ABSTRACT
We report herein a case of delayed bowel stenosis after surgery for non-occlusive mesenteric ischemia (NOMI), which was successfully treated with endoscopic stenting. The patient was a 78-year-old woman who underwent an emergency laparotomy for NOMI and duodeno-ileal anastomosis. Necrosis was observed in almost all areas of the small intestine except for the beginning of the jejunum and the end of the ileum. Postoperatively, the patient was discharged with central venous nutrition, but was readmitted on postoperative day 54 with a diagnosis of postoperative ileus. The patient failed to respond to conservative treatment. Fluoroscopic endoscopy revealed wall stiffness and circumferential stenosis in the ascending colon at a different site from that of the anastomosis. Based on this finding, delayed stenosis of the ascending colon after NOMI treatment was diagnosed. Bougie dilatation was performed for the stenosis, leading to temporary improvement. However, stenosis along with ileus soon recurred. To prevent restenosis, a metallic stent was endoscopically implanted at the stenotic site. Thereafter, the patient was discharged without any further episodes of restenosis. Delayed bowel stenosis may occur after a subtotal resection of the small intestine for NOMI. Endoscopic stenting is an effective treatment option if resection is difficult.
Subject(s)
Ileus , Intestinal Obstruction , Mesenteric Ischemia , Female , Humans , Aged , Mesenteric Ischemia/diagnostic imaging , Mesenteric Ischemia/etiology , Mesenteric Ischemia/surgery , Constriction, Pathologic/etiology , Constriction, Pathologic/surgery , Intestine, Small/surgery , Intestinal Obstruction/diagnostic imaging , Intestinal Obstruction/etiology , Intestinal Obstruction/surgery , Ischemia/etiology , Ischemia/surgeryABSTRACT
Introduction: The pathogenic gene CDH23 plays a pivotal role in tip links, which is indispensable for mechanoelectrical transduction in the hair cells. However, the underlying molecular mechanism and signal regulatory networks that influence deafness is still largely unknown. Methods: In this study, a congenital deafness family, whole exome sequencing revealed a new mutation in the pathogenic gene CDH23, subsequently; the mutation has been validated using Sanger sequencing method. Then CRISPR/Cas9 technology was employed to knockout zebrafish cdh23 gene. Startle response experiment was used to compare with wide-type, the response to sound stimulation between wide-type and cdh23-/-. To further illustrate the molecular mechanisms underlying congenital deafness, comparative transcriptomic profiling and multiple bioinformatics analyses were performed. Results: The YO-PRO-1 assay result showed that in cdh23 deficient embryos, the YO-PRO-1 signal in inner ear and lateral line neuromast hair cells were completely lost. Startle response experiment showed that compared with wide-type, the response to sound stimulation decreased significantly in cdh23 mutant larvae. Comparative transcriptomic showed that the candidate genes such as atp1b2b and myof could affect hearing by regulating ATP production and purine metabolism in a synergetic way with cdh23. RT-qPCR results further confirmed the transcriptomics results. Further compensatory experiment showed that ATP treated cdh23-/- embryos can partially recover the mutant phenotype. Conclusion: In conclusion, our study may shed light on deciphering the principal mechanism and provide a potential therapeutic method for congenital hearing loss under the condition of CDH23 mutation.
