Drosophila immune cells transport oxygen through PPO2 protein phase transition.

Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells1,2. Here we describe that Drosophila crystal cells-myeloid-like immune cells called haemocytes-control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, we propose that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.

Dephosphorylation of T517 on Hemocyanin Is Required for Antibacterial Activity in Penaeus vannamei.

Posttranslational modifications expand the functions of immune-related proteins, especially during infections. The respiratory glycoprotein, hemocyanin, has been implicated in many other functions, but the role of phosphorylation modification in its functional diversity is not fully understood. In this study, we show that Penaeus vannamei hemocyanin (PvHMC) undergoes phosphorylation modification during bacterial infection. Dephosphorylation of PvHMC mediated by P. vannamei protein phosphatase 2A catalytic increases its in vitro antibacterial activity, whereas phosphorylation by P. vannamei casein kinase 2 catalytic subunit α decreases its oxygen-carrying capacity and attenuates its in vitro antibacterial activity. Mechanistically, we show that Thr517 is a critical phosphorylation modification site on PvHMC to modulate its functions, which when mutated attenuates the action of P. vannamei casein kinase 2 catalytic subunit α and P. vannamei protein phosphatase 2A catalytic, and hence abolishes the antibacterial activity of PvHMC. Our results reveal that phosphorylation of PvHMC modulates its antimicrobial functions in penaeid shrimp.

Function of hemocyanin-mediated succinate dehydrogenase in glucose metabolism and immunity of Penaeus vannamei.

Succinate dehydrogenase (SDH) is a crucial enzyme in the tricarboxylic acid cycle (TCA) and has established roles in immune function. However, the understanding of SDH in Penaeus vannamei, particularly its involvement in immune responses, is currently limited. Through affinity proteomics, a potential interaction between hemocyanin (HMC) and SDH in shrimp has been identified. The successful cloning of PvSDH in this study has revealed a high degree of evolutionary conservation. Additionally, it has been found that hemocyanin regulates SDH not only at the transcriptional and enzymatic levels but also through confirmed protein-protein interactions observed via Co-immunoprecipitation (CoIP) assay. Moreover, by combining PvHMC knockdown and Vibrio parahaemolyticus challenge, it was demonstrated that fumaric acid, a product of SDH, enhances the host's immune resistance to pathogen infection by modulating the expression of antimicrobial peptides. This research provides new insights into HMC as a crucial regulator of SDH, potentially impacting glycometabolism and the dynamics of immune responses.

The Microbial Composition of Penaeid Shrimps' Hepatopancreas Is Modulated by Hemocyanin.

Aquatic organisms have to produce proteins or factors that help maintain a stable relationship with microbiota and prevent colonization by pathogenic microorganisms. In crustaceans and other aquatic invertebrates, relatively few of these host factors have been characterized. In this study, we show that the respiratory glycoprotein hemocyanin is a crucial host factor that modulates microbial composition and diversity in the hepatopancreas of penaeid shrimp. Diseased penaeid shrimp (Penaeus vannamei), had an empty gastrointestinal tract with atrophied hepatopancreas, expressed low hemocyanin, and high total bacterial abundance, with Vibrio as the dominant bacteria. Similarly, shrimp depleted of hemocyanin had mitochondrial depolarization, increased reactive oxygen species (ROS) levels, and dysregulation of several energy metabolism-related genes. Hemocyanin silencing together with ROS scavenger (N-acetylcysteine) treatment improved microbial diversity and decreased Vibrio dominance in the hepatopancreas. However, fecal microbiota transplantation after hemocyanin knockdown could not restore the microbial composition in the hepatopancreas. Collectively, our data provide, to our knowledge, new insight into the pivotal role of hemocyanin in modulating microbial composition in penaeid shrimp hepatopancreas via its effect on mitochondrial integrity, energy metabolism, and ROS production.

Modulation of SREBP Expression and Fatty Acid Levels by Bacteria-Induced ER Stress Is Mediated by Hemocyanin in Penaeid Shrimp.

