Fibrinogen-related Protein Research Articles

Molecular characterization, expression and in-silico analysis of fibrinogen-related protein 1 (frep1) in malaria vector Anopheles stephensi.

Fibrinogen-related protein 1 (frep1) is a member of the pattern-recognizing receptor family (PRR) which generates an innate immune response after recognizing the pattern associated molecular pattern (PAMP) that occurs on the surface of microorganisms. The main objective of this study is to characterize frep1 and its in-silico analysis in Anopheles stephensi. The DNA was extracted from female Anopheles stephensi. PCR was performed for complete analysis of frep1 using specific primers. The gene sequence of frep1 was identified by Sanger sequencing. The bioinformatics structure analysis approach revealed the presence of 3 exons and 4 introns in the frep1. The sequence of frep1 was submitted to NCBI GeneBank with accession number ON817187.1. Quantitative real-time PCR was performed to analyze frep1 expression. At the developmental stage, frep1 is highly expressed in the L1 stage, egg, and adult female mosquito. In addition, frep1 is highly expressed in the tissue fat body, midgut, and salivary gland. After blood-fed, an upregulation of frep1 at 48h in the midgut, and downregulation in fat body were observed at different time intervals. The genomic data of frep1 is encoded by 12,443bp. The frep1 has a significant role in the early metamorphosis. Its expression in fat body and midgut suggests it could be important for fat metabolism and post-blood digestion. The conserved domain could be targeted for vector control. Further study is required to elucidate its function against malaria parasites to confirm its agonist role in malaria transmission.

Comparative transcriptome analysis reveals the different responding mechanisms of ovary and hepatopancreas following polyI:C challenge in Macrobrachium nipponense

The ovary in mammals has developed specialized mechanisms for protection against pathogen infections; however, the understanding of the innate immune system in the ovary of crustaceans is still limited. To elucidate the ovary's defense mechanisms in response to viral challenges, we subjected oriental river prawns (Macrobrachium nipponense) to poly I:C, a double-stranded RNA analog that emulates viral dsRNA, and analyzed the ovary's transcriptome profiles. Concurrently, RNA-seq analysis was performed on the hepatopancreas, a well-recognized immune-related tissue, following poly I:C challenge to investigate the distinct response mechanisms of the ovary and hepatopancreas and to gain a comprehensive understanding of the immune responses in both tissues. The results indicate that 1368 genes are differentially expressed in the ovary, with 903 genes upregulated and 465 genes downregulated. Subsequent analysis reveals that these differentially expressed genes (DEGs) include numerous genes associated with innate immunity, such as members of the C-type lectin, fibrinogen-related protein (Frep), Toll-like receptor, and NOD-like receptor (NLR) gene families, as well as acid phosphatase, scavenger receptor, crustin, Down syndrome cell adhesion molecule (Dscam), hemocyanin, and lipopolysaccharide and beta-1,3-glucan binding protein (LGBP). Furthermore, the DEGs include several genes related to ovary development, such as sox8, vitellogenin, progranulin, cyclin-dependent kinase, ecdysone receptor, frizzled, and members of the Fox gene family. In the hepatopancreas, a total of 729 DEGs were identified. Comparison of the DEGs in both tissues indicates that only 91 genes are common to both groups, highlighting significant tissue-specific responses to poly I:C stimulation. This study aims to enhance our understanding of the immune protective mechanisms employed by the ovary in response to pathogen exposure and establishes a foundation for investigating ovarian reproductive immunity in crustaceans.

Full-length transcriptome analysis provides new insights into the diversity of immune-related genes in the threatened freshwater shellfish Solenaia oleivora.

