Nucleic Acid Sensing by STING Induces an IFN-like Antiviral Response in a Marine Invertebrate.

The cytosolic detection of pathogen-derived nucleic acids has evolved as an essential strategy for host innate immune defense in mammals. One crucial component in this process is the stimulator of IFN genes (STING), which acts as a vital signaling adaptor, connecting the cytosolic detection of DNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) to the downstream type I IFN signaling pathway. However, this process remains elusive in invertebrates. In this study, we present evidence demonstrating that STING, an ortholog found in a marine invertebrate (shrimp) called Litopenaeus vannamei, can directly detect DNA and initiate an IFN-like antiviral response. Unlike its homologs in other eukaryotic organisms, which exclusively function as sensors for cyclic dinucleotides, shrimp STING has the ability to bind to both double-stranded DNA and cyclic dinucleotides, including 2'3'-cGAMP. In vivo, shrimp STING can directly sense DNA nucleic acids from an infected virus, accelerate IFN regulatory factor dimerization and nuclear translocation, induce the expression of an IFN functional analog protein (Vago4), and finally establish an antiviral state. Taken together, our findings unveil a novel double-stranded DNA-STING-IKKε-IRF-Vago antiviral axis in an arthropod, providing valuable insights into the functional origins of DNA-sensing pathways in evolution.

Intestine bacterial community affects the growth of the Pacific white shrimp (Litopenaeus vannamei).

Increasing evidence suggests that intestine microorganisms are closely related to shrimp growth, but there is no existing experiment to prove this hypothesis. Here, we compared the intestine bacterial community of fast- and slow-growing shrimp at the same developmental stage with a marked difference in body size. Our results showed that the intestine bacterial communities of slow-growing shrimp exhibited less diversity but were more heterogeneous than those of fast-growing shrimp. Uncultured_bacterium_g_Candidatus Bacilloplasma, Tamlana agarivorans, Donghicola tyrosinivorans, and uncultured_bacterium_f_Flavobacteriaceae were overrepresented in the intestines of fast-growing shrimp, while Shimia marina, Vibrio sp., and Vibrio campbellii showed the opposite trends. We further found that the bacterial community composition was significantly correlated with shrimp length, and some bacterial species abundances were found to be significantly correlated with shrimp weight and length, including T. agarivorans and V. campbellii, which were chosen as indicators for a reverse gavage experiment. Finally, T. agarivorans was found to significantly promote shrimp growth after the experiment. Collectively, these results suggest that intestine bacterial community could be important factors in determining the growth of shrimp, indicating that specific bacteria could be tested in further studies against shrimp growth retardation. KEY POINTS: • A close relationship between intestine bacterial community and shrimp growth was proven by controllable experiments. • The bacterial signatures of the intestine were markedly different between slow- and fast-growing shrimp, and the relative abundances of some intestine bacterial species were correlated significantly with shrimp body size. • Reverse gavage by Tamlana agarivorans significantly promoted shrimp growth.

A predictive nomogram for surgical site infection in patients who received clean orthopedic surgery: a retrospective study.