Many environmental and pathogenic insults induce endoplasmic reticulum (ER) stress in animals, especially in aquatic ecosystems, where these factors are crucial for life. In penaeid shrimp, pathogens and environmental stressors induce hemocyanin expression, but the involvement of hemocyanin in ER stress response is unknown. We demonstrate that in response to pathogenic bacteria (Vibrio parahaemolyticus and Streptococcus iniae), hemocyanin, ER stress proteins (Bip, Xbp1s, and Chop), and sterol regulatory element binding protein (SREBP) are induced to alter fatty acid levels in Penaeus vannamei. Interestingly, hemocyanin interacts with ER stress proteins to modulate SREBP expression, while ER stress inhibition with 4-Phenylbutyric acid or hemocyanin knockdown attenuates the expression of ER stress proteins, SREBP, and fatty acid levels. Contrarily, hemocyanin knockdown followed by tunicamycin treatment (ER stress activator) increased their expression. Thus, hemocyanin mediates ER stress during pathogen challenge, which consequently modulates SREBP to regulate the expression of downstream lipogenic genes and fatty acid levels. Our findings reveal a novel mechanism employed by penaeid shrimp to counteract pathogen-induced ER stress.

Effects of chronic exposure to cadmium and copper on the proteome profile of hemolymph in false widow spider Steatoda grossa (Theridiidae).

The aim of this study was to evaluate the quantitative and qualitative changes in the proteome of the hemolymph of female Steatoda grossa spiders (Theridiidae) that were chronically exposed to cadmium and copper in food and were additionally immunostimulated (phorbol 12-myristate 13-acetate (PMA); bacterial suspensions: Staphylococcus aureus (G+), Pseudomonas fluorescens (G-). It was found that the expression of nearly 90 proteins was altered in cadmium-intoxicated spiders and more than 60 in copper-exposed individuals. Regardless of the type of metal used, these proteins were mainly overexpressed in the hemolymph of the exposed spiders. On the other hand, immunostimulation did not significantly change the number of proteins with altered expression in metal-intoxicated individuals. Hemocyanin (Hc) was found to be the most abundant of the proteins identified with altered expression. In copper-intoxicated spiders, immunostimulation increased the expression of A-, E-, F-, and G-chain-containing proteins, while in the case of cadmium-intoxicates spiders, it decreased the expression of E- and A-chain-containing Hc and increased the expression of G-chain-containing Hc. Regardless of the type of metal and immunostimulant used, there was an increase in the expression of actin. In addition, cadmium increased the expression of cullin, vimentin, and ceruloplasmin. The changes observed in the expression of hemolymph proteins indicate their protective function in S. grossa (Theridiidae) spiders under conditions of metal exposure.

Lipoprotein-induced cell growth and hemocyanin biosynthesis in rhogocytes.

Rhogocyte is a unique molluscan cell that synthesises a supramolecular respiratory protein known as hemocyanin. Its ability to synthesise the protein has eluded the scientists despite hemocyanin's importance as a carrier protein and complex molecule with anti-viral activity. Although a hypothetical model of hemocyanin release from the rhogocytes lacunae was proposed based on colloid-osmotic pressure mechanism, lack of in vitro studies limits further validation of this model. In this study, we aim to investigate the impact of cell culture conditions and nature of hemocyanin biosynthesis of rhogocyte cells dissociated from Haliotis laevigata mantle tissue. Population of cells with different hemocyanin expression levels was profiled using flow cytometry, while hemocyanin concentrations in the media were elucidated by ELISA assay. We demonstrated that addition of lipoprotein supplement into the media resulted in a burst secretion of hemocyanin into the culture media. Over 7 days of culture, the population of cells tagged with hemocyanin antibody increased steadily while hemocyanin release in the media decreased significantly. Variation of culture medium, temperature, growth supplement type and concentration also impacted the cell growth and hemocyanin biosynthesis. These results indicated the possibility of an active process triggered by the addition of supplement to synthesise the protein at the highest amount during the first hour. The current study provides a glimpse of the hemocyanin biosynthesis by rhogocyte that may be significant to understand the cell ability to synthesise supramolecular protein and secretion through lacunae.

LvHemB1, a novel cationic antimicrobial peptide derived from the hemocyanin of Litopenaeus vannamei, induces cancer cell death by targeting mitochondrial voltage-dependent anion channel 1.