Solenaia oleivora, a valuable and rare bivalve endemic to China, is becoming a threatened freshwater sepcies. However, the lack of research on its genome and immune system will hinder advances in its conservation and artificial breeding. In this study, we obtained the full-length transcriptome of S. oleivora using PacBio sequencing. A total of 21,415 transcripts with an average length of 1,726 bp were generated. Among these transcripts, 12,084 had coding sequences (CDS), of which 8,639 were annotated in 6 databases. The structure analysis identified 625 transcript factors (TFs), 8,005 long non-coding RNAs (lncRNAs), and 5,288 simple sequences repeat (SSRs). Meanwhile, massive immune genes were identified from the transcriptome of S. oleivora. In terms of non-self-identification, 97 transcripts of pattern recognition receptors (PRRs) were discovered, including peptidoglycan recognition proteins (PGRPs), gram-negative bacteria binding proteins (GNBPs), toll-like receptors (TLRs), scavenger receptors (SRs), galectins (GALs), C-type lectins (CLTs), and fibrinogen-related protein (FREPs). For pathogen elimination, 7 transcripts related to antimicrobial peptides, lysozymes, and lysosomal enzymes were identified. Moreover, 33 complement-associated transcripts were found. This study enriched the genome resources of S. oleivora and provided new insights for the study of the immune system of S. oleivora.

Targeting plasmodium α-tubulin-1 to block malaria transmission to mosquitoes.

Plasmodium ookinetes use an invasive apparatus to invade mosquito midguts, and tubulins are the major structural proteins of this apical complex. We examined the role of tubulins in malaria transmission to mosquitoes. Our results demonstrate that the rabbit polyclonal antibodies (pAb) against human α-tubulin significantly reduced the number of P. falciparum oocysts in Anopheles gambiae midguts, while rabbit pAb against human β-tubulin did not. Further studies showed that pAb, specifically against P. falciparum α-tubulin-1, also significantly limited P. falciparum transmission to mosquitoes. We also generated mouse monoclonal antibodies (mAb) using recombinant P. falciparum α-tubulin-1. Out of 16 mAb, two mAb, A3 and A16, blocked P. falciparum transmission with EC50 of 12 μg/ml and 2.8 μg/ml. The epitopes of A3 and A16 were determined to be a conformational and linear sequence of EAREDLAALEKDYEE, respectively. To understand the mechanism of the antibody-blocking activity, we studied the accessibility of live ookinete α-tubulin-1 to antibodies and its interaction with mosquito midgut proteins. Immunofluorescent assays showed that pAb could bind to the apical complex of live ookinetes. Moreover, both ELISA and pull-down assays demonstrated that insect cell-expressed mosquito midgut protein, fibrinogen-related protein 1 (FREP1), interacts with P. falciparum α-tubulin-1. Since ookinete invasion is directional, we conclude that the interaction between Anopheles FREP1 protein and Plasmodium α-tubulin-1 anchors and orients the ookinete invasive apparatus towards the midgut PM and promotes the efficient parasite infection in the mosquito.

Fibrinogen-Related Protein, Fgl2, of Hamster Cauda Epididymal Fluid: Enzymatic Characterization, and Identification of Fgl2-Binding Proteins and Ligand of Defective Hamster Sperm Organelles.

The luminal environment of the mammalian epididymidis performs a dual function; sperm maturation and maintaining sperm viability. We previously identified a secretory protein (260/280KDa oligomers) of hamster cauda epididymal principal cells that binds to nonviable sperm. The 260/280KDa oligomers are composed of 64kDa FGL2 (fibrinogen-like protein-2) and 33kDa FGL1) (fibrinogen-like protein-1). The potential mechanism by which FGL2 binds to degenerative sperm is not clearly demonstrated. In this study, we report the downstream sequence of prothrombinase activity of FGL2, the identification of organelles, and characterize candidate proteins that bind FGL2. The following reaction sequence confirms that FGL2 is a phospholipid-activated serine protease; the conversion of prothrombin to thrombin by FGL2, followed by the conversion of soluble fibrinogen to insoluble fibrin polymers by thrombin. FGL2 binds intensely to tails than heads of de-membranated sperm. A spectrum of polypeptides of cauda sperm tails binds to FGL2. Proteomic analyses of 65KDa, 16kDa, and 13kDa polypeptides of tails correspond to a-Kinase anchor protein 4, glutathione peroxidase 4, and cytochrome c oxidase subunit 4, respectively. Annexin V, a calcium-dependent phosphatidylserine-binding protein localized to the flagellum and co-precipitated with FGL2. We have demonstrated a novel protective mechanism for recognizing and eliminating defective spermatozoa from viable sperm population.