BACKGROUND: Surgical site infection (SSI) is a common and serious complication of elective clean orthopedic surgery that can lead to severe adverse outcomes. However, the prognostic efficacy of the current staging systems remains uncertain for patients undergoing elective aseptic orthopedic procedures. This study aimed to identify high-risk factors independently associated with SSI and develop a nomogram prediction model to accurately predict the occurrence of SSI. METHODS: A total of 20,960 patients underwent elective clean orthopedic surgery in our hospital between January 2020 and December 2021, of whom 39 developed SSI; we selected all 39 patients with a postoperative diagnosis of SSI and 305 patients who did not develop postoperative SSI for the final analysis. The patients were randomly divided into training and validation cohorts in a 7:3 ratio. Univariate and multivariate logistic regression analyses were conducted in the training cohort to screen for independent risk factors of SSI, and a nomogram prediction model was developed. The predictive performance of the nomogram was compared with that of the National Nosocomial Infections Surveillance (NNIS) system. Decision curve analysis (DCA) was used to assess the clinical decision-making value of the nomogram. RESULTS: The SSI incidence was 0.186%. Univariate and multivariate logistic regression analysis identified the American Society of Anesthesiology (ASA) class (odds ratio [OR] 1.564 [95% confidence interval (CI) 1.029-5.99, P = 0.046]), operative time (OR 1.003 [95% CI 1.006-1.019, P < 0.001]), and D-dimer level (OR 1.055 [95% CI 1.022-1.29, P = 0.046]) as risk factors for postoperative SSI. We constructed a nomogram prediction model based on these independent risk factors. In the training and validation cohorts, our predictive model had concordance indices (C-indices) of 0.777 (95% CI 0.672-0.882) and 0.732 (95% CI 0.603-0.861), respectively, both of which were superior to the C-indices of the NNIS system (0.668 and 0.543, respectively). Calibration curves and DCA confirmed that our nomogram model had good consistency and clinical predictive value, respectively. CONCLUSIONS: Operative time, ASA class, and D-dimer levels are important clinical predictive indicators of postoperative SSI in patients undergoing elective clean orthopedic surgery. The nomogram predictive model based on the three clinical features demonstrated strong predictive performance, calibration capabilities, and clinical decision-making abilities for SSI.

Identification of an Ortholog of MALT1 from Shrimp That Induces NF-κB-Mediated Antiviral Immunity.

MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) serves as a pivotal mediator for NF-κB activation in response to a wide spectrum of transmembrane receptor stimuli. In the present study, a homolog of MALT1, named LvMALT1, is cloned from the Pacific white shrimp (Litopenaeus vannamei) and its potential function in shrimp innate immunity is explored. The open reading frame of LvMALT1 is 2364 bp that encodes 787 amino acids. The predicted LvMALT1 protein structure comprises a death domain, three immunoglobulin domains, and a caspase-like domain, exhibiting remarkable similarity to other homologs. LvMALT1 is a cytoplasmic-localized protein and could interact with LvTRAF6. Overexpression of LvMALT1 induces the activation of promoter elements governing the expression of several key antimicrobial peptides (AMPs), including penaeidins (PENs) and crustins (CRUs). Conversely, silencing of LvMALT1 leads to a reduction in the phosphorylation levels of Dorsal and Relish, along with a concomitant decline in the in vivo expression levels of multiple AMPs. Furthermore, LvMALT1 is prominently upregulated in response to a challenge by the white spot syndrome virus (WSSV), facilitating the NF-κB-mediated expression of AMPs as a defense against viral infection. Taken together, we identified a MALT1 homolog from the shrimp L. vannamei, which plays a positive role in the TRAF6/NF-κB/AMPs axis-mediated innate immunity.

Rapid adaptive and acute stimulatory responses to low salinity stress in Pacific white shrimp (Litopenaeus vannamei): Insights from integrative transcriptomic and proteomic analysis.

The Pacific white shrimp (Litopenaeus vannamei) is a euryhaline crustacean capable of tolerating a wide range of ambient salinity, but the strategies of hepatopancreas to rapid adaptive or acute stimulatory responses to extremely low salinity fluctuations remains unclear. In this study, we integrated transcriptomic and proteomic analyses on the hepatopancreas derived from rapid adaptative (RA) and acute stimulatory (AS) responses to extremely low salinity stress (0.3 ppt) to unveil specific regulatory mechanisms. The RA group displayed normal epithelial cells and tubule structures, while the AS group showed histological changes and lesions. A total of 754 and 649 differentially expressed genes (DEGs) were identified in RA and AS treatments, respectively. For proteome, a total of 206 and 66 differentially expressed proteins (DEPs) were obtained in the RA/CT and AS/CT comparison groups, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were conducted among the DEGs and DEPs, revealing that metabolic related pathways were significantly enriched pathways in both comparison groups. In addition, correlation analysis of transcriptomic and proteomic results showed that 20 and 3 pairs of DEGs/DEPs were identified in RA vs. CT and AS vs. CT comparison groups, respectively. This study is the first report on the rapid adaptive and acute stimulatory transcriptomic and proteomic responses of L. vannamei to extremely low salinity, shedding light on the mechanisms underlying osmoregulation in euryhaline crustaceans.