Current cancer treatment regimens such as chemotherapy and traditional chemical drugs have adverse side effects including the appearance of drug-resistant tumor cells. For these reasons, it is imperative to find novel therapeutic agents that overcome these factors. To this end, we explored a cationic antimicrobial peptide derived from Litopenaeus vannamei hemocyanin (designated LvHemB1) that induces cancer cell death, but sparing normal cells. LvHemB1 inhibits the proliferation of human cervical (HeLa), esophageal (EC109), hepatocellular (HepG2), and bladder (EJ) cancer cell lines, but had no significant effect on normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE-3) cells). In addition to its antiproliferative effects, LvHemB1 induced apoptosis, by permeating cells and targeting mitochondrial voltage-dependent anion channel 1 (VDAC1). Colocalization studies revealed the localization of LvHemB1 in mitochondria, while molecular docking and pull-down analyses confirmed LvHemB1-VDAC1 interaction. Moreover, LvHemB1 causes loss in mitochondrial membrane potential and increases levels of reactive oxygen species (ROS) and apoptotic proteins (caspase-9, caspase-3, and Bax (Bcl-2-associated X)), which results in mitochondrial-mediated apoptosis. Thus, peptide LvHemB1 has the potential of being used as an anticancer agent due to its antiproliferation effect and targeting to VDAC1 to cause mitochondrial dysfunction in cancer cells, as well as its ability to induce apoptosis by increasing ROS levels, and the expression of proapoptotic proteins.

Functional Characterization, Antimicrobial Effects, and Potential Antibacterial Mechanisms of NpHM4, a Derived Peptide of Nautilus pompilius Hemocyanin.

Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of Nautilus pompilius have not been reported. Here, the bactericidal activity of six predicted peptides from N. pompilius hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), NpHM4, showed relatively high antibacterial activity. NpHM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, NpHM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 µM for Vibrio alginolyticus), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with V. alginolyticus cells, we confirmed that the bactericidal activity of NpHM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.

Ammonia stress affects the structure and function of hemocyanin in Penaeus vannamei.

Anthropogenic factors and climate change have serious effects on the aquatic ecosystem and aquaculture. Among water pollutants, ammonia has the greatest impact on aquaculture organisms such as penaeid shrimp because it makes them more susceptible to infections. In this study, we explored the effects of ammonia stress (0, 50, 100, and 150 mg/L) on the molecular structure and functions of the multifunctional respiratory protein hemocyanin (HMC) in Penaeus vannamei. While the mRNA expression of Penaeus vannamei hemocyanin (PvHMC) was up-regulated after ammonia stress, both plasma hemocyanin protein and oxyhemocyanin (OxyHMC) levels decreased. Moreover, ammonia stress changed the molecular structure of hemocyanin, modulated the expression of protein phosphatase 2 A (PP2A) and casein kinase 2α (CK2α) to regulate the phosphorylation modification of hemocyanin, and enhanced its degradation into fragments by trypsin. Under moderate ammonia stress conditions, hemocyanin also undergoes glycosylation to improve its in vitro antibacterial activity and binding with Gram-negative (Vibrio parahaemolyticus) and Gram-positive (Staphylococcus aureus) bacteria, albeit differently. The current findings indicate that P. vannamei hemocyanin undergoes adaptive molecular modifications under ammonia stress enabling the shrimp to survive and counteract the consequences of the stress.

Embryonic and post-embryonic development of the spider Polybetes pythagoricus (Sparassidae): A biochemical point of view.

Analysis of energy expense during development has achieved special interest through time on account of the crucial role of the consumption of resources required for offspring survival. Spider eggs have a fixed composition as well as some initial energy that is supplied by mothers. These resources are necessary to support the metabolic expense not only through the embryonic period but also during the post-embryonic period, as well as for post emerging activities before spiderlings become self-sustaining. Depletion of these resources would be critical for spiders since it could give rise to prey competition as well as filial cannibalism. Even though spiders represent a megadiverse order, information regarding the metabolic requirements during spiders development is very scarce. In this study, we analyse the changes in protein, lipid and carbohydrate content as well as the variation in lipovitellin reserves and hemocyanin content during Polybetes pythagoricus development. Our results show that lipovitellins and phospholipids represent the major energy source throughout embryonic and post-embryonic development. Lipovitellin apolipoproteins are gradually consumed but are later depleted after dispersion. Phosphatidylethanolamine is mainly consumed during the post-embryonic period, while triacylglycerides are consumed after juveniles' dispersion. Finally, hemocyanin concentration starts to increase in postembryonic stages.