A pattern recognition receptor ficolin from Portunus trituberculatus (Ptficolin) regulating immune defense and hemolymph coagulation

Ficolins, belonging to the fibrinogen-related protein superfamily, are important pattern recognition receptors in innate immunity. Here, a ficolin gene Ptficolin was characterized from the swimming crab Portunus trituberculatus. The completed cDNA sequence of Ptficolin encoded a signal peptide, a coiled-coil region and a fibrinogen-like domain but without the typical collagen region of vertebrate ficolins. Ptficolin showed higher expression in stomach and hepatopancreas, and presented a time-dependent response after pathogen challenge and injury stimulation. The recombinant Ptficolin (rPtficolin) could bind to various PAMPs and microorganisms, and agglutinate microorganisms and rabbit erythrocytes in a Ca2+-dependent manner, with strong binding ability to N-acetyl sugars. Meanwhile, rPtficolin promoted the hemocyte phagocytosis and clearance activity of Vibrio, while Ptficolin knockdown impaired the bacterial phagocytosis and clearance ability, suggesting the opsonin activity of Ptficolin. Knockdown of Ptficolin could downregulate the transcription of most complement-like genes and AMPs, but enhance the expression of most proPO system-related genes and key genes of Toll, IMD and JNK pathways. Moreover, knockdown of Ptficolin led to the increased hemolymph clotting time and the decreased expression of clotting-related genes. Our results indicate that Ptficolin could recognize and eliminate invading pathogens, and might be a prominent component in hemolymph coagulation of crab.

The correlation of fibrinogen-like protein-1 expression with the progression and prognosis of hepatocellular carcinoma

BackgroundFibrinogen-like-protein 1 (FGL1), a member of the fibrinogen-related protein (FREP) family, is a major ligand of the immune inhibitory receptor lymphocyte-activation gene 3 (LAG-3). While FGL1 is strongly implicated in the development and prognosis of a variety of diseases, its role in hepatocellular carcinoma (HCC) is still disputed. Therefore, the role of FGL1 expression in the progression and prognosis of HCC was investigated.Methods and resultsIn the present study, bioinformatics analysis was first used to probe the expression profile of FGL1 in multiple malignant tumor tissues and paired normal tissues, and to explore the possible relationship between FGL1 and prognosis of HCC patients. Thereafter, the expression levels of FGL1 were determined and compared in human HCC cell lines, HCC tissues, peri-tumor tissues and normal liver tissues by western blot analysis. Furthermore, tissue microarrays were used to detect the expression of FGL1 through immunohistochemical staining and to verify whether the FGL1 expression level was associated with clinicopathological features and the prognosis of HCC patients. The results showed that FGL1 was downregulated significantly in most of the HCC cells lines and HCC tissues, corresponding to the results of the bioinformatics and western blot analyses. FGL1 expression level in HCC was found to be correlated to Edmondson grade and metastasis of the HCC. Additionally, high FGL1 expression was associated with better overall survival in HCC patients, suggesting that FGL1 could function as a tumor suppressor.ConclusionsThe expression level of FGL1 can be correlated with the progression and prognosis of HCC, suggesting its potential as a prognostic biomarker.