ABSTRACT
INTRODUCTION: Pneumocystis pneumonia (PCP) is an opportunistic lung infection and has been reported among patients with rheumatoid arthritis (RA). An animal study revealed that sulfasalazine enhances Pneumocystis clearance from the lung by accelerating macrophage activity. METHODS: The self-controlled case series (SCCS) method was used to investigate the association between sulfasalazine use and PCP development in patients with RA without the effect of time-invariant, interpatient confounders. PCP episodes which developed in patients with RA at five hospitals between 2003 and 2019 were identified. PCP was defined by the following criteria: 1) detection of Pneumocystis jirovecii in respiratory specimens by polymerase chain reaction; 2) clinical symptoms (pyrexia, dry cough, dyspnea or hypoxia); 3) diffuse interstitial infiltrate on chest imaging; and 4) absence of PCP prophylaxis. The PCP incidence rate ratio (IRR) was compared between periods with and without sulfasalazine use by conditional Poisson regression. RESULTS: Fifty episodes of PCP were identified in 49 patients. Thirty patients received sulfasalazine at some point during their observation. While 49 episodes of PCP developed in 170.3 person-years without sulfasalazine use, only one episode of PCP developed in 103.7 person-years with sulfasalazine use. Sulfasalazine use was associated with a decreased PCP risk (adjusted IRR <0.01; 95% confidence interval <0.01-0.03) after adjusting for age and glucocorticoid, methotrexate, and tumor necrosis factor inhibitor administration. CONCLUSION: Our study demonstrated a preventive effect of sulfasalazine against PCP in patients with RA.
Subject(s)
Arthritis, Rheumatoid , Opportunistic Infections , Pneumonia, Pneumocystis , Sulfasalazine , Arthritis, Rheumatoid/complications , Arthritis, Rheumatoid/drug therapy , Methotrexate , Opportunistic Infections/drug therapy , Opportunistic Infections/prevention & control , Pneumocystis carinii , Pneumonia, Pneumocystis/drug therapy , Pneumonia, Pneumocystis/prevention & control , Retrospective Studies , Sulfasalazine/therapeutic use , HumansABSTRACT
Charge mobility is an essential factor of organic crystalline materials. Although many investigators have made important progress, the exact relationship between the crystal structure and carrier mobility remains to be clarified. Fortunately, a series of bis-1,3,4-oxadiazole derivatives have been successfully prepared and reported. They have similar main molecular fragments but different crystal packing modes, which provide an ideal research objective for studying the effect of molecular packing on charge mobility in organic photoelectric conversion systems. In this work, the charge mobilities of these molecules are systematically evaluated from the perspective of first-principles calculation, and the effect of a molecular overlap on orbital overlap integral and final charge carrier mobility is fully discussed. It can be seen that the small intermolecular distance (less than 6 Å) is the decisive factor to achieve high electron mobility in π stacking, and better mobility can be obtained by increasing the hole migration distance appropriately. A larger dihedral angle of anisotropy is an important point limiting the charge mobility in the herringbone arrangement. It is hoped that the correlation results between the crystal structure and mobility can assist the experimental study and provide an effective way to improve the photoelectric conversion efficiency of the organic semiconductor devices and multiple basis for multiscale material system characterization and material information.
ABSTRACT
Benzo[a]pyrene (BaP), a common environmental pollutant, can modulate the immune-associated signal pathway NF-κB, which is one of the critical signal pathways involved in various immune responses. BaP exposure usually generates reactive oxygen species (ROS), but whether ROS are predominantly involved in the modulation mechanism of the NF-κB pathway has not been clearly understood. In this study, an in vivo examination of Oryzias melastigma demonstrated that BaP exposure led to a down-regulation of the NF-κB pathway and increased levels of ROS. Conversely, in vitro results using the medaka liver cell line DIT-29 and a widely applied H2O2 method showed the opposite: up-regulation of the NF-κB pathway. However, the down-regulation of NF-κB upon BaP exposure in vitro was inhibited by the addition of a ROS inhibitor, indicating ROS are involved in the modulation of NF-κB. The discrepancy between in vivo and in vitro results of ROS impacts on NF-κB activation might be related to the concentration and persistence of ROS. Using a modified luminol detection system, BaP was found to generate sustained physiological concentrations of ROS for 24â¯h, while an H2O2 bolus generated ROS for less than 30â¯min. Furthermore, a steady-state sub-micromolar H2O2 system (H2O2ss) was developed in parallel as a positive control of ROS, by which H2O2 could be maintained for 24â¯h. Comparative evaluation using H2O2, H2O2ss and BaP exposures on the medaka cell line with pGL4.32 demonstrated that the persistent physiological concentrations of ROS generated upon BaP exposure or treatment with H2O2ss inhibited the NF-κB pathway, but direct H2O2 exposure had the opposite effect. Moreover, a western-blot assay and EMSA detection further confirmed the modulation of the NF-κB pathway in DIT-29. Taken together, this study shows that BaP exposure inhibits the NF-κB pathway by generating sustained physiological concentrations of ROS.