Molecular isolation and expression analysis of hemocyanin isoform 2 of Macrobrachium rosenbergii.

OBJECTIVE: Hemocyanin is a copper-bearing protein in the hemolymph of many arthropods and mollusks and functions as an oxygen transport and important nonspecific immune protein. METHODS: In this study, complementary DNA of hemocyanin isoform 2 of the prawn Macrobrachium rosenbergii (MrHc2) was isolated by rapid amplification of cDNA ends and mRNA expression was characterized to elucidate molecular basis of its function. RESULT: With a molecular mass of 77.3 kDa, MrHc2 contained three domains: hemocyanin-all-alpha, hemocyanin-copper-containing, and hemocyanin-immunoglobulin-like domains. Molecular phylogenetic analysis revealed that MrHc2 belongs to the γ-type subunit and is closely related to hemocyanin subunit 1 of the palaemonid shrimp Macrobrachium nipponense. In addition, MrHc2 resided in a different clade relative to hemocyanin (MrHc) of M. rosenbergii (α-type subunit) and in a different subclade relative to the hemocyanin proteins of penaeid shrimp. The messenger RNA transcript of MrHc2 was highly expressed in the hepatopancreas and weakly expressed in the gills, intestine, stomach, muscle, and hemocytes. Upon challenge with M. rosenbergii nodavirus (MrNV), the expression of MrHc2 was 1.96-, 2.93-, and 1.96-fold on days 3, 4, and 5, respectively, and then gradually declined to basal levels on day 7. CONCLUSION: This study suggests that MrHc2 plays an important role in the innate immune response of M. rosenbergii to MrNV.

trans-3-Methylglutaconyl CoA isomerization-dependent protein acylation.

3-methylglutaconic (3MGC) aciduria is associated with a growing number of discrete inborn errors of metabolism. Herein, an antibody-based approach to detection/quantitation of 3MGC acid has been pursued. When trans-3MGC acid conjugated keyhole limpet hemocyanin (KLH) was inoculated into rabbits a strong immune response was elicited. Western blot analysis provided evidence that immune serum, but not pre-immune serum, recognized 3MGC-conjugated bovine serum albumin (BSA). In competition ELISAs using isolated immune IgG, the limit of detection for free trans-3MGC acid was compared to that for cis-3MGC acid and four structurally related short-chain dicarboxylic acids. Surprisingly, cis-3MGC acid yielded a much lower limit of detection (∼0.1 mg/ml) than trans-3MGC acid (∼1.0 mg/ml) while all other dicarboxylic acids tested were poor competitors. The data suggest trans-3MGC- isomerized during, or after, conjugation to KLH such that the immunogen was actually comprised of KLH harboring a mixture of cis- and trans-3MGC haptens. To investigate this unexpected isomerization reaction, trans-3MGC CoA was prepared and incubated at 37 °C in the presence of BSA. Evidence was obtained that non-enzymatic isomerization of trans-3MGC CoA to cis-3MGC CoA precedes intramolecular catalysis to form cis-3MGC anhydride plus CoASH. Anhydride-dependent acylation of BSA generated 3MGCylated BSA, as detected by anti-3MGC immunoblot. The results presented provide an explanation for the unanticipated detection of 3MGCylated proteins in a murine model of primary 3MGC aciduria. Furthermore, non-enzymatic hydrolysis of cis-3MGC anhydride represents a potential source of cis-3MGC acid found in urine of subjects with 3MGC aciduria.

Molluscan Hemocyanins.