FGL2 expression and relation with survival and prognosis in patients treated with concurrent temozolamide and radiotherapy in high-grade glioma.

e14019 Background: FGL2 is a member of the fibrinogen-related protein, Literature suggests that FGL2 contributes to the progression of glioblastoma multiforme (GBM) through inducing multiple immunosuppression mechanisms. The present study aimed at evaluation of the expression and the prognostic value of FGL2 in high grade glioma. Methods: In our study, we analysed sixty specimens retrieved at diagnosis of high grade glioma patients. They received concurrent Temozolamide and radiotherapy, as a standard of care, during the period from October 2019 to December 2020 and and were followed up for 12 months . Evaluation of FGL2 expression done by using FGL2 Immunohistochemistry .ClinicalTrials.gov Identifier: NCT04113278. Results: Mean age of studied patients was (48.88± 12.45) . Majority (75%) of patients were males. Mean FGL2 expression was 61 ± 31.80 (%) with range between 3% and 95%. It was found that 7 (11.7%) patients had negative FGL2. Over the period of follow up, incidence of disease progression was significantly higher among the patients with higher percentage of FGL2 in comparison to those with lower FGL2 percentages (76.54 ± 18.52 vs. 56.17 ± 33.41(%); P= 0.04). number of deaths was also significantly higher among the high FGL2 percentage group (64.86 ± 31.58 vs. 54.16 ± 31.71(%); P= 0.03). Intensity of FGL2 was mild 16 (26.7%), moderate 20 (33.3%) and strong 17 (28.3%) . Intensity of FGL2 was more among patients with disease progression. All patients with progression had either moderate intensity (46.2%) or strong intensity (53.8%). Seven (14.9%) patients without disease progression had negative FGL2 expression with ( P= 0.01). Disease free progression (DFP) was significantly negatively correlated with increasing in the intensity of FGL2. Patients with strong intensity had the lowest DFP (14 month) while patients with negative FGL2 and mild intensity had the highest DFP (25 month) with ( P <0.001). Overall survival was significantly worse with increasing in the intensity of FGL2 where patients with strong intensity had the lowest overall survival (19 month), while patients with negative FGL2 and mild intensity had the highest overall survival (27and 26 month, respectively) with ( P <0.001). For prediction of progression among patients with high grade glioma, FGL2 at cut off point > 60% has 92.3% sensitivity and 28% specificity with ( P= 0.04). For prediction of mortality among patients with high grade glioma, FGL2 at cut off point > 70% has 67% sensitivity and 61% specificity with ( P= 0.03). Conclusions: FGL2 expression in high grade glioma patients can be used as a prognostic factor but additional studies are required to clarify the prognostic value.

Identification and diversity of fibrinogen-related protein (FREP) gene family in Haliotis discus hannai, H. rufescens, and H. laevigata and their responses to Vibrio parahemolyticus infection

Fibrinogen-related proteins (FREPs) are distributed universally in vertebrates and invertebrates. These proteins contain fibrinogen-like (FBG) domains in their C-terminal region and involve in immune responses and other aspects of physiology in invertebrates. In this study, 54 proteins that contain FBG domains or a fibrinogen_c domain were identified in Haliotis discus hannai. Comparatively, 88 and 63 FREPs were identified from the genomes of H. rufescens and H. laevigata. Most FREPs of abalones had a conserved motif containing a bound calcium ion site and a second conserved motif containing a polymerization pocket site. By sequence analysis, 394 SNPs and 11 Indels were identified in 20 FREP genes of the whole genome of H. discus hannai; 992 SNPs and 42 Indels were found in 64 FREPs of H. rufescens, and 192 SNPs and 12 Indels were found in 21 FREPs of H. laevigata. Among these SNPs, 92 missense mutation sites were identified in 26 FREP genes of H. rufescens, and 12 were identified in 8 FREP genes of H. laevigata. Due to the poor genomic integrity, annotations of the SNPs or Indels in H. discus hannai did not yield missense mutant sites. FREP genes with polymorphisms were ubiquitously expressed in all the tested tissues; however, the expression is lowest in the hemolymph. In response to Vibrio parahemolyticus infection, expression of FREP genes was significantly upregulated at different exposure times in gills, hepatopancreas, and hemolymph in H. discus hannai. Overall, this study documented the FREP genes of abalones and shed light on the role of FREPs in the innate immune system of these aquaculture species for the prevention and control of diseases.