Subject(s)
Benzo(a)pyrene/toxicity , Environmental Pollutants/toxicity , NF-kappa B/metabolism , Oryzias/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/drug effects , Animals , Antioxidants/metabolism , Cell Line , Down-Regulation , Hydrogen Peroxide/pharmacology , Liver/drug effects , Liver/enzymology , Male , Oryzias/immunologyABSTRACT
Our previous study revealed that an antimicrobial peptide hepcidin, can be significantly up-regulated either with LPS challenge or upon exposure to Benzo[a]pyrene (BaP) in red sea bream, but the molecular mechanism involved in whether the transcriptional expression of hepcidin induced by LPS or BaP is regulated through a similar signaling pathway is not yet known. To elucidate the underlying molecular mechanism, the marine model fish Oryzias melastigma was exposed to 1 µg/L BaP as well as challenged with 5 µg of LPS per fish. Samples at 3 h post-LPS challenge, and 2 d and 3 d post-BaP exposure were separately collected for transcriptome analysis. General analysis of the predicted immune-associated unigenes based on the transcriptomic data showed that the percentages of modulated immune-associated genes were 7% with LPS challenge, and 3% and 7% with BaP exposure at 2 and 3 days, respectively. Genes involved in functions like antimicrobial activity, neutrophil activation, and leukocyte chemotaxis were up-regulated with LPS challenge, whereas more than half of the immune associated genes including the KLF family were down-regulated upon BaP exposure, indicating a difference in the modulated immune genes between LPS challenge and BaP exposure. Specific comparative analyses of the immune-associated signal pathways NOD, TOLL, NF-κB and JAK-STAT with LPS challenge or upon exposure to BaP, indicated that most of the modulated genes in association with the NOD, TOLL and NF-κB pathways were induced with LPS challenge but only a few after exposure to BaP, suggesting that BaP exposure was generally not associated with any of the three signal pathways. Interestingly, further transcriptomic analysis revealed that 5 of the 8 modulated genes associated with the JAK-STAT pathway were down-regulated, while 2 inhibiting genes were up-regulated after BaP exposure for 2 days whereas LPS challenge resulted in only less than half modulated, suggesting the possibility of down-regulation caused by BaP exposure through JAK-STAT pathway. Further testing using an EPC cell culture demonstrated that expression of the hepcidin1 gene was less involved in the known signal pathways, such as c/EBP, BMP, and NF-κB, but instead mostly in association with the JAK-STAT pathway upon BaP exposure.
Subject(s)
Benzo(a)pyrene/toxicity , Hepcidins/genetics , Oryzias , Transcriptome , Animals , Down-Regulation/drug effects , Gene Expression Profiling , Signal Transduction/drug effects , Transcriptome/drug effects , Water Pollutants, Chemical/toxicityABSTRACT
17α-ethinylestradiol (EE2) exerts endocrine disrupting effect and immunotoxic effect on marine animals, including modulation of hepcidin expression. The antimicrobial peptide hepcidin displays a crucial role in innate immunity in fish against invading pathogens. It is known that the transcription of hepcidin in mammals is individually regulated by many stimuli, including inflammation, iron overload, anemia or hypoxia, through several distinct molecular pathways. The canonical mechanism for endocrine disrupting effects is mediated by an estrogen receptor (ER) and estrogen responsive element (ERE), whereas the underlying mechanism for immunotoxic effect is still unclear. In this study, a hepcidin from Oryzias melastigma (OM-