Instead of the red blood of vertebrates, most molluscs have blue hemolymph containing hemocyanin, a type-3 copper-containing protein. The hemoglobin of vertebrate blood is replaced in most molluscs with hemocyanin, which plays the role of an  oxygen transporter. Oxygen-binding in hemocyanin changes its hue from colorless deoxygenated hemocyanin into blue oxygenated hemocyanin. Molecules of molluscan hemocyanin are huge, cylindrical multimeric proteins-one of the largest protein molecules in the natural world. Their huge molecular weight (from 3.3 MDa to more than 10 MDa) are the defining characteristic of molluscan hemocyanin, a property that has complicated structural analysis of the molecules for a long time. Recently, the structural analysis of a cephalopod (squid) hemocyanin has succeeded using a hybrid method employing both X-ray crystallography and cryo-EM. In a biochemical breakthrough for molluscan hemocyanin, the first quaternary structure with atomic resolution is on the verge of solving the mystery of molluscan hemocyanin. Here we describe the latest information about the molecular structure, classification and evolution of the molecule, and the physiology of molluscan hemocyanin.

Arachnid Hemocyanins.

Hemocyanin (Hc), a copper-containing extracellular multimeric protein, is the major protein component of hemolymph in different arachnid groups. Hc possesses 7 or 8 very well-characterized types of monomers with molecular weights ranging from 70 to 85 kDa, organized in hexamers or multiple of hexamers. The present chapter compiles the existing data with relation to the function of this protein in the arachnids. Hc has as main function the reversible transport of O2, but it shows many secondary though not less important functions. With reference to this, it has been described that Hc can transport hydrophobic molecules (lipid-derived hormones and lipids) to the different organs, having a key role in the lipid transport system. In arachnids, like in other arthropods and invertebrates, Hc has phenoloxidase function which is related to different metabolic processes such as melanin formation and defense against pathogens. In addition, Hc has additional defensive functions since it can serve as precursor for the production of antimicrobial peptides. In short, the evolution of this protein has led to the development of multiple functions essential for organisms possessing this protein.

Multifunctional Roles of Hemocyanins.

The copper-containing hemocyanins are proteins responsible for the binding, transportation and storage of dioxygen within the blood (hemolymph) of many invertebrates. Several additional functions have been attributed to both arthropod and molluscan hemocyanins, including (but not limited to) enzymatic activity (namely phenoloxidase), hormone transport, homeostasis (ecdysis) and hemostasis (clot formation). An important secondary function of hemocyanin involves aspects of innate immunity-such as acting as a precursor of broad-spectrum antimicrobial peptides and microbial/viral agglutination. In this chapter, we present the reader with an up-to-date synthesis of the known functions of hemocyanins and the structural features that facilitate such activities.

Hemocyanin and hexamerins expression in response to hypoxia in stoneflies (Plecoptera, Insecta).

Many freshwater ecosystems worldwide undergo hypoxia events that can trigger physiological, behavioral, and molecular responses in many organisms. Among such molecular responses, the regulation of the hemocyanin (Hc) protein expression which plays a major role in oxygen transportation within aquatic insects remains poorly understood. The stoneflies (Plecoptera) are aquatic insects that possess a functional Hc in the hemolymph similar to crustacean that co-occurs with a nonfunctional Hc protein, hexamerins (Hx). However, the role of both proteins during hypoxia remains undetermined. Here, we evaluated the effect of hypoxia on the expression of Hc and Hx proteins via a comparison between hypoxia and normoxia amino acid sequence variation and protein expression pattern within 23 stonefly species. We induced short-term hypoxia in wild-caught stoneflies species, sequenced the target region of Hc and Hx by complementary DNA synthesis, characterized the protein biochemistry using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ultrafiltration, and polarographic fluorometric method, and amplified the genome region of the hypoxia-inducible factor (HIF) transcriptional response element that regulated Hc using genome walking library approach. We found a lack of Hc expression in all examined species during hypoxia conditions, despite recognition of the HIF gene region as a possible regulatory factor of Hc, suggesting that compensatory responses as metabolic changes or behavioral tracheal movements to enhance respiratory efficiency could be possible mechanics to compensate for hypoxia. A short Hc-like novel isoform was detected instead in these 23 species, possibly due to either protein degradation or alternative splicing mechanisms, suggesting that the protein could be performing a different function other than oxygen transportation. Hx during hypoxia was expressed and exhibited species-level amino acid changes, highlighting a possible role during hypoxia. Our results demonstrate that hypoxia could enable a similar potential adaptive response of multiple species regarding specific physiological requirements, thereby shedding light on community behavior in stress environments that may help us to improve conservation practices and biomonitoring.