A fibrinogen-related protein mediates the recognition of various bacteria and haemocyte phagocytosis in oyster Crassostrea gigas

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like (FBG) domains, which play important roles as pattern recognition receptors (PRRs) in the innate immune responses. In the present study, a fibrinogen-like protein was identified from the oyster Crassostrea gigas (defined as CgFREP1). The open reading frame of CgFREP1 was of 966 bp that encoded a predicted polypeptide of 321 amino acids comprising a signal peptide and a fibrinogen-like domain. The mRNA expression of CgFREP1 was detected in all the examined tissues. The recombinant CgFREP1 (rCgFREP1) displayed binding activities to lipopolysaccharide (LPS), mannose (MAN), as well as Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram-negative bacteria (Vibrio splendidus and Escherichia coli). The rCgFREP1 displayed the agglutinating activity towards M. luteus, V. splendidus and E. coli in the presence of Ca2+. rCgFREP1 was able to enhance the phagocytic activity of haemocytes towards V. splendidus, and exhibited binding activity to the CUB domain of CgMASPL-1. These results suggest that CgFREP1 not only serves as a PRR to recognize and agglutinate different bacteria but also mediates the haemocytes phagocytosis towards V. splendidus.

Circulating hepassocin level in patients with stable angina is associated with fatty liver and renal function.

Background: Chronic kidney disease (CKD) is a major risk factor for coronary artery disease and it is often associated with hepatic steatosis. Hepassocin (also known as hepatocyte-derived fibrinogen related protein or fibrinogen-like 1) is a novel hepatokine that causes hepatic steatosis and induces insulin resistance. However, the role of hepassocin in renal function status remains unclear. Our objective was to investigate the association of plasma hepassocin level with fatty liver and renal function status in patients with stable angina.Methods: Plasma hepassocin levels were determined by enzyme-linked immunosorbent assays in 395 consecutive patients with stable angina. Renal function was defined as an estimated glomerular filtration rate (eGFR). Fatty liver was defined by ultrasonography and fibrosis-4 (FIB-4) index.Results: With increasing hepassocin tertiles, patients had higher prevalence of fatty live, an increased waist-to-hip ratio, and neutrophil count, monocyte count, and FIB-4 index, higher levels of uric acid, blood urine nitrogen and higher sensitivity C-reactive protein. They also had incrementally lower eGFR, serum hemoglobin and albumin levels. In multiple linear stepwise regression analysis, only eGFR was significantly independent negatively associated with plasma hepassocin levels.Conclusion: Our results indicate that circulating hepassocin in patients with stable angina is associated with fatty liver and renal function, which suggests that increased plasma hepassocin may be involved in the pathogenesis of fatty liver and CKD.

Characterization and functional analysis of fibrinogen-related protein (FreP) in the black tiger shrimp, Penaeus monodon

Ficolin is classified as an immune related protein containing collagen-like and fibrinogen-related domain (FreD). In invertebrates, the functions of fibrinogen-related proteins (FrePs) are of importance to innate immunity. In this study, a FreP in the black tiger shrimp Penaeus monodon was identified and characterized. The PmFreP cDNA is 1,007 bp long with a 921 bp-open reading frame that encodes for 306 amino acids. The deduced PmFreP sequence consists of a signal peptide, an unknown region and the FreD. Phylogenetic analysis showed that PmFreP was clustered with fibrinogen-like proteins in crustaceans which was separated from vertebrate ficolin-like proteins. The deduced fibrinogen-like domain contains four conserved cysteine residues (Cys96, Cys127, Cys249, and Cys262) that are responsible for the formation of disulfide bridges. Gene expression analysis shows that Pmfrep is mainly expressed in the intestine and the expression is significantly upregulated after Vibrio harveyi and white spot syndrome virus (WSSV) challenge. Recombinant PmFreP (rPmFreP) were successfully expressed and purified, and forms a trimeric structure as judged by native-PAGE. Bacterial binding assay showed that the rPmFreD can bind and agglutinate Gram-negative and Gram-positive bacteria in the presence of calcium (Ca2+) ions. Moreover, the rPmFreP facilitates the clearance of V. harveyi in vivo. Overall, our results suggested that the PmFreP may serve as pattern recognition receptors implicated in shrimp innate immunity.