Transcriptomic analysis reveals the role of Glycolysis pathway in Litopenaeus vannamei during DIV1 infection.

In recent years, shrimp farming has experienced significant losses due to the emergence of DIV1 (Decapod iridescent virus 1), an infectious virus with a high fatality rate among shrimp. In this study, we conducted transcriptomic analyses on shrimp Litopenaeus vannamei hemocytes following DIV1 infection and focused on the function of genes in the Glycolysis pathway during DIV1 infection. A total of 2197 differentially expressed genes (DEGs) were identified, comprising 1506 up-regulated genes and 691 down-regulated genes. These genes were primarily associated with Phagosome, ECM-Receptor Interaction, Drug Metabolism-Other Enzymes, and the AGE-RAGE signaling pathway in diabetic complications. KEGG pathway enrichment analysis of the DEGs revealed a noteworthy correlation with metabolic pathways, with a specific focus on glucose metabolism. Specifically, the Glycolysis/Gluconeogenesis pathway exhibited significant upregulation following DIV1 infection. In line with this, we observed an augmented accumulation of glycolytic-related metabolites in the hemolymph following DIV1 challenge along with upregulation of the relative mRNA expression of several glycolytic-related genes. Moreover, we found that the inhibition of lactate dehydrogenase (LDH) activity through RNAi or the use of an inhibitor resulted in reduced lactate production, effectively safeguarding shrimp from DIV1 infection. These findings not only provide a comprehensive dataset for further investigation into DIV1 pathogenesis but also offer valuable insights into the immunometabolism mechanisms that govern shrimp responses to DIV1 infection.

A simple sequence repeats marker of disease resistance in shrimp Litopenaeus vannamei and its application in selective breeding.

The polymorphism of the simple sequence repeat (SSR) in the 5' untranslated coding region (5'-UTR) of the antiviral gene IRF (LvIRF) has been shown to be implicated in the resistance to viral pathogens in shrimp Litopenaeus vannamei (L. vannamei). In this study, we explored the potential of this (CT)n-SSR marker in disease resistance breeding and the hereditary property of disease resistance traits in offspring. From 2018 to 2021, eight populations were generated through crossbreeding by selecting individuals according to microsatellite genotyping. Our results demonstrated that shrimp with the shorter (CT)n repeat exhibited higher resistance to white spot syndrome virus (WSSV) or Decapod iridescent virus 1 (DIV1); meanwhile, these resistance traits could be inherited in offspring. Interestingly, we observed that the longer (CT)n repeats were associated with bacterial resistance traits. Accordingly, shrimp with longer (CT)n repeats exhibited higher tolerance to Vibrio parahaemolyticus infection. Taken together, these results indicate that the single (CT)n-SSR marker could be used to selective breeding for both resistance to virus and bacteria in shrimps.

A fluorescent probe based on an enhanced ICT effect for Hg2+ detection and cell imaging.

The mercury ion (Hg2+) has hindered society to some extent due to its high biological toxicity, and a rapid method for Hg2+ detection is urgently needed. In the present work, two fluorescent probes, YF-Hg and YF-Cl-Hg, were developed. YF-Cl-Hg was produced by introducing an electron-withdrawing substituent (-Cl) into the structure of YF-Hg. The probe YF-Cl-Hg possesses a larger Stokes shift and a more pronounced UV-vis absorption redshift compared to YF-Hg in a pH = 7.4 environment. The reasons for the superior spectral performance of YF-Cl-Hg over YF-Hg were explored by density functional theory (DFT) calculations and UV-vis absorption spectroscopy. Furthermore, the good biocompatibility suggests that YF-Cl-Hg possesses the potential to be a tool for Hg2+ detection in cells.

Identification of a Double-ß-Defensin with Multiple Antimicrobial Activities in a Marine Invertebrate.