Developmental expression and evolution of hexamerin and haemocyanin from Folsomia candida (Collembola).

Haemocyanins constitute a group of copper-containing respiratory proteins, and hexamerins were derived from hexapod haemocyanin but lost the ability to transport oxygen and serve as storage proteins. Although hexamerins have been reported in most insect species, none of them has been identified in Collembola, one of the most primitive hexapod lineages, thereby preventing us from exploring relevant evolutionary scenarios regarding the origin and evolution of hexamerins in hexapods. Here we report on collembolan hexamerins for the first time, and investigated the temporal expression profiles of hexamerin and haemocyanin in the collembolan Folsomia candida. Haemocyanin was expressed over the entire life cycle, with higher expression at the embryonic stage than at other stages, whereas hexamerin expression was restricted to embryos, unlike insect hexamerins, which are generally expressed from larval to adult stages. A phylogenetic analysis and molecular clock estimation suggested that all investigated hexapod hexamerins have a single and ancient origin (~423 Ma), coincident with the rise of atmospheric oxygen levels in the Silurian-Devonian period, indicating a physiological link between molecular evolution and Palaeozoic oxygen changes.

Formulation and Characterization of Conjugate Vaccines to Reduce Opioid Use Disorders Suitable for Pharmaceutical Manufacturing and Clinical Evaluation.

This study focused on formulating conjugate vaccines targeting oxycodone and heroin for technology transfer, good manufacturing practice (GMP), and clinical evaluation. Lead vaccines used the highly immunogenic carrier protein keyhole limpet hemocyanin (KLH), which poses formulation problems because of its size. To address this barrier to translation, an oxycodone-based hapten conjugated to GMP-grade subunit KLH (OXY-sKLH) and adsorbed on alum adjuvant was studied with regard to carbodiimide coupling reaction time, buffer composition, purification methods for conjugates, conjugate size, state of aggregation, and protein/alum ratio. Vaccine formulations were screened for post-immunization antibody levels and efficacy in reducing oxycodone distribution to the brain in rats. While larger conjugates were more immunogenic, their size prevented characterization of the haptenation ratio by standard analytical methods and sterilization by filtration. To address this issue, conjugation chemistry and vaccine formulation were optimized for maximal efficacy, and conjugate size was measured by dynamic light scattering prior to adsorption to alum. An analogous heroin vaccine (M-sKLH) was also optimized for conjugation chemistry, formulated in alum, and characterized for potency against heroin in rats. Finally, this study found that the efficacy of OXY-sKLH was preserved when co-administered with M-sKLH, supporting the proof of concept for a bivalent vaccine formulation targeting both heroin and oxycodone. This study suggests methods for addressing the unique formulation and characterization challenges posed by conjugating small molecules to sKLH while preserving vaccine efficacy.

c-Jun regulates the promoter of small subunit hemocyanin gene of Litopenaeus vannamei.

Hemocyanin (HMC) is a respiratory glycoprotein, which also plays multifunctional non-specific innate immune defense functions in shrimp. However, the transcriptional regulatory mechanisms of the hemocyanin gene expression have not been reported. In the present study, we cloned a 4324 bp fragment of small subunit hemocyanin (HMCs) gene of Litopenaeus vannamei including the 5'-flanking region, from upstream 2475 bp to downstream 1849 bp (exon 1-intron 1-exon 2) by genome walking method. Four deletion constructs were then generated and their promoter activity assessed using the luciferase reporter system. Interestingly, we identified an alternative promoter (+1516/+1849 bp) located in exon 2, which has stronger promoter activity than the full-length or the other constructs. Bioinformatics analyses revealed that the alternative promoter region contains two conserved binding sites of the transcription factor c-Jun. Mutational analysis and electrophoretic mobility shift assay showed that Litopenaeus vannamei c-Jun (Lvc-Jun) binds to the region +1582/+1589 bp and +1831/+1837 bp of the alternative promoter. Furthermore, overexpression of Lvc-Jun significantly increased the alternative promoter activity, while co-transfection with dsRNA-Lvc-Jun significantly reduced the alternative promoter activity of HMCs. Taken together, our present data indicate that the transcription factor Lvc-Jun is essential for the transcriptional regulation of the HMCs gene expression.