Microfibril-associated glycoprotein 4 (Mfap4) regulates haematopoiesis in zebrafish

Microfibril-associated glycoprotein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen-related protein superfamily. MFAP4 is produced by vascular smooth muscle cells and is highly enriched in the blood vessels of the heart and lung, where it is thought to contribute to the structure and function of elastic fibers. Genetic studies in humans have implicated MFAP4 in the pathogenesis of Smith-Magenis syndrome, in which patients present with multiple congenital abnormalities and mental retardation, as well as in the severe cardiac malformation left-sided congenital heart disease. Comprehensive genetic analysis of the role of MFAP4 orthologues in model organisms during development and tissue homeostasis is however lacking. Here, we demonstrate that zebrafish mfap4 transcripts are detected embryonically, resolving to the macrophage lineage by 24 h post fertilization. mfap4 null mutant zebrafish are unexpectedly viable and fertile, without ostensible phenotypes. However, tail fin amputation assays reveal that mfap4 mutants have reduced numbers of macrophages, with a concomitant increase in neutrophilic granulocytes, although recruitment of both cell types to the site of injury was unaffected. Molecular analyses suggest that loss of Mfap4 alters the balance between myeloid and lymphoid lineages during both primitive and definitive haematopoiesis, which could significantly impact the downstream function of the immune system.

A fibrinogen-related protein (Mnfico3) acts as a novel pattern recognition receptor in Macrobrachium nipponense

Fibrinogen-related proteins (FREPs) are widely found in both vertebrates as well as invertebrates, and they play a crucial role in host immunity. In this study, we isolated a novel ficolin gene (Mnfico3) from the oriental river prawn Macrobrachium nipponense. The complete cDNA sequence of Mnfico3 was 1133 bp long, containing an open reading frame of 765 bp coding for Mnfico3, a protein consisting of 254 amino acids. The Mnfico3 protein contained a putative N-terminal signal peptide and a fibrinogen-related protein domain present at the C-terminal. Phylogenetic analysis indicated that Mnfico3 had a closer evolutionary relationship with vertebrate ficolins than with its invertebrate homologues. Tissue distribution analysis indicated that Mnfico3 was predominantly expressed in muscle, in which its transcription was increased following bacterial challenge by Aeromonas veronii. Function analysis using recombinant protein revealed that rMnFico3 had broad-spectrum binding capacity to a variety of microorganisms and pathogen-associated molecular pattern (PAMP) ligands. Furthermore, rMnFico3 exhibited Ca2+-dependent agglutinating activity against microbes in vitro, and ability to attach to the hemocyte surface which promoted phagocytosis and subsequent clearance of invasive bacteria in vivo. Silencing rMnFico3 in prawn through RNAi did not alter the expression of antimicrobial peptide genes (ALF and Crustin). These results manifested that MnFico3 functioned as a potential pattern recognition receptor (PPR) to mediate cellular immune response by recognizing PAMPs, agglutinating invasive microbes, and promoting phagocytosis of hemocytes.

Transcriptomics of Cherax quadricarinatus hepatopancreas during infection with Decapod iridescent virus 1 (DIV1)

