaIFRD1 plays a crucial role in the antiviral immune response and immunological regulation in E. akaara., (Copyright © 2024. Published by Elsevier Ltd.)

Background: Poor mental health among young adults in higher education is a growing concern. In recent years, the visibility of racism has sharply risen. Vicarious discrimination is defined as the secondhand witnessing of racism, and given society's increased accessibility to social media and the Internet, addressing indirect violence is urgently needed to inform anti-racism and mental health efforts. The current study examined associations between vicarious discrimination and mental health across a large sample of young college students in the United States., Methods: We analyzed data from the Healthy Minds Study (HMS; 2020-2021; N=130,566) and used multivariable logistic regression to examine whether past-year vicarious discrimination was associated with various mental health outcomes, adjusting for age, gender, race/ethnicity, and direct discrimination., Results: The sample was predominantly white (n=31,438, 63.66%) and female-identifying (n=34,313, 69.49%) with an average age of 21.1 years. Approximately 35.9% of the sample endorsed experiencing vicarious discrimination. Vicarious discrimination was associated with greater depression (OR:1.97; 95% CI: [1.86,2.09], p< 0.001), anxiety (OR:1.82; 95% CI: [1.72,1.92], p<0.001), languishing (OR:1.75; 95% CI: [1.65,1.87], p<0.001), perceived need for treatment (OR:2.24; 95% CI: [2.10,2.40], p<0.001), suicidal ideation (OR:1.86; 95% CI: [1.73,2.01], p<0.001), suicide plan (OR:1.91; 95% CI: [1.71,2.14], p<0.001), suicide attempt (OR:1.89; 95% CI: [1.51,2.36], p<0.001), self-injurious behavior (OR:2.0; 95% CI: [1.88,2.12], p<0.001), and loneliness (OR:1.67; 95% CI: [1.58,1.77], p<0.001)., Discussion: Consistent with growing literature, vicarious discrimination was associated with poorer mental health among young college students. Additional research should investigate moderators, mediators, and interventions to support those who may be impacted indirectly by discrimination., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest or competing interests that may inappropriately influence or bias this work., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Opsoclonus-myoclonus-ataxia syndrome (OMAS) is a rare acquired neurological disorder characterized by opsoclonus, focal or diffuse myoclonus, truncal instability and associated other cerebellar signs and ataxia. While predominantly affecting children, it can rarely manifest in adults and could be associated with infections, paraneoplastic syndrome, drugs or other neurological disorders. We present a case of an elderly gentleman presenting with OMAS associated with a culture-positive urinary tract infection with Escherichia coli , successfully treated with antibiotics and immunoglobulins resulting in significant recovery., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Galectin 8 belongs to the tandem repeat subclass of the galectin superfamily. It possesses two homologous carbohydrate recognition domains linked by a short peptide and preferentially binds to β-galactoside-containing glycol-conjugates in a calcium-independent manner. This study identified Galectin-8-like isoform X1 (PhGal8X1) from red-lip mullet (Planiliza haematocheilus) and investigated its role in regulating fish immunity. The open reading frame of PhGal8X1 was 918bp, encoding a soluble protein of 305 amino acids. The protein had a theoretical isoelectric (pI) point of 7.7 and an estimated molecular weight of 34.078 kDa. PhGal8X1 was expressed in various tissues of the fish, with prominent levels in the brain, stomach, and intestine. PhGal8X1 expression was significantly (p < 0.05) induced in the blood and spleen upon challenge with different immune stimuli, including polyinosinic:polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. The recombinant PhGal8X1 protein demonstrated agglutination activity towards various bacterial pathogens at a minimum effective concentration of 50 μg/mL or 100 μg/mL. Subcellular localization observations revealed that PhGal8X1 was primarily localized in the cytoplasm. PhGal8X1 overexpression in fathead minnow cells significantly (p < 0.05) inhibited viral hemorrhagic septicemia virus (VHSV) replication. The expression levels of four proinflammatory cytokines and two chemokines were significantly (p < 0.05) upregulated in PhGal8X1 overexpressing cells in response to VHSV infection. Furthermore, overexpression of PhGal8X1 exhibited protective effects against oxidative stress induced by H 2 O 2 through the upregulation of antioxidant enzymes. Taken together, these findings provide compelling evidence that PhGal8X1 plays a crucial role in enhancing innate immunity and promoting cell survival through effective regulation of antibacterial, antiviral, and antioxidant defense mechanisms in red-lip mullet., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Peroxiredoxin-1 (Prdx1) is a thiol-specific antioxidant enzyme that detoxifies reactive oxygen species (ROS) and regulates the redox status of cells. In this study, the Prdx1 cDNA sequence was isolated from the pre-established Amphiprion clarkii (A. clarkii) (AcPrdx1) transcriptome database and characterized structurally and functionally. The AcPrdx1 coding sequence comprises 597 bp and encodes 198 amino acids with a molecular weight of 22.1 kDa and a predicted theoretical isoelectric point of 6.3. AcPrdx1 is localized and functionally available in the cytoplasm and nucleus of cells. The TXN domain of AcPrdx1 comprises two peroxiredoxin signature VCP motifs, which contain catalytic peroxidatic (C p -C 52 ) and resolving cysteine (C R -C 173 ) residues. The constructed phylogenetic tree and sequence alignment revealed that AcPrdx1 is evolutionarily conserved, and its most closely related counterpart is Amphiprion ocellaris. Under normal physiological conditions, AcPrdx1 was ubiquitously detected in all tissues examined, with the most robust expression in the spleen. Furthermore, AcPrdx1 transcripts were significantly upregulated in the spleen, head kidney, and blood after immune stimulation by polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharide (LPS), and Vibrio harveyi injection. Recombinant AcPrdx1 (rAcPrdx1) demonstrated antioxidant and DNA protective properties in a concentration-dependent manner, as evidenced by insulin disulfide reduction, peroxidase activity, and metal-catalyzed oxidation (MCO) assays, whereas cells transfected with pcDNA3.1 (+) /AcPrdx1 showed significant cytoprotective function under oxidative and nitrosative stress. Overexpression of AcPrdx1 in fathead minnow (FHM) cells led to a lower viral copy number following viral hemorrhagic septicemia virus (VHSV) infection, along with upregulation of several antiviral genes. Collectively, this study provides insights into the function of AcPrdx1 in defense against oxidative stressors and its role in the immune response against pathogenic infections in A. clarkii., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Galectin 9 (Gal9) is a tandem repeat type ß-galactoside-binding galectin that mediates various cellular biochemical and immunological functions. Many studies have investigated the functional properties of Gal9 in mammals; however, knowledge of fish Gal9 is limited to antibacterial studies. In this context, our aim was to clone Gal9 from Planiliza haematocheilus (PhGal9) and investigate its structural and functional characteristics. We discovered the PhGal9 open reading frame, which was 969 base pairs long and encoded a 322 amino acid protein. PhGal9 had a projected molecular weight of 35.385 kDa but no signal peptide sequence. PhGal9 mRNA was ubiquitously produced in all investigated tissues but was predominant in the intestine, spleen, and brain. Its mRNA expression was increased in response to stimulation by Poly(I:C), LPS, and L. garvieae. The rPhGal9 exhibited a dose-dependent agglutination potential toward gram-positive and gram-negative bacteria at a minimum concentration of 50 μg/mL. Overexpression of PhGal9 promoted M2-like phenotype changes in mouse macrophages, and RT-qPCR analysis of M1 and M2 marker genes confirmed M2 polarization with upregulation of M2 marker genes. In the antiviral assay, the expression levels of Viral Hemorrhagic Septicemia Virus (VHSV) glycoproteins, phosphoproteins, nucleoproteins, non-virion proteins, matrix proteins, and RNA polymerase were significantly reduced in PhGal9-overexpressed cells. Furthermore, the mRNA expression of autophagic genes (sqstm1, tax1bp1b, rnf13, lc3, and atg5) and antiviral genes (viperin) were upregulated in PhGal9 overexpressed cells. For the first time in teleosts, our study demonstrated that PhGal9 promotes M2 macrophage polarization by upregulating M2-associated genes (egr2 