ß-Defensins are a family of cysteine-rich antimicrobial peptides that are generally monodomain. Interestingly, the avian ß-defensin 11 (AvBD11) is unique, with two ß-defensin motifs with a broad range of antimicrobial activities. However, a double-sized ß-defensin has not been identified and functionally characterized in invertebrates. In this study, we cloned and identified a double-ß-defensin in shrimp Litopenaeus vannamei (named LvDBD) and explored its potential roles during infection with shrimp pathogens Vibrio parahaemolyticus and white spot syndrome virus (WSSV). LvDBD is an atypical double-sized defensin, which is predicted to possess two motifs related to ß-defensin and six disulfide bridges. The RNA interference-mediated knockdown of LvDBD in vivo results in phenotypes with increased bacterial loads, rendering the shrimp more susceptible to V. parahaemolyticus infection, which could be rescued by the injection of recombinant LvDBD protein. In vitro, rLvDBD could destroy bacterial membranes and enhance hemocyte phagocytosis, possibly attributable to its affinity to the bacterial wall components LPS and peptidoglycan. In addition, LvDBD could interact with several viral envelope proteins to inhibit WSSV proliferation. Finally, the NF-κB transcription factors (Dorsal and Relish) participated in the regulation of LvDBD expression. Taken together, these results extend the functional understanding of a double-ß-defensin to an invertebrate and suggest that LvDBD may be an alternative agent for the prevention and treatment of diseases caused by V. parahaemolyticus and WSSV in shrimp.

Signaling events induced by lipopolysaccharide-activated Toll in response to bacterial infection in shrimp.

Toll-like receptors (TLR) play a crucial role in the detection of microbial infections in vertebrates and invertebrates. Mammalian TLRs directly recognize a variety of structurally conserved microbial components. However, invertebrates such as Drosophila indirectly recognize microbial products by binding to the cytokine-like ligand Spätzle, which activates signaling cascades that are not completely understood. In this study, we investigated the signaling events triggered by Toll in response to lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria, and Vibrio parahaemolyticus infection in the arthropod shrimp Litopenaeus vannamei. We found that five of the nine Tolls from L. vannamei bound to LPS and the RNAi of LvToll1, LvToll2, LvToll3, LvToll5, and LvToll9 weakened LvDorsal-L phosphorylation induced by V. parahaemolyticus. All nine Tolls combined with MyD88 via the TIR domain, thereby conferring signals to the tumor necrosis factor receptor-associated factor 6 (TRAF6)-transforming growth factor-ß activated kinase 1 binding protein 2 (TAB2)-transforming growth factor-ß activated kinase 1 (TAK1) complex. Further examination revealed that the LvTRAF6-LvTAB2-LvTAK1 complex contributes to Dorsal-L phosphorylation and nuclear translocation during V. parahaemolyticus infection. Overall, shrimp Toll1/2/3/5/9-TRAF6/TAB2/TAK1-Dorsal cascades protect the host from V. parahaemolyticus infection, which provides a better understanding of how the innate immune system recognizes and responds to bacterial infections in invertebrates.

Dual colorimetric and near-infrared fluorescence probe for Hg2+ detection and cell imaging.

Hg2+ in the environment endangers human health, and a convenient monitoring method is needed for the detection of Hg2+. In this study, we constructed a dual colorimetric near-infrared fluorescent probe (E)-2-(3-(3-(1,3-dithian-2-yl)-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (YF-Hg), based on the malononitrile isophorone. YF-Hg can detect Hg2+ rapidly and sensitively, with fluorescence emission in the near-infrared region (659 nm) with an obvious color change from violet to red in the visible light range. In addition, the low toxicity and large Stokes shift (191 nm) of YF-Hg also suggest that it is a potential tool for live-cell fluorescence imaging.