Cherax quadricarinatus is a large-sized, highly fecund, and fast-growing species of freshwater crayfish, and has become one of the world's most intensely studied crustaceans. Decapod iridescent virus 1 (DIV1), a newly described species in the family Iridoviridae, is known to infect various crustaceans, including C. quadricarinatus, and may pose a new threat in the shrimp-farming industry. The present study performed de novo transcriptome sequencing of C. quadricarinatus hepatopancreas during DIV1 infection. A total of 114,784 transcripts and 56,418 genes were obtained; 1070 genes were upregulated and 775 genes were downregulated when compared with the uninfected samples (controls). Three pattern recognition receptor genes (fibrinogen-related protein, C-type lectin, and beta-1,3-glucan-binding protein) were upregulated during DIV1 infection. Among the top-30 upregulated unigenes, 9 unigenes were identified as vitellogenin (Vg) genes, and the top-3 upregulated unigenes were identified as involved in Vg lipid transport, lipid localization, and lipid transporter activity, which were all significantly over-representative GO terms in the GO enrichment analysis of total and upregulated differentially expressed genes (DEGs). Many genes associated with Jak-STAT signaling pathway, Endocytosis, Phagosome, MAPK signaling pathway, Apoptosis and Lysosome were positively modified after DIV1 infection. The predicted protein–protein interaction (PPI) analysis showed NF1 and TUBA, CRM1 and TUBB were involved in protein interactions. This research showed that DIV1 infection has a significant impact on the transcriptome profile of C. quadricarinatus hepatopancreas, and the results enhance our understanding of virus–host interactions. Furthermore, the high number of transcripts generated in the present study will provide information for identifying novel genes in the absence of a full C. quadricarinatus genome sequence.

Molecular characterization and expression analysis of fibrinogen related protein (FREP) genes of Manila clam (Ruditapes philippinarum) after lipopolysaccharides challenge

Ruditapes philippinarum has high economic value and is distributed all over the world. Fibrinogen associated protein (FREP) is a type of pattern recognition receptor, participates in the innate immune response to eliminate pathogens after the invasion of pathogenic microorganisms. In this study, three FREP genes (FREP-1, FREP-2, and FREP-3) were identified and characterized from R. philippinarum. The protein sequence of FREPs were highly conserved with those homologous in vertebrates, and FBG domain possessed significantly high structural conservation in polypeptide binding site and Ca2+ binding site. The tissues expression analysis of FREPs in three shell color strains and two population of R. philippinarum were examined, with the highest expression level in gill and hepatopancreas. Besides, FREP genes were demonstrated to be induced by lipopolysaccharides injection, the significantly changes were observed after LPS injected. Our results suggest the involvement of FREPs in response to LPS injection, and it might exert a significant function on the innate immune defense of the Manila clam.

Microfibrillar‐associated protein 4 in serum is associated with asthma in Danish adolescents and young adults

BackgroundMicrofibrillar‐associated protein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen‐related protein superfamily, which plays multifaceted roles in innate immunity and normal endothelial function. It has been proposed that MFAP4 promotes the development of asthma in vivo and proasthmatic pathways of bronchial smooth muscle cells in vitro. The aim of this study was to investigate the significance of serum MFAP4 in adolescents and young adolescents with persistent asthma.MethodsProspective, observational study including adolescents and young adults (age 11‐27 years) previously diagnosed with asthma during childhood 2003 to 2005 (0‐15 years) at the four pediatric outpatient clinics in the Region of Southern Denmark (n = 449). Healthy controls were recruited at follow‐up (n = 314). Detection of serum MFAP4 was performed by AlphaLISA technique.ResultsCurrent asthma was associated to a 14% higher mean level of serum MFAP4 compared with controls (expβ 1.14, 95% confidence intervals [CI], 1.05‐1.23) and a 6% higher mean level compared with subjects with no current asthma (expβ 1.06, 95% CI, 0.99‐1.13). No association was found at follow‐up between serum MFAP4 and self‐reported atopic symptoms (other than asthma), Asthma Control Test‐score, fractional exhaled nitric oxide (FeNO), nor to flow rate at 1 second, forced vital capacity, and forced expiratory flow 25% to 75%, response to short‐acting beta 2 agonist or mannitol.ConclusionsWe found a significantly higher mean level of serum MFAP4 in adolescent and young adults with mild to moderate asthma compared with healthy controls but no association to FeNO and lung function nor to the response to short‐acting beta 2 agonist or mannitol. The result supports the hypothesis that MFAP4 plays a role in the pathogenesis of asthma although the marker did not demonstrate any obvious potential as an asthma biomarker in adolescents and young adults with asthma. To understand the possible proasthmatic functions of MFAP4, further investigation in specific asthma phenotypes and the underlying molecular mechanisms is warranted.