and cmyc) and suppressing M1-associated genes (iNOS and IL-6). Furthermore, our results show that exogenous and endogenous PhGal9 prevented VHSV attachment and replication by neutralizing virion and autophagy, respectively. Gal9 may be a potent modulator of the antimicrobial immune response in teleost fish., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Thioredoxin-like protein 1 (TXNL1) is a redox-active protein belonging to the thioredoxin family, which mainly controls the redox status of cells. The TXNL1 gene from Amphiprion clarkii (AcTXNL1) was obtained from a pre-established transcriptome database. The AcTXNL1 is encoded with 289 amino acids and is predominantly localized in the cytoplasm and nucleus. The TXN domain of AcTXNL1 comprises a 34 CGPC 37 motif with redox-reactive thiol (SH-) groups. The spatial distribution pattern of AcTXNL1 mRNA was examined in different tissues, and the muscle was identified as the highest expressed tissue. AcTXNL1 mRNA levels in the blood and gills were significantly increased in response to different immunostimulants. In vitro antioxidant capacity of the recombinant AcTXNL1 protein (rACTXNL1) was evaluated using the ABTS free radical-scavenging activity assay, cupric ion reducing antioxidant capacity assay, turbidimetric disulfide reduction assay, and DNA nicking protection assay. The potent antioxidant activity of rAcTXNL1 exhibited a concentration-dependent manner in all assays. Furthermore, in the cellular environment, overexpression of AcTXNL1 increased cell viability under H 2 O 2 stress and reduced nitric oxide (NO) production induced by lipopolysaccharides (LPS). Collectively, the experimental results revealed that AcTXNL1 is an antioxidant and immunologically important gene in A. clarkii., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cystatins are natural inhibitors of lysosomal cysteine proteases, including cathepsins B, L, H, and S. Cystatin C (CSTC) is a member of the type 2 cystatin family and is an essential biomarker in the prognosis of several diseases. Emerging evidence suggests the immune regulatory roles of CSTC in antigen presentation, the release of different inflammatory mediators, and apoptosis in various pathophysiologies. In this study, the 390-bp cystatin C (HaCSTC) cDNA from big-belly seahorse (Hippocampus abdominalis) was cloned and characterized by screening the pre-established cDNA library. Based on similarities in sequence, HaCSTC is a homolog of the teleost type 2 cystatin family with putative catalytic cystatin domains, signal peptides, and disulfide bonds. HaCSTC transcripts were ubiquitously expressed in all tested big-belly seahorse tissues, with the highest expression in ovaries. Immune challenge with lipopolysaccharides, polyinosinic:polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae caused significant upregulation in HaCSTC transcript levels. Using a pMAL-c5X expression vector, the 14.29-kDa protein of recombinant HaCSTC (rHaCSTC) was expressed in Escherichia coli BL21 (DE3), and its protease inhibitory activity against papain cysteine protease was determined with the aid of a protease substrate. Papain was competitively blocked by rHaCSTC in a dose-dependent manner. In response to viral hemorrhagic septicemia virus (VHSV) infection, HaCSTC overexpression strongly decreased the expression of VHSV transcripts, pro-inflammatory cytokines, and pro-apoptotic genes; while increasing the expression of anti-apoptotic genes in fathead minnow (FHM) cells. Furthermore, HaCSTC overexpression protected VHSV-infected FHM cells against VHSV-induced apoptosis and increased cell viability. Our findings imply the profound role of HaCSTC against pathogen infections by modulating fish immune responses., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Thioredoxins are small ubiquitous redox proteins that are involved in many biological processes. Proteins with thiol-disulfide bonds are essential regulators of cellular redox homeostasis and diagnostic markers for redox-dependent diseases. Here, we identified and characterized the thioredoxin domain-containing protein 12 (EaTXNDC12) gene in red spotted grouper (Epinephelus akaara), evaluated transcriptional responses, and investigated the activity of the recombinant protein using functional assays. EaTXNDC12 is a 19.22-kDa endoplasmic reticulum (ER)-resident protein with a 522-bp open reading frame and 173 amino acids, including a signal peptide. We identified a conserved active motif ( 66 WCGAC 70 ) and ER retention motif ( 170 GDEL 173 ) in the EaTXNDC12 amino acid sequence. Relative EaTXNDC12 mRNA expression was analyzed using 12 different tissues, with the highest expression seen in brain tissue, while skin tissue showed the lowest expression level. Furthermore, mRNA expression in response to immune challenges was analyzed in the head kidney, blood, and gill tissues. EaTXNDC12 was significantly modulated in response to bacterial endotoxin lipopolysaccharide (LPS), nervous necrosis virus (NNV), and polyinosinic:polycytidylic acid (poly(I:C)) challenges in all of the tested tissues. Recombinant EaTXNDC12 (rEaTXNDC12) displayed antioxidant ability in an insulin reductase assay, and a capacity for free radical inhibition in a 2,2-diphenyl-1-picryl-hydrazyl-hydrate assay. In addition, a DNA nicking assay revealed that purified rEaTXNDC12 exhibited concentration-dependent DNA protection activity, while results from 2-hydroxyethyl disulfide and L-dehydroascorbic assays indicated that rEaTXNDC12a possesses reducing ability. Furthermore, fathead minnow (FHM) cells transfected with EaTXNDC12-pcDNA demonstrated significantly upregulated cell survival against H 2 O 2 -induced apoptosis. Collectively, the results of this study strengthen our knowledge of EaTXNDC12 with respect to cellular redox hemostasis and immune regulation in Epinephelus akaara., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Red-spotted grouper (Epinephelus akaara) is a popular aquaculture species with high commercial value in the food industry. However, some infectious diseases may cause mass mortality in cultural practice. Therefore, it is important to understand the immune responses of red-spotted groupers upon pathogenic invasion to develop successful disease prevention mechanisms. Here, we analyzed the transcriptomic profiles of red-spotted grouper head kidney stimulated with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (poly I:C), and nervous necrosis virus (NNV) and identified differentially expressed genes (DEGs) using RNA-sequencing technology. Cluster analysis of the identified DEGs showed DEG distribution in nine separate clusters based on their expression patterns. However, significant upregulation of most DEGs was observed 6 h after poly I:C stimulation. The DEGs were functionally annotated using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, which revealed significant expression of many immune-related signaling pathways, including antiviral, protein translation, cellular protein catabolic process, inflammatory responses, DNA repair, and cell division. Furthermore, selected DEGs were validated by quantitative real-time PCR, confirming the reliability of our findings. Collectively, this study provides insight into the immune responses of red-spotted groupers, thereby expanding the understanding of fish immunity., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Caveolin-1 (Cav-1), a major structural component of caveolae, is involved in various biological functions, such as endocytosis, cholesterol trafficking, transcytosis, signal transduction, and immunity. To date, three caveolin members have been identified in mammals: Cav-1, Cav-2, and Cav-3. In this study, we identified the Cav-1 sequence from Amphiprion clarkii (AcCav-1). The protein is 181 amino acids long, with a molecular weight of 20.73 kDa and a predicted isoelectric point of 5.48. The phylogenetic tree disclosed that AcCav-1 is closely related to teleost fish orthologs and clusters together with vertebrates. It shares the highest identity (99.4%) and similarity (100%) with Amphiprion ocellaris. Subcellular localization assays showed that Cav-1 expressed in the endoplasmic reticulum and cytoplasm. Further, AcCav-1 was ubiquitously expressed in all examined tissues, but most highly in the skin and the spleen. The up and downregulation of AcCav-1 was observed throughout the testing period after in-vivo immunostimulation with lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (poly (I:C), and Vibrio harveyi (V. harveyi). The antiviral assay showed that AcCav-1 overexpression suppresses the replication of the viral hemorrhagic septicemia virus (VHSV) in Fathead minnow cells by activating antiviral genes. Further, LPS induced NO production and H 2 O 2 -mediated oxidative stress assays showed that AcCav-1 is involved in the regulation of