An AhR-Caspase Axis Mediated Antiviral Apoptosis in an Arthropod.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates immune modulation following exposure of animals to many environmental xenobiotics. However, its role in innate immune responses during viral infection is not fully understood, especially in invertebrates. In this study, a cDNA encoding an AhR homolog was cloned from an arthropod Litopenaeus vannamei (LvAhR). The expression of LvAhR was strongly upregulated in response to the challenge of white spot syndrome virus, a pathogen of highly contagious and fatal infectious disease of shrimp. The relevance of LvAhR to host defense was underlined by heightened susceptibility and elevated virus loads after AhR-silenced shrimp exposure to white spot syndrome virus. LvAhR could induce an apoptosis response through regulating the expression of L. vannamei caspase-1 (homologous to human caspase-3) by directly targeting its promoter that was required to couple with AhR nuclear translocator. Additionally, knockdown of L. vannamei caspase-1 resulted in elevated virus titers and a lower cell apoptotic rate. Thus, we demonstrate that an AhR-caspase axis restrains virus replication by promoting antiviral apoptosis, supporting a previously unidentified direct link between AhR signaling and caspase-mediated apoptosis signaling and, furthermore, suggests that the AhR-caspase axis could be a potential therapeutic target for enhancing antiviral responses in arthropods.

Stimulator of interferon genes defends against bacterial infection via IKKß-mediated Relish activation in shrimp.

Stimulator of interferon genes (STING) is crucial for the innate immune to defend against pathogenic infections. Our previous study showed that a STING homolog from Litopenaeus vannamei (LvSTING) was involved in antibacterial response via regulating antimicrobial peptides (AMPs). Nevertheless, how LvSTING induces AMPs expression to inhibit bacterial infection remains unknown. Herein, we revealed that the existence of a STING-IKKß-Relish-AMPs axis in shrimp that was essential for opposing to Vibrio parahaemolyticus invasion. We observed that LvRelish was essential for host defense against V. parahaemolyticus infection via inducing several AMPs, such as LvALF1, LvCRU1, LvLYZ1 and LvPEN4. Knockdown of LvSTING or LvIKKß in vivo led to the attenuated phosphorylation and diminished nuclear translocation of LvRelish, as well as the impaired expression levels of LvRelish-regulated AMPs. Accordingly, shrimps with knockdown of LvSTING or LvIKKß or both were vulnerable to V. parahaemolyticus infection. Finally, LvSTING could recruit LvRelish and LvIKKß to form a complex, which synergistically induced the promoter activity of several AMPs in vitro. Taken together, our results demonstrated that the shrimp STING-IKKß-Relish-AMPs axis played a critical role in the defense against bacterial infection, and provided some insights into the development of disease prevention strategies in shrimp culture.

The MIP-T3 from shrimp Litopenaeus vannamei restricts white spot syndrome virus infection via regulating NF-κB activation.

In vertebrate, MIP-T3 (microtubule-interacting protein associated with TRAF3) functions as a regulator of innate immune response that involves many cellular processes. However, the immune response regulated by shrimp (an arthropod) MIP-T3 remains unrevealed. In the present study, a MIP-T3 homolog from shrimp Litopenaeus vannamei (named as LvMIP-T3) was cloned and identified. LvMIP-T3 had a 2076 bp open reading frame (ORF), encoding a polypeptide of 691 amino acids that contained a classic coiled-coil domain in the C-terminal that showed a high degree of conservation to other homologs. LvMIP-T3 could interact with LvTRAF6, a member of the canonical NF-κB pathway, but not LvTRAF3, which implies that LvMIP-T3 is able to regulate NF-κB activity via its interaction with LvTRAF6. In addition, LvMIP-T3 was substantially inducted in response to white spot syndrome virus (WSSV) challenge, and we demonstrated that LvMIP-T3 facilitated the expression of NF-κB-mediated several Penaeidins (antimicrobial peptides, AMPs) to oppose infection. Taken together, we identified a MIP-T3 homolog from shrimp L. vannamei that played a positive role in the TRAF6/NF-κB/AMPs axis mediated defense response, which will contribute to better understand the regulator relationship among members of the canonical NF-κB pathway in shrimp, and provides some insights into disease resistance breeding.

The MRE11 opposes white spot syndrome virus infection through the IRF and Dorsal mediated antiviral pathways in shrimp.