A feedback loop involving FREP and NF-κB regulates the immune response of sea cucumber Apostichopus japonicus

Our previous work indicated that fibrinogen-related protein (AjFREP) from sea cucumber (Apostichopus japonicus) plays important roles in innate immunity. To understand AjFREP expression regulation in A. japonicus, we cloned the AjFREP promoter and characterized the putative transcription-factor binding motifs. The AjFREP promoter region spans 1365bp, containing several transcription-factor binding sites. The full-length sequence and all truncated fragments exhibited high promoter activity in HEK-293 cells. Luciferase activity significantly increased for P1(−1365/+16) after LPS exposure, suggesting that the promoter responded to LPS. We also found that two potential NF-κB binding sites were involved in the promoter region, and co-transfection assay demonstrated that the first binding site was necessary for AjFREP transcription. The expression of AjNF-κB/Rel after AjFREP knock-down followed by LPS injection in vivo was further investigated. We found that AjNF-κB/Rel transcript significantly decreased after silencing AjFREP with LPS challenge compared with the control at 4 and 8h, suggesting that activated AjFREP in turn affected the NF-κB pathway under immune responses. These results provided novel insights into the activation mechanism of AjFREP, i.e., that selectively changing AjFREP expression may prevent pathogen infection in A. japonicus.

Alcohol Activates Scabrous-Notch to Influence Associated Memories

Drugs of abuse, like alcohol, modulate gene expression in reward circuits and consequently alter behavior. However, the invivo cellular mechanisms through which alcohol induces lasting transcriptional changes are unclear. We show that Drosophila Notch/Su(H) signaling and the secreted fibrinogen-related protein Scabrous in mushroom body (MB) memory circuitry are important for the enduring preference of cues associated with alcohol's rewarding properties. Alcohol exposure affects Notch responsivity in the adult MB and alters Su(H) targeting at the dopamine-2-like receptor (Dop2R). Alcohol cue training also caused lasting changes to the MB nuclear transcriptome, including changes in the alternative splicing of Dop2R and newly implicatedtranscripts like Stat92E. Together, our data suggestthat alcohol-induced activation of the highly conserved Notch pathway and accompanying transcriptional responses in memory circuitry contribute to addiction. Ultimately, this provides mechanistic insight into the etiology and pathophysiology of alcohol use disorder.

Novel fibrinogen-related protein with single FReD contributes to the innate immunity of Macrobrachium rosenbergii

Fibrinogen-related proteins (FREPs) are widely found in vertebrates and invertebrates, and they play crucial roles in innate immunity. Here, a new FREP named as MrFREP was identified from giant freshwater prawn (Macrobrachium rosenbergii). The full-length cDNA of MrFREP measures 1649 bp in length and consists of a 1086 bp open reading frame encoding a polypeptide composed of 361 amino acids. The MrFREP sequence has a signal peptide with 20 amino acids and a fibrinogen-related domain (FReD) with 223 amino acids. Phylogenetic analysis showed that MrFREP was grouped with FREPs from Marsupenaeus japonicus and Litopenaeus vannamei. BLASTp results showed that it had 43% identity with a FREP from M. japonicus. The expression of MrFREP was higher in gills, intestine, and hepatopancreas than in hemocytes, heart, stomach, and muscles. The expression levels of MrFREP in gills and intestine were obviously upregulated after they were exposed to Vibrio parahaemolyticus or White spot syndrome virus infection. Recombinant MrFReD protein (rMrFReD) could bind to Gram-positive and Gram-negative bacteria and agglutinate the tested bacteria in the presence of calcium. rMrFReD demonstrated lipopolysaccharide and peptidoglycan binding activities. rMrFReD could accelerate the clearance of V. parahaemolyticus in vivo. These results suggested that MrFREP could function as a pattern recognition receptor contributing to the innate immunity of M. rosenbergii.