oxidative stress. Collectively, these findings suggest the indispensable role of Cav-1 in the immune system of A.clarkii., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Interleukin 17D (IL-17D), a pro-inflammatory cytokine, is a signature cytokine of T helper 17 (Th17) cells. However, studies characterizing the functions of IL-17D in teleost are scarce. Therefore, we aimed to characterize the properties of IL-17D in Amphiprion clarkii. We performed spatial and temporal expression, AcIL-17D-mediated antibacterial and inflammatory gene expression, NFκB pathway-related gene expression analyses, and bacterial colony counting and cell protection assays. We found that AcIL-17D contains a 630 bp coding sequence and encodes 210 amino acids. The spatial expression analysis of AcIL-17D in 12 tissues showed ubiquitous expression, with the highest expression in the brain, followed by blood and skin. Temporal expression analysis of AcIL-17D in blood showed upregulated expression at 6 and 24 h (polyinosinic: polycytidylic acid and lipopolysaccharide), 12 h (all stimulants), and 48 h (polyinosinic: polycytidylic acid and Vibrio harveyi). AcIL-17D expression in the blood gradually decreased at later hours in response to all the stimulants. After treatment of fathead minnow (FHM) cells with different recombinant AcIL-17D concentrations, the downstream gene expression analysis showed increased expression of antimicrobial genes in the FHM cells, namely [NK-Lysin (NKL), Hepcidin antimicrobial peptide-1 (HAMP-1), Defensin-β (DEFB1)] and some inflammatory genes such as IL-1β, TNF-α, IL-11, and STAT3. Further nuclear factor κB (NFκB) subunits (NFκB1, NFκB2, RelA, and Rel-B) showed upregulated gene expression at 12 and 24 h. The bacterial colony counting assay using FHM cells showed lower bacterial colony counts in rAcIL-17D-treated cells than in control. Furthermore, the Water-Soluble Tetrazolium Salt (WST -1) assay confirmed the ability of rAcIL-17D in the protection of FHM cells from bacterial infection and conducted the Hoechst 33342 staining upon treatment with rAcIL-17D and rMBP. Therefore, our findings provide important insights into the activation of IL-17D pathway genes in FHM cells, the protective role of AcIL-17D against bacterial infection, and host defense mechanisms in teleost., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Apoptosis plays a vital role in maintaining cellular homeostasis in multicellular organisms. Caspase-9 (casp-9) is one of the major initiator caspases that induces apoptosis by activating downstream intrinsic apoptosis pathway genes. Here, we isolated the cDNA sequence (1992 bp) of caspase-9 from Amphiprion clarkii (Accasp-9) that consists of a 1305 bp coding region and encodes a 434 aa protein. In silico analysis showed that Accasp-9 has a theoretical isoelectric point of 5.81 and a molecular weight of 48.45 kDa. Multiple sequence alignment revealed that the CARD domain is located at the N-terminus, whereas the large P-20 and small P-10 domains are located at the C-terminus. Moreover, a highly conserved pentapeptide active site ( 296 QACGG 301 ), as well as histidine and cysteine active sites, are also retained at the C-terminus. In phylogenetic analysis, Accasp-9 formed a clade with casp-9 from different species, distinct from other caspases. Accasp-9 was highly expressed in the gill and intestine compared with other tissues analyzed in healthy A. clarkii. Accasp-9 expression was significantly elevated in the blood after stimulation with Vibrio harveyi and polyinosinic:polycytidylic acid (poly I:C; 12-48 h), but not with lipopolysaccharide. The nucleoprotein expression of the viral hemorrhagic septicemia virus was significantly reduced in Accasp-9 overexpressed fathead minnow (FHM) cells compared with that in the control. In addition, other in vitro assays revealed that cell apoptosis was significantly elevated in poly I:C and UV-B-treated Accasp-9 transfected FHM cells. However, H 248P or C 298S mutated Accasp-9 significantly reduced apoptosis in UV-B irradiated cells. Collectively, our results show that Accasp-9 might play a defensive role against invading pathogens and UV-B radiation and H 248 and C 298 active residues are significantly involved in apoptosis in teleosts., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Reactive oxygen species modulator 1 (Romo1) is a mitochondrial inner membrane protein that induces mitochondrial reactive oxygen species (ROS) generation. In this study, we identified the Romo1 homolog from the black rockfish (Sebastes schlegelii), named it as SsRomo1, and characterized it at the molecular as well as functional levels. An open reading frame consisting of 240 bp was identified in the SsRomo1 complementary DNA (cDNA) sequence that encodes a 79 amino acid-long polypeptide with a molecular weight of 8,293 Da and a theoretical isoelectric point (pI) of 9.89. The in silico analysis revealed the characteristic features of SsRomo1, namely the presence of a transmembrane domain and the lack of a signal peptide. Homology analysis revealed that SsRomo1 exhibits the highest sequence identity with its fish counterparts (>93%) and shares a similar percentage of sequence identity with mammals (>92%). Additionally, it is closely clustered together with the fish clade in the constructed phylogenetic tree. The subcellular localization analysis confirmed its mitochondrial localization within the fathead minnow (FHM) cells. Under normal physiological conditions, the SsRomo1 mRNA is highly expressed in the rockfish ovary, followed by the blood and testis, indicating the abundance of mitochondria in these tissues. Furthermore, the significant upregulation of SsRomo1 in cells treated with lipopolysachharide (LPS), polyinosinic:polycytidylic acid, and Streptococcus iniae suggest that the increased ROS production is induced by SsRomo1 to eliminate pathogens during infections. Incidentally, we believe that this study is the first to determine the involvement of SsRomo1 in LPS-mediated nitric oxide (NO) production in RAW267.4 cells, based on their higher NO production as compared to that in the control. Moreover, overexpression of SsRomo1 enhanced the wound healing ability of FHM cells, indicating its high invasion and migration properties. We also determined the hydrogen peroxide-mediated cell viability of SsRomo1-overexpressed FHM cells and observed a significant reduction in viability, which is possibly due to increased ROS production. Collectively, our observations suggest that SsRomo1 plays an important role in oxidative stress modulation upon immune stimulation and in maintenance of tissue homeostasis in black rockfish., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In flounder aquaculture, selective breeding plays a vital role in the development of disease-resistant traits and animals with high growth rates. Moreover, superior animals are required to achieve high profits. Unlike growth-related traits, disease-resistant experiments need to be conducted in a controlled environment, as the improper measurement of traits often leads to low genetic correlation and incorrect estimation of breeding values. In this study, viral hemorrhagic septicemia virus (VHSV) resistance was studied using a genome-wide association study (GWAS), and the genetic parameters were estimated. Genotyping was performed using a high-quality 70 K single nucleotide polymorphism (SNP) Affymetrix® Axiom® myDesign™ Genotyping Array of olive flounder. A heritability of ∼0.18 for resistance to VHSV was estimated using genomic information of the fish. According to the GWAS, significant SNPs were detected in chromosomes 21, 24, and contig AGQT02032065.1. Three SNPs showed significance at the genome-wide level (p < 1 × 10 -6 ), while others showed significance above the suggestive cutoff (p < 1 × 10 -4 ). The 3% phenotypic variation was explained by the highest significant SNP, named AX-419319631. Of the important genes for disease resistance, SNPs were associated with plcg1, epha4, clstn2, pik3cb, hes6, meis3, prx6, cep164, siae, and kirrel3b. Most of the genes associated with these SNPs have been previously reported with respect to viral entry, propagation, and immune mechanisms. Therefore, our study provides helpful information regarding VHSV resistance in olive flounder, which can be used for breeding applications., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cystatins are a diverse group of cysteine protease inhibitors widely present among various organisms. Beyond their protease inhibitor function, cystatins play a crucial role in diverse pathophysiological conditions in animals, including neurodegenerative disorders, tumor progression, inflammatory diseases, and immune response. However, the role of cystatins in immunity against viral and bacterial infections in fish remains to be elucidated. In this study, the cystatin B from big-belly seahorse, Hippocampus abdominalis, designated as HaCSTB, was identified and characterized. HaCSTB shared the highest homology with type 1 cystatin family members of teleosts and had three cystatin catalytic domains with no signal peptides or disulfide bonds. HaCSTB transcripts were mainly expressed in peripheral blood cells (PBCs), followed by the testis and pouch of healthy big-belly seahorses. Immune challenge with lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (Poly I:C), and Streptococcus iniae induced upregulation of relative HaCSTB mRNA expression in PBCs. Subcellular localization analysis revealed the distribution of HaCSTB in the cytosol, mitochondria, and nuclei of fathead minnow cells (FHM). Recombinant HaCSTB (rHaCSTB) exhibited potent in vitro inhibitory activity against papain, a cysteine protease, in a concentration-, pH-, and temperature-dependent manner. Overexpression of HaCSTB in viral hemorrhagic septicemia virus (VHSV)-susceptible FHM cells increased cell viability and reduced VHSV-induced apoptosis. Collectively, these results suggest that HaCSTB might engage in the teleostean immune protection against bacteria and viruses., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Galectin-8 is a typical β-galactoside binding lectin, which primarily functions as a pattern recognition receptor and/or danger receptor that is engaged in pathogen recognition by the host innate immune system. Although several fish galectins have been identified, the role of galectin-8 in teleost immunity is still not fully understood. In this study, molecular, transcriptional, and immune-related functions of galectin-8 (EaGal8) from red-spotted grouper (Epinephelus akaara) were analyzed. The open reading frame of EaGal8 comprised 960 bp encoding 319 amino acids of a ∼35 kDa protein, composed of the N- and C-terminal carbohydrate recognition domains joined by a short hinge peptide. Phylogenetic analysis revealed that EaGal8 was closely related to the Epinephelus lanceolatus galectin-8-like protein. Although EaGal8 showed ubiquitous tissue expression, the highest expression level was observed in the blood. Immunostimulants, including lipopolysaccharide, poly(I:C), and nervous necrosis virus, significantly upregulated the EaGal8 transcription level in a time-dependent manner (p < 0.05). Furthermore, recombinant EaGal8 (rEaGal8) showed a binding affinity toward seven different carbohydrates in a concentration-dependent manner. In addition, rEaGal8 caused strong agglutination of fish red blood cells and several gram-positive and gram-negative bacteria, including Streptococcus iniae, Streptococcus parauberis, Lactococcus garvieae, Escherichia coli, Edwardsiella tarda, Vibrio alginolyticus, Vibrio parahaemolyticus, and Pseudomonas aeruginosa. For the first time in teleosts, we report the wound healing ability of galectin-8 in this study. At low concentrations, rEaGal8 showed potential wound healing responses in FHM cells, in vitro. Thus, this study reinforces the role of EaGal8 in innate immune responses against bacterial and viral infections and wound healing in red-spotted grouper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Superoxide dismutases (SODs) are metalloenzymes that convert superoxide radicals to H 2 O 2 and O 2 . Although SODs have been extensively studied in mammals and other species, comparative studies in invertebrates, such as abalones, are lacking. Here, we aimed to characterize manganese superoxide dismutase in disk abalone (Haliotis discus discus) (AbMnSOD) by assessing its transcriptional levels at different embryonic developmental stages. Additionally, the temporal expression of AbMnSOD in different abalone tissues in response to bacterial, viral, and pathogen-associated molecular pattern (PAMP) stimuli was investigated. SOD activity was measured at various recombinant protein concentrations via the xanthine oxidase/WST-1 system. Cell viability upon exposure to H 2 O 2 , wound healing ability, and subcellular localization were determined in AbMnSOD-transfected cells. AbMnSOD was 681 bp long and contained the SOD-A domain. AbMnSOD expression was higher at the trochophore stage than at the other stages. When challenged with immune stimulants, AbMnSOD showed the highest expression at 6 h post-injection (p.i.) for all stimulants except lipopolysaccharides. In the gills, the highest AbMnSOD expression was observed at 6 h p.i., except for the Vibrio parahaemolyticus challenge. Recombinant AbMnSOD showed concentration-dependent xanthine oxidase activity. Furthermore, AbMnSOD-transfected cells survived H 2 O 2 -induced apoptosis and exhibited significant wound gap closure. As expected, AbMnSOD was localized in the mitochondria of the cells. Our findings suggest that AbMnSOD is an essential antioxidant enzyme that participates in regulating developmental processes and defense mechanisms against oxidative stress in hosts., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Thioredoxin, a highly conserved class of proteins involved in redox signaling, is found in a range of organisms from bacteria to higher-level eukaryotes. Thioredoxin acts as an active regulatory enzyme to eliminate excessive reactive oxygen species, thereby preventing cellular damage. In this study, the cDNA sequence of thioredoxin domain-containing 5 (AbTXNDC5) from the disk abalone transcriptomic database was characterized. An in silico analysis of AbTXNDC5 was performed, and its spatial and temporal expression patterns in hemocytes and gills in response to bacteria (Vibrio parahaemolyticus, Listeria monocytogenes), viral hemorrhagic septicemia virus, and pathogen-associated molecular pattern molecules were observed. Furthermore, AbTXNDC5 expression was examined in different developmental stages. Functional assays to explore insulin disulfide reduction, anti-apoptotic activity, and protection against hypoxic cell death of AbTXNDC5 were conducted through recombinant proteins or overexpression in cells. AbTXNDC5 contains a 1179-bp open reading frame coding for 392 amino acids. Conserved thiol-disulfide cysteine residues within two Cys-X-X-Cys motifs were found in AbTXNDC5. Quantitative real-time polymerase chain reaction indicated that healthy digestive tract and hemocyte tissues expressed high levels of AbTXNDC5 mRNA, which may protect the host from invading pathogens. Immune-challenged abalone hemocytes and gills exhibited upregulated expression of AbTXNDC5 at different time points. rAbTXNDC5 also exhibited a functional insulin disulfide reductase activity. AbTXNDC5 conferred protection to cultured cells from apoptosis and hypoxia-induced stress , compared to the pcDNA3.1 (+) transfected control cells. Therefore, AbTXNDC5 can be considered an important gene in abalones in relation to the primary immune system and regulation of redox homeostasis and confers protection from stress., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Interleukin 17C (IL17C) is a cytokine that regulates innate immunity by recruiting antimicrobial peptides and pro-inflammatory cytokines. In this study, we characterized properties of IL-17C from Amphiprion clarkii also known as yellowtail clownfish (AcIL-17C). The AcIL-17C gene is 489 base pairs long and encodes a 163 amino acid long protein. AcIL-17C includes a signal peptide for localization in the extracellular space and comprises the IL-17 domain. The transcription analysis revealed that AcIL-17C mRNA was ubiquitously expressed in 12 tested tissues. Blood cells treated with polyinosinic:polycytidylic acid (poly (I:C)), lipopolysaccharides (LPS), and Vibrio harveyi, AcIL-17C mRNA expression was upregulated at 6 h (following poly (I:C) and LPS treatments) and at 24 h post-injection (following all treatments). The downstream gene analysis of the epithelial fathead minnow (FHM) cells showed upregulated expression of genes, such as FHM&#95;NK-Lysin, FHM&#95;Hepcidin-1, FHM&#95;Defensin-β, encoding antimicrobial peptides, as well as of FHM&#95;IL-1β, FHM&#95;TNF-A, FHM&#95;IL-11, and FHM&#95;STAT3 genes encoding inflammation-related proteins and IL-17C receptor genes FHM&#95;IL-17RA, and FHM&#95;IL-17RE at 12 and 24 h after treatment with AcIL-17C. The bacterial colony counting assay showed lower colony counts of Escherichia coli grown on FHM cells transfected with AcIL-17C carrying vector compared to those grown on control FHM cells. Further, AcIL-17C had a concentration-dependent positive effect on the survival of FHM cells infected with E. coli compared to the percentage of survived control cells. There has been a lack of studies characterizing the functions of teleost IL-17C. Therefore, these findings provide important information about the teleost host defense mechanisms and insights on the IL-17C-mediated antibacterial immunity., (Copyright © 2021. Published by Elsevier Inc.)

CD63, a member of the tetraspanin family, is involved in the activation of immune cells, antiviral immunity, and signal transduction. The economically important anemonefishes Amphiprion sp. often face disease outbreaks, and the present study aimed to characterize CD63 in Amphiprion clarkii (denoted AcCD63) to enable better disease management. The in-silico analysis revealed that the AcCD63 transcript is 723 bp long and encodes 240 amino acids. The 26.2 kDa protein has a theoretical isoelectric point of 5.51. Similar to other tetraspanins, AcCD63 consists of four domains: short N-/C-terminal domains and small/large extracellular loops. Pairwise sequence alignment revealed that AcCD63 has the highest identity (100%) and similarity (99.2%) with CD63 from Amphiprion ocellaris. Multiple sequence alignment identified a conserved tetraspanin CCG motif, PXSCC motif, and C-terminal lysosome-targeting GYEVM motif. The quantitative polymerase chain reaction analysis showed that AcCD63 was highly expressed in the spleen and head kidney tissue, with low levels of expression in the liver. Temporal expression patterns of AcCD63 were measured in the head kidney and blood tissue after injection of polyinosinic:polycytidylic acid (poly (I:C)), lipolysacharides (LPS), or Vibrio harveyi (V. harveyi). AcCD63 was upregulated at 12 h post-injection with poly (I:C) or V. harveyi, and at 24 h post-injection with all stimulants in the head kidney. At 24 h post-injection, poly (I:C) and LPS upregulated, whereas V. harveyi downregulated AcCD63 expression in the blood. All viral hemorrhagic septicemia virus transcripts (M, G, N, RdRp, P, and NV) were downregulated in response to AcCD63 overexpression, and removal of viral particles occurred via the involvement of AcCD63. The expression of antiviral genes MX dynamin-like GTPase 1, interferon regulatory factor 3, interferon-stimulated gene 15, interferon-gamma, and viperin in CD63-overexpressing fathead minnow cells was downregulated. Collectively, our findings suggest that AcCD63 is an immunologically important gene involved in the A. clarkii pathogen stress response., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Glutathione-S-transferase (GST) is a key enzyme in the phase-II detoxification process and is a biomarker of oxidative stress. In this study, we analyzed the molecular, biochemical, and antioxidant properties of GST alpha-4 from Hippocampus abdominalis (HaGSTA-4). Also, the spatial and temporal expression of HaGSTA-4 upon immune challenge with abiotic and biotic stimulants were evaluated. The HaGSTA-4 ORF encodes 223 amino acids with a molecular weight of 25.7 kDa, and an estimated isoelectric point (pI) of 8.47. It consists of the GST&#95;C superfamily and thioredoxin-like superfamily domain. The phylogenetic tree revealed that HaGSTA-4 is evolutionarily conserved with its GST alpha class counterparts. From pairwise alignment, the highest values of identity (78.5%) and similarity (85.7%) were with Parambassis ranga GSTA-4. Protein rHaGSTA-4 exhibited the highest conjugation activity towards 1-chloro-2,4-dinitrobenzene (CDNB) at pH 7 and 20 °C. A disk diffusion assay showed that rHaGSTA-4 significantly protects cells from the stress of exposure to ROS inducers such as CuSO 4 , CdCl 2 , and ZnCl 2 . Furthermore, overexpressed HaGSTA-4 defended cells against oxidative stress caused by H 2 O 2 ; evidence of selenium-independent peroxidase activity. From qPCR, the tissue-specific expression profile demonstrates that HaGSTA-4 is most highly expressed in the kidney, followed by the intestine and stomach, among fourteen different tissues extracted from healthy seahorses. The mRNA expression profile of HaGSTA-4 upon immune challenge varied depending on the tissue and the time after challenge. Altogether, this study suggests that HaGSTA-4 may be involved in protection against oxidative stress, in immune defense regulation, and xenobiotic metabolism., (Copyright © 2020. Published by Elsevier Inc.)

Glutathione S-transferases (GSTs) are important enzymes involved in phase II detoxification and function by conjugating with the thiol group of glutathione. In this study, we isolated an omega class GST from the big-belly seahorse (Hippocampus abdominalis; HaGSTO1) to study the putative xenobiotic responses and defense ability against viral and bacterial infections in this animal. The isolated HaGSTO1 gene, with a cording sequence of 720 bp, encodes a peptide of 239 amino acids. The predicted molecular mass and theoretical isoelectric point of HaGSTO1 was 27.47 kDa and 8.13, respectively. In-silico analysis of HaGSTO1 revealed a characteristic N-terminal thioredoxin-like domain and a C-terminal domain. Unlike other GSTs, the C-terminal of HaGSTO1 reached up to the N-terminal, and the N-terminal functional group was cysteine rather than tyrosine or serine, as observed in other GSTs. Phylogenetic analysis showed the evolutionary proximity of HaGSTO1 with other identified vertebrate and invertebrate GST orthologs. For the first time, we demonstrated the viral defense capability of HaGSTO1 against viral hemorrhagic septicemia virus (VHSV) infection. All six nucleoproteins of VHSV were significantly downregulated in HaGSTO1-overexpressing FHM cells at 24 h after infection compared with those in the control. Moreover, arsenic toxicity was significantly reduced in HaGSTO1-overexpressing FHM cells, and cell viability increased. Real-time polymerase chain reaction analysis showed that HaGSTO1 transcripts were highly expressed in the pouch and gill when compared with those in other tissues. Blood HaGSTO1 transcripts were significantly upregulated after Edwardsiella tarda, Streptococcus iniae, lipopolysaccharide, and polyinosinic:polycytidylic acid challenge experiments. Collectively, these findings suggest the involvement of HaGSTO1 in the host defense mechanism of seahorses., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Peroxiredoxins (Prxs) are cysteine-dependent antioxidant proteins that play a leading part in oxidative stress response. Peroxiredoxin 4 (Prx4) is located in the endoplasmic reticulum, where it is primarily involved in regulating the concentration of H 2 O 2 , generated during protein folding. Prx4 is also located in the extracellular space, where it activates the JAK/STAT-mediated stress response. Here, we focus on the identification and characterization of the sequence and function of Prx4 from the big-belly seahorse (Hippocampus abdominalis) (HaPrx4). The size of the HaPrx4 coding sequence was 777 bp, which encoded a 258 amino acid protein of 28.8 kDa molecular weight. The amino acid sequence comprises a signal peptide, two active sites with peroxidatic cysteine and resolving cysteine, catalytic triad, and peroxiredoxin superfamily domain. According to the tissue distribution results, ovaries exhibited the highest HaPrx4 expression level within fourteen examined tissues. The HaPrx4 transcriptional responses to four immune stimulants (lipopolysaccharides, polyinosinic: polycytidylic acid, Edwardsiella tarda, and Streptococcus iniae) were evaluated in the blood and liver tissues. Additionally, the functions of recombinant HaPrx4 protein were evaluated by metal ion-catalyzed oxidation assay, peroxidase activity assay, insulin reduction assay, cell viability assay, and Hoechst staining. The assay results confirmed that the functions of HaPrx4 involved DNA protection, hydrogen peroxide (H 2 O 2 ) elimination, oxidoreductase activity, enhancing cell survival, and cell protection. The results of the current study propose that HaPrx4 is effectively involved in H 2 O 2 scavenging activity during stress conditions and in innate immune responses of the big-belly seahorse., (Copyright © 2020. Published by Elsevier Inc.)

Glutaredoxins (Grxs) are well-known oxidoreductases involved in a wide range of redox activities in organisms. In this study, two invertebrate Grxs (AbGrx1-like and AbGrx2) from disk abalone were identified and characterized in an effort to gain a deeper understanding into their immune and redox regulatory roles. Both AbGrxs share typical thioredoxin/Grx structures. AbGrx1-like and AbGrx2 were identified as monothiol and diothiol Grxs, respectively. AbGrxs were significantly expressed at the egg and 16-cell stage of early abalone development. Although the expression of both AbGrxs demonstrated similar patterns, the expression of AbGrx1-like was higher than AbGrx2 during development stages. In contrast, AbGrx2 expression was significantly higher than that of AbGrx1-like in adult tissues. Highest AbGrx1-like expression was observed in the hepatopancreas and digestive tract, while highest AbGrx2 expression was found in the gills, followed by the mantle, in healthy adult abalone tissues. The highest expression of AbGrx1-like was observed in the gills at 12 h and 6 h post injection (p.i) of Vibrio parahemolyticus and other stimulants, respectively. The highest expression of AbGrx2 in the gills were observed at 120 h, 6 h, 24 h, and 12 h post injection of V. parahaemolyticus, Listeria monocytogenes, Viral hemorrhagic septicemia virus, and Polyinosinic:polycytidylic acid, respectively. AbGrxs possessed significant 2-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) reduction activity, but AbGrx2 exhibited higher redox activity than AbGrx1-like. Altogether, our results suggest an important role of AbGrx1-like and AbGrx2 in redox homeostasis, as well as in the invertebrate immune defense system. Our findings will aid the development of new disease management strategies for this economically valuable species., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Glutaredoxins are a group of heat stable oxidoreductases ubiquitously found in prokaryotes and eukaryotes. They are widely known for GSH (glutathione)-dependent protein disulfide reduction and cellular redox homeostasis. This study was performed to identify and characterize rockfish (Sebastes schlegelii) glutaredoxin 1 (SsGrx1) at molecular, transcriptional, and functional levels. The coding sequence of SsGrx1 was 318 bp in length and encoded a protein containing 106 amino acids. The molecular weight and theoretical isoelectric point of the putative SsGrx1 protein were 11.6 kDa and 6.71 kDa, respectively. The amino acid sequence of SsGrx1 comprised a CPYC redox active motif surrounded by several conserved GSH binding sites. The modeled protein structure was found to consist of five α-helices and four β-sheets, similar to human Grx1. SsGrx1 showed a tissue specific expression in all the tissues tested, with the highest expression in the kidney. Immune stimulation by lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (polyI:C), and Streptococcus iniae (S. iniae) could significantly modulate the SsGrx1 expression pattern in the blood and gills. Analysis of its subcellular localization disclosed that SsGrx1 was prominently localized in the cytosol. Recombinant SsGrx1 (rSsGrx1) exhibited significant activity in insulin disulfide reduction assay and HED (β-Hydroxyethyl Disulfide) assay. Furthermore, transient overexpression of SsGrx1 in FHM (fathead minnow) cells significantly enhanced cell survival upon H 2 O 2 -induced apoptosis. Collectively, our findings strongly suggest that SsGrx1 plays a crucial role in providing rockfish immune protection against pathogens and oxidative stress., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interests, (Copyright © 2020 Elsevier Inc. All rights reserved.)

Thioredoxin (Trx) is a small ubiquitous multifunctional protein with a characteristic WCGPC thiol-disulfide active site that is conserved through evolution. Trx plays a crucial role in the antioxidant defense system. Further, it is involved in a variety of biological functions including gene expression, apoptosis, and growth regulation. Trx exists in several forms, with the cytosolic (Trx-1) and mitochondrial (Trx-2) forms being the most predominant. In this study, the mitochondrial Trx protein (HaTrx-2), from the big-belly seahorse (Hippocampus abdominalis) was characterized, and its molecular features and functional properties were investigated. The cDNA sequence of HaTrx-2 consists of a 519 bp ORF, and it encodes a polypeptide of 172 amino acids. This protein has a calculated molecular mass of 18.8 kDa and a calculated isoelectric point (pI) of 7.80. The highest values of identity (78.7%) and similarity (86.2%) were observed with Fundulus heteroclitus Trx-2 from the pairwise alignment results. The phylogenetic analysis revealed that HaTrx-2 is closely clustered with teleost fishes. The qPCR results showed that HaTrx-2 was prevalently expressed at various levels in all the tissues examined. The ovary showed the highest expression, followed by the brain and kidney. HaTrx-2 showed varying mRNA expression levels during the immune challenge experiment, depending on the type of tissue and the time interval. Our results confirmed the antioxidant property of HaTrx-2 by performing the MCO assay, DPPH radical scavenging activity, and cell viability assays. Further, an insulin disulfide reduction assay revealed the dithiol remove the enzymatic activity of HaTrx-2. Altogether these results indicate that HaTrx-2 plays indispensable roles in the regulation of oxidative stress and immune response in the seahorse., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Malectin is a carbohydrate-binding lectin protein found in the endoplasmic reticulum (ER). It selectivity binds to Glc 2 -N-glycan and is involved in a glycoprotein quality control mechanism. Even though malectin may play a role in immunity, its role in innate immunity is not fully known. In the present study, we identified and characterized the malectin gene from Hippocampus abdominalis (HaMLEC). We analyzed sequence features, spatial expression levels, temporal expression profiles upon immune responses, bacterial and carbohydrate binding abilities and anti-viral properties to investigate the potential role of HaMLEC in innate immunity. The molecular weight and isoelectric point (pI) were estimated to be 31.99 kDa and 5.17, respectively. The N-terminal signal peptide, malectin superfamily domain and C-terminal transmembrane region were identified from the amino acid sequence of HaMLEC. The close evolutionary relationship of HaMLEC with other teleosts was identified by phylogenetic analysis. According to quantitative PCR (qPCR) results, HaMLEC expression was observed in all the examined tissues and high expression was observed in the ovary and brain, compared to other tested tissues. Temporal expression of HaMLEC in liver and blood tissues were significant modulated upon exposure to immunogens Edwardasiella tarda, Streptococcus iniae, polyinosinic:polycytidylic and lipopolysaccharide. The presence of carbohydrate binding modules (CBMs) of bacterial glycosyl hydrolases were functionally confirmed by a bacterial binding assay. Anti-viral activity significantly reduced viral hemorrhagic septicemia virus (VHSV) replication in cells overexpressing HaMLEC. The observed results suggested that HaMLEC may have a significant role in innate immunity in Hippocampus abdominalis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Syndecan-2, also known as CD362, is a transmembrane heparan sulfate proteoglycan which regulates cell growth, proliferation, cell adhesion, wound healing, and recruits immune cells. In the present study, we performed bioinformatics, spatial and temporal expression analyses of Hippocampus abdominalis syndecan-2 (HaSDC-2). Additionally, functional assays were conducted. HaSDC-2 has five major domains; an extracellular heparan sulfate attachment domain, a co-receptor binding domain, a transmembrane domain, two conserved domains (C1 domain, C2 domain), and a variable (V) domain. The ectodomain contained a signal peptide and GAG attachment sites. In-silico analysis revealed that HaSDC-2 contained a 798 bp long ORF and protein sequence of 265 amino acid residues. Further analysis of the amino acid sequence predicted a 28.9 kDa molecular weight and a 4.13 theoretical isoelectric point. The spatial expression of HaSDC-2 was ubiquitous in all tested tissues. HaSDC-2 expression in the liver was upregulated 24 h post-injection in response to all stimuli. Further, HaSDC-2 expression in blood cells was upregulated at 12 and 72 h post-injection in response to all the stimuli. HaSDC-2 + pcDNA™3.1(+) transfected cells exhibited significant survival in response to cell stressors such as H 2 O 2 and HED. The ectodomain of recombinant HaSDC-2 treated cells showed significant cell proliferation in a concentration-dependent manner. The scratch wound healing assay showed significant Δ gap closures with increasing concentrations of HaSDC-2. Collectively, these results indicated that syndecan-2 was involved in regulating immune responses and cell stress conditions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The interferon-induced GTP-binding protein Mx is responsible for a specific antiviral state against a broad spectrum of viral infections that are induced by type-I interferons (IFN α/β) in different vertebrates. In this study, the Mx gene was isolated from the constructed mullet cDNA database. Structural features of mullet Mx (MuMx) were analyzed using different in-silico tools. The pairwise comparison revealed that the MuMx sequence was related to Stegastes partitus Mx with an 83.7% sequence identity, whereas MuMx was clustered into the teleost category in the phylogentic analysis. Sequence alignment showed that the dynamin-type guanine nucleotide-binding domain (G&#95;DYNAMIN&#95;2), central interactive domain (CID), and GTPase effector domain (GED) were conserved among Mx counterparts. The transcriptional expression of MuMx was the highest in blood cells from unchallenged fish. The temporal mRNA profile showed that MuMx expression was significantly elevated in all tissues, including blood, spleen, head kidney, liver, and gills after the injection of polyinosinic-polycytidylic acid (poly I:C) at many time points. Moreover, MuMx expression increased slightly, in the blood, spleen, and head kidney at a few time points after the injection of lipopolysaccharide (LPS) and Lactococcus garvieae (L. garvieae). Results of the subcellular localization analysis confirmed that the MuMx protein was highly expressed in the cytoplasm. The analysis of the gene expression of the viral hemorrhagic septicemia virus (VHSV) under conditions of MuMx overexpression confirmed the significant inhibition of viral transcripts. The cell viability (MTT) assay and VHSV titer quantification with the presence of MuMx indicated a significant reduction in virus replication. Collectively, these findings suggest that Mx is a specific immune-related gene that elicits crucial antiviral functions against viral antigens in the mullet fish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glutaredoxins are oxidoreductases present in almost all living organisms. They belong to the thioredoxin superfamily and share the thioredoxin structure and catalytic motif. Glutaredoxin 2 has been identified as a mitochondrial protein in vertebrates. In this study, the sequence of Glutaredoxin 2 from Hippocampus abdominalis (HaGrx2) was analyzed by molecular, transcriptional, and functional assays. In-silico analysis revealed that HaGrx2 shows the highest homology with Hippocampus comes, while distinctly cluster with fish Grx2 orthologs. Tissue distribution analysis showed that HaGrx2 is ubiquitously expressed in all tissues tested, and the highest expression was observed in the brain and skin. Significant HaGrx2 transcript modulation was identified in blood and liver upon injecting bacterial and Pathogen Associated Molecular Patterns. The redox activity of HaGrx2 was revealed by Dehydroascorbic reduction and insulin disulfide reduction activity assays. Further, the deglutathionylation activity of 1 nM HaGrx2 