Meiotic recombination 11 (MRE11), a key component of the MRE11-RAD50-NBS1 (MRN) complex, plays important roles in damaged DNA repair and immune response. In this study, we described the molecular cloning of a new member of MRE11 from Litopenaeus vannamei named as LvMRE11. The full length of LvMRE11 was 2999 bp, including a 1947 bp open reading frame (ORF) that encoded a putative protein of 648 amino acids with a calculated molecular weight of ∼73.2 kDa LvMRE11 was universally expressed in all tested tissues and its expression in intestine was responsive to the challenge of white spot syndrome virus (WSSV), poly (I: C), poly [dA:dT], CpG-ODN 2006 and IFN stimulatory DNA (ISD). The dsRNA-mediated knockdown of LvMRE11 enhanced the susceptibility of shrimps to WSSV infection, as manifested by a higher mortality and viral loads observed in LvMRE11 silenced shrimps. Besides, silencing of LvMRE11 resulted in decreased expression levels of IRF-Vago-JAK/STAT pathway components, and Dorsal but not the Relish, as well as several antimicrobial peptides (AMPs). In conclusion, we provided some evidences that the involvement of LvMRE11 in innate immune against virus infection probably through regulating the IRF and Dorsal mediated antiviral pathways.

Interferon-Induced Protein 6-16 (IFI6-16) from Litopenaeus vannamei Regulate Antiviral Immunity via Apoptosis-Related Genes.

A growing number of evidence shows that some invertebrates possess an antiviral immunity parallel to the interferon (IFN) system of higher vertebrates. For example, the IRF (interferon regulatory factor)-Vago-JAK/STAT regulatory axis in an arthropod, shrimp Litopenaeus vannamei (whiteleg shrimp) is functionally similar to the IRF-IFN-JAK/STAT axis of mammals. IFNs perform their cellular immunity by regulating the expression of target genes collectively referred to as IFN-stimulated genes (ISGs). However, the function of invertebrate ISGs in immune responses is almost completely unclear. In this study, a potential ISG gene homologous to the interferon-induced protein 6-16 (IFI6-16) was cloned and identified from L. vannamei, designated as LvIFI6-16. LvIFI6-16 contained a putative signal peptide in the N-terminal, and a classic IFI6-16-superfamily domain in the C-terminal that showed high conservation to other homologs in various species. The mRNA levels of LvIFI6-16 were significantly upregulated after the stimulation of poly (I:C) and challenges of white spot syndrome virus (WSSV). Moreover, silencing of LvIFI6-16 caused a higher mortality rate and heightened virus loads, suggesting that LvIFI6-16 could play a crucial role in defense against WSSV. Interestingly, we found that the transcription levels of several caspases were regulated by LvIFI6-16; meanwhile, the transcription level of LvIFI6-16 self was regulated by the JAK/STAT cascade, suggesting there could be a JAK/STAT-IFI6-16-caspase regulatory axis in shrimp. Taken together, we identified a crustacean IFI6-16 gene (LvIFI6-16) for the first time, and provided evidence that the IFI6-16 participated in antiviral immunity in shrimp.

TAK1 confers antibacterial protection through mediating the activation of MAPK and NF-κB pathways in shrimp.

MAPK and NF-κB pathways are important components of innate immune system in multicellular animals. In some model organisms, the MAP3-kinase TGF-beta-activated kinase 1 (TAK1) have been shown to regulate both MAPK and NF-κB pathways activation to tailor immune responses to pathogens or infections. However, this process is not fully understood in shrimp. In this study, we investigated the effect of TAK1 on MAPK and NF-κB activation in shrimp Litopenaeus vannamei following Vibrio parahaemolyticus infection. We found that shrimp TAK1 could activate c-Jun and Relish, the transcription factors of MAPK pathway and NF-κB pathway, respectively. Specifically, over-expression of shrimp TAK1 was able to strongly induce the activities of both AP-1 and NF-κB reporters. TAK1 was shown to bind several MAP2-kinases, including MKK4, MKK6 and MKK7, and induced their phosphorylations, the hallmarks for MAPK pathways activation. TAK1 knockdown in vivo also inhibited the nuclear translocation of c-Jun and Relish during V. parahaemolyticus infection. Accordingly, ectopic expression of shrimp TAK1 in Drosophila S2 cells increased the cleavage of co-expressed shrimp Relish, and induced the promoter activity of Relish targeted gene Diptericin (Dpt). Furthermore, knockdown of c-Jun and Relish enhanced the sensitivity of shrimp to V. parahaemolyticus infection. These findings indicated that shrimp TAK1 conferred antibacterial protection through regulating the activation of both MAPK pathway and NF-κB pathway, and suggested that the TAK1-MAPK/NF-κB axis could be a potential therapeutic target for enhancing antibacterial responses in crustaceans.