was found to be equivalent to that of 0.84 nM HaGrx1. HaGrx2 exhibited antiapoptotic activity against H 2 O 2 -induced oxidative stress in FHM cells. Altogether, the results of this study suggest that HaGrx2 plays a role in redox homeostasis and innate immune responses in fish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glutathione S-transferases (GSTs) are essential enzymes for the bioactivation of xenobiotics through the conjugation of the thiol group of glutathione (GSH). In this study, a kappa class of GST was identified from the big belly seahorse (Hippocampus abdominalis) (HaGSTκ1) and its biochemical and functional properties were analyzed. HaGSTκ1 has 231 amino acids encoded by a 696 bp open reading frame (ORF). The protein has a predicted molecular mass of 26.04 kDa and theoretical isoelectric point (pI) of 8.28. It comprised a thioredoxin domain, disulfide bond formation protein A (DsbA) general fold, and Ser15 catalytic site as well as GSH-binding and polypeptide-binding sites. Phylogenetic analysis revealed that HaGSTκ1 is closely clustered with the kappa class of GSTs from teleost fishes. The recombinant (rHaGSTκ1) protein exhibited activity toward 1-chloro-2,4-dinitrobenzene (CDNB), 4-nitrobenzyl (4-NBC), and 4-nitrophenethyl bromide (4-NPB) but not 1,2-dichloro-4-nitrobenzene (DCNB). The optimum pH and temperature were 8 and 30 °C, respectively, for the catalysis of CDNB and the universal substrate of GSTs. The rHaGSTκ1 activity was efficiently inhibited in the presence of Cibacron blue (CB) as compared with hematin. Most prominent expression of HaGSTκ1 was observed in the liver and kidney among the fourteen different tissues of normal seahorse. After challenge with lipopolysaccharide (LPS), polyinosinic-polycytidylic (poly I:C), gram-negative Edwardsiella tarda, and gram-positive Streptococcus iniae, HaGSTκ1 expression was significantly modulated in the liver and blood tissues. Altogether, our study proposes the plausible important role of HaGSTκ1 in innate immunity and detoxification of harmful xenobiotics., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glutaredoxins (Grx) are redox enzymes conserved in viruses, eukaryotes, and prokaryotes. In this study, we characterized glutaredoxin 1 (HaGrx1) from big-belly seahorse, Hippocampus abdominalis. In-silico analysis showed that HaGrx1 contained the classical glutaredoxin 1 structure with a CSYC thioredoxin active site motif. According to multiple sequence alignment and phylogenetic reconstruction, HaGrx1 presented the highest homology to the Grx1 ortholog from Hippocampus comes. Transcriptional studies demonstrated the ubiquitous distribution of HaGrx1 transcripts in all the seahorse tissues tested. Significant modulation (p < 0.05) of HaGrx1 transcripts were observed in blood upon stimulation with pathogen-associated molecular patterns and live pathogens. The β-hydroxyethyl disulfide reduction assay confirmed the antioxidant activity of recombinant HaGrx1. Further, dehydroascorbate reduction and insulin disulfide reduction assays revealed the oxidoreductase activity of HaGrx1. HaGrx1 utilized 1,4-dithiothreitol, l-cysteine, 2-mercaptoethanol, and reduced l-glutathione as reducing agent with different dehydroascorbate reduction activity levels. Altogether, our results suggested a vital role of HaGrx1 in redox homeostasis as well as the host innate immune defense system., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Endoplasmic reticulum resident protein 44 (ERp44) is a protein disulfide isomerase (PDI) and a member of thioredoxin family, which is involved in several functions such as oxidative folding and polymerization of molecules, carrier protein activity, regulation of the Ca 2+ ion levels in the endoplasmic reticulum (ER), cellular signaling, and maintenance of lumen redox homeostasis. In this study, ERp44 from Hippocampus abdominalis, commonly known as the big belly seahorse, was characterized. ERp44 possessed three PDI-like domains and one thioredoxin fold with a CXXC conserved motif. The open reading frame consisted of 1233 bp encoding 410 amino acids. Additionally, it contained a C-terminal RDEL motif, which suggests a localization of ERp44 to the endoplasmic reticulum. ShERp44 showed highest mRNA expression in the ovary, brain, and gills. Temporal expression of ShERp44 in blood showed significant upregulation against bacterial, LPS, and PolyI:C stimuli at 24 and 72 h. Trunk kidney tissue exhibited upregulated ShERp44 expression at 24 h in response to lipopolysaccharides and Streptococcus iniae and at 72 h in response to Edwardsiella tarda and poly I:C. NADPH turnover was observed as 0.06122 ± 0.0075 μmol/s protein/μg through the HED assay. Insulin aggregation assay showed a significant reduction ability of rShERp44 by precipitating insulin rapidly, beginning at 5 min. Moreover, rShERp44-treated fathead minnow cells showed significant cell survival against 2-hydroxyethyl disulfide and thus exhibited capability to resist oxidative stress. Taken together, these findings provide insight into teleost defense mechanisms and functional properties of ERp44 in controlling redox homeostasis at the molecular level., (Copyright © 2019. Published by Elsevier Inc.)

Thioredoxin domain-containing protein 17 (TXNDC17) is a small protein (∼14 kDa) involved in maintaining cellular redox homeostasis via a thiol-disulfide reductase activity. In this study, TXNDC17 was identified and characterized from Hippocampus abdominalis. The open reading frame (ORF) consisted of 369 bp and 123 amino acids. Similar to the other thioredoxins, TXNDC17 contained a conserved WCXXC functional motif. The highest spatial mRNA expressions of HaTXNDC17 were observed in the muscle, brain, and intestine. Interestingly, the mRNA expression of HaTXNDC17 in blood showed significant upregulation at 48 h against all the pathogen associated molecular patterns (PAMPs) and bacteria. Further, HaTXNDC17 transcripts in the trunk kidney were significantly upregulated at 24-48 h by bacterial endotoxin lipopolysaccharides (LPS), viral mimic polyinosinic: polycytidylic acid (poly I:C), and gram-negative bacteria (Edwardsiella tarda). The DPPH assay showed that the radical scavenging activity varies in a concentration-dependent manner. The insulin reduction assay demonstrated a significant logarithmic relationship with the concentration of rHaTXNDC17. Moreover, FHM cells treated with recombinant HaTXNDC17 significantly enhanced cellular viability under oxidative stress. Together, these results show that HaTXNDC17 function is important for maintaining cellular redox homeostasis and that it is also involved in the immune mechanism in seahorses., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The structural and evolutionary linkage between tumor necrosis factor (TNF) and the globular C1q (gC1q) domain defines the C1q and TNF-related proteins (CTRPs), which are involved in diverse functions such as immune defense, inflammation, apoptosis, autoimmunity, and cell differentiation. In this study, red-lip mullet (Liza haematocheila) CTRP4-like (MuCTRP4-like), CTRP5 (MuCTRP5), CTRP6 (MuCTRP6), and CTRP7 (MuCTRP7) were identified from the red-lip mullet transcriptome database and molecularly characterized. According to in silico analysis, coding sequences of MuCTRP4-like, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of 1128, 753, 729, and 888 bp open reading frames (ORF), respectively and encoded 375, 250, 242, and 295 amino acids, respectively. All CTRPs possessed a putative C1q domain. Additionally, MuCTRP5, MuCTRP6, and MuCTRP7 consisted of a collagen region. Phylogenetic analysis exemplified that MuCTRPs were distinctly clustered with the respective CTRP orthologs. Tissue-specific expression analysis demonstrated that MuCTRP4-like was mostly expressed in the blood and intestine. Moreover, MuCTRP6 was highly expressed in the blood, whereas MuCTRP5 and MuCTRP7 were predominantly expressed in the muscle and stomach, respectively. According to the temporal expression in blood, all MuCTRPs exhibited significant modulations in response to polyinosinic:polycytidylic acid (poly I:C) and Lactococcus garvieae (L. garvieae). MuCTRP4-like, MuCTRP5, and MuCTRP6 showed significant upregulation in response to lipopolysaccharides (LPS). The results of this study suggest the potential involvement of Mullet CTRPs in post-immune responses., (Copyright © 2018. Published by Elsevier Ltd.)

The redlip mullet (Liza haematocheila) is one of the most economically important fish in Korea and other East Asian countries; it is susceptible to infections by pathogens such as Lactococcus garvieae, Argulus spp., Trichodina spp., and Vibrio spp. Learning about the mechanisms of the complement system of the innate immunity of redlip mullet is important for efforts towards eradicating pathogens. Here, we report a comprehensive study of the terminal complement complex (TCC) components that form the membrane attack complex (MAC) through in-silico characterization and comparative spatial and temporal expression profiling. Five conserved domains (TSP1, LDLa, MACPF, CCP, and FIMAC) were detected in the TCC components, but the CCP and FIMAC domains were absent in MuC8β and MuC9. Expression analysis of four TCC genes from healthy redlip mullets showed the highest expression levels in the liver, whereas limited expression was observed in other tissues; immune-induced expression in the head kidney and spleen revealed significant responses against Lactococcus garvieae and poly I:C injection, suggesting their involvement in MAC formation in response to harmful pathogenic infections. Furthermore, the response to poly I:C may suggest the role of TCC components in the breakdown of the membrane of enveloped viruses. These findings may help to elucidate the mechanisms behind the complement system of the teleosts innate immunity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Glutathione S-transferase (GST; EC 2.5.1.18) isoenzymes represent a complex group of proteins that are involved in phase II detoxification in several organisms. In this study, GST kappa (GSTκ) from the disk abalone (Haliotis discus discus; AbGSTκ) was characterized at both the transcriptional and functional levels to determine its potential capacity to perform as a detoxification agent under conditions of different stress. The predicted AbGSTκ protein consists of 227 amino acids, with a predicted molecular weight of 25.6 kDa and a theoretical isoelectric point (pI) of 7.78. In silico analysis reveals that AbGSTκ is a disulfide bond formation protein A (DsbA), consisting of a thioredoxin domain, GSH binding sites (G-sites), and a catalytic residue. In contrast, no hydrophobic ligand binding site (H-site), or signal peptides, were detected. AbGSTκ showed the highest sequence identity with the orthologue from pufferfish (Takifugu obscurus) (60.0%). In a phylogenetic tree, AbGSTκ clustered closely together with other fish GSTκs, and was evolutionarily distanced from other cytosolic GSTs. The predicted three-dimensional structure clearly demonstrates that the dimer adopts a butterfly-like shape. A tissue distribution analysis revealed that GSTκ was highly expressed in the digestive tract, suggesting it has detoxification ability. Depending on the tissue and time, AbGSTκ showed different expression patterns, and levels of expression, following challenge of the abalone with immune stimulants. Enzyme kinetics of the purified recombinant proteins demonstrated its conjugating ability using 1-Chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) as substrates, and suggested it has a low affinity for both substrates. The optimum temperature and pH for the rAbGSTκ GSH: CDNB conjugating activity were found to be 35 °C and pH 8, respectively indicating that the abalone is well adapted to a wide range of environmental conditions. Cibacron blue (100 μM) was capable of completely inhibiting rAbGSTκ (100%) with an IC 50 (half maximal inhibitory concentration) of 0.05 μM. A disk diffusion assay revealed that rAbGSTκ could significantly protect cells from H 2 O 2 , CdCl 2, and ZnCl 2 . Altogether, this current study suggests that AbGSTκ is involved in detoxification and immunological host defense mechanisms and allows abalones to overcome stresses in order for them to have an increased chance of survival., (Copyright © 2018. Published by Elsevier Ltd.)

Thioredoxin is a highly conserved protein found in both prokaryotes and eukaryotes. Reactive oxygen species (ROS) are produced in response to metabolic processes, radiation, metal oxidation, and pathological infections. High levels of ROS lead to cell death via autophagy. However, thioredoxin acts as an active regulatory enzyme in response to excessive ROS. Here, we performed in-silico analysis, immune challenge experiments, and functional assays of seahorse thioredoxin-like protein 1 (ShTXNL1). Evolutionary identification showed that ShTXNL1 protein belongs to the thioredoxin superfamily comprising 289 amino acids. It possesses an N-terminal active thioredoxin domain and C-terminal proteasome-interacting thioredoxin domain (PITH) of ShTXNL1 which is a component of 26S proteasome and binds to the matrix or cell. Pairwise alignment results showed 99.0% identity and 99.7% similarity with the sequence of Hippocampus species. Conserved thiol-disulfide cysteine residue containing Cys-X-X-Cys motif may be found in the first few amino acids in the second beta sheet starting from the N-terminus. This motif can be discovered in ShTXNL1 as 14 CRPC 17 and comprised two N-linked glycosylation sites at 72 NISA 75 and 139 NESD 142 . According to the quantitative real-time polymerase chain reaction analysis from healthy seahorses, highest ShTXNL1 mRNA expression was observed in muscle, followed by ovary, brain, gill, and blood tissues. Moreover, significant temporal expression of ShTXNL1 was observed in gill and blood tissues after bacterial stimuli. Thus, the ShTXNL1 gene may be identified as an immunologically important gene in seahorse., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Infectious disease is the leading cause of death worldwide, and diagnosis of polymicrobial and fungal infections is increasingly challenging in the clinical setting. Conventionally, molecular detection is still the best method of species identification in clinical samples. However, the limitations of Sanger sequencing make diagnosis of polymicrobial infections one of the biggest hurdles in treatment. The development of massively parallel sequencing or next generation sequencing (NGS) has revolutionized the field of metagenomics, with wide application of the technology in identification of microbial communities in environmental sources, human gut and others. However, to date there has been no commercial application of this technology in infectious disease diagnostic settings., Methods: Credence Genomics Rapid Infection Detection™ test, is a molecular based diagnostic test that uses next generation sequencing of bacterial 16S rRNA gene and fungal ITS1 gene region to provide accurate identification of species within a clinical sample. Here we present a study comparing 16S and ITS1 metagenomic identification against conventional culture for clinical samples. Using culture results as gold standard, a comparison was conducted using patient specimens from a clinical microbiology lab., Results: Metagenomics based results show a 91.8% concordance rate for culture positive specimens and 52.8% concordance rate with culture negative samples. 10.3% of specimens were also positive for fungal species which was not investigated by culture. Specificity and sensitivity for metagenomics analysis is 91.8 and 52.7% respectively., Conclusion: 16S based metagenomic identification of bacterial species within a clinical specimen is on par with conventional culture based techniques and when coupled with clinical information can lead to an accurate diagnostic tool for infectious disease diagnosis.

Delta-sultones, prepared by C-H insertion, can be oxidatively converted to gamma-lactones by treatment with t-BuOK/t-BuOOH. An intermediate in the synthesis of (-)-eburnamonin was prepared using this approach.

Intramolecular C-H insertion on diazosulfone and diazosulfonate substrates was used to prepare synthetically useful intermediates from easily available starting materials.

Background: Cryptococcus neoformans is saprophytic encapsulated yeast. Infection is acquired by inhalation of the organism and could be asymptomatic or limited to the lungs, specially in the immunocompetent host. Cryptococcal meningitis is a serious opportunistic infection among post transplant recipients. Cranial nerve palsies and ophthalmoplegia are well known complications of this disease, but bilateral complete ophthalmoplegia is a very rare presentation., Case Presentation: A Sri Lankan young male, who is a post kidney transplant recipient, presented with bilateral complete ophthalmoplegia and subsequently was diagnosed to have cryptococcal meningitis based on Indian ink stain and culture of cerebrospinal fluid (CSF). His magnetic resonance imaging (MRI) showed bilateral multiple nodular lesions in both basal ganglia and thalami. Brainstem imaging was normal., Conclusions: Cryptococcal meningitis is a serious fungal infection in post transplant patients. It should be suspected in any immunocompromised patient with fever, headache and focal neurological signs. Bilateral thalamic lesions, inflammation and invasion of the cranial nerves and raised intracranial pressure were thought to be possible mechanisms resulting in bilateral complete ophthalmoplegia in this patient.