Identification and function of an Arasin-like peptide from Litopenaeus vannamei.

Antimicrobial peptides (AMPs) play an important role in the host defense system of shrimps. In this study, an Arasin-like peptide, named as LvArasin-like, was identified from the hemocytes of the pacific white shrimp, Litopenaeus vannamei. The complete open reading frame (ORF) of LvArasin-like was 213 bp, encoding 70 amino acid residues with a predicted molecular mass of 5.68 kDa and a theoretical isoelectric point (pI) of 6.73. The predicted peptide consisted of a signal peptide, an N-terminal Pro/Arg-rich domain, and a C-terminal cysteine-rich domain. LvArasin-like expression was most abundant in the gills and was up-regulated in hemocytes after LPS or Poly I:C injection as well as challenges by Vibrio parahaemolyticus or Staphylococcus aureus infection. In the heterologous expression system, LvArasin-like protein (rLvArasin-like) was recombinantly expressed in the forms of a dimer or both a monomer and dimer. The rLvArasin-like could directly bind to gram-positive and gram-negative bacteria and exhibited broad-spectrum antimicrobial activity towards them, with 50 % of minimal inhibitory concentrations (MIC50) of 6.25-50 µM. Moreover, dsRNA-mediated knockdown of LvArasin-like enhanced the susceptibility of shrimp to V. parahaemolyticus. In addition, the transcriptional level of LvArasin-like was downregulated when silencing of the transcription factors LvDorsal and LvRelish using RNAi in vivo. All of these results suggest that LvArasin-like is involved in host defense against bacterial infection. Therefore, it is a potential therapeutic agent for disease control in shrimp aquaculture.

Elaborating the excited-state double proton transfer mechanism and multiple fluorescent characteristics of 3,5-bis(2-hydroxypheny)-1H-1,2,4-triazole.

Recently, Krishnamoorthy and coworkers reported a new type of proton transfer, which was labeled as 'proton transfer triggered proton transfer', in 3,5-bis(2-hydroxypheny)-1H-1,2,4-triazole (bis-HPTA). In this work, the excited-state double proton transfer (ESDPT) mechanism and multiple fluorescent characteristics of bis-HPTA were investigated. Upon photo-excitation, the intramolecular hydrogen bonding strength changed and the electron density of bis-HPTA redistributed. These changes will affect the proton transfer process. In S0 state, the proton transfer processes of bis-HPTA were prohibited on the stepwise and concerted pathways. After vertical excitation to the S1 state, the ESIPT-II process was more likely to occur than the ESIPT-I process, which was contrary to the conclusion that the ESIPT-II process is blocked and the ESIPT-II process takes place after the ESIPT-I process proposed by Krishnamoorthy and coworkers. When the K2 tautomer was formed through the ESIPT-II process, the second proton transfer process on the stepwise pathway was prohibited. On another stepwise pathway, after the ESIPT-I process (form the K1 tautomer), the second proton transfer process should overcome a higher potential barrier than the ESIPT-I process to form ESDPT tautomer. On the concerted pathway, the bis-HPTA can synchronous transfer double protons to form the ESDPT tautomer. The ESDPT tautomer was unstable and immediately converted to the K2 tautomer via a barrierless reverse proton transfer process. Thus, the fluorescent maximum at 465 nm from the ESDPT tautomer reported by Krishnamoorthy and coworkers was ascribed to the K2 tautomer. Most of the fluorophores show dual fluorescent properties, while the bis-HPTA undergoing ESDPT process exhibited three well-separated fluorescent peaks, corresponding to its normal form (438 nm), K1 tautomer (462 nm) and K2 tautomer (450 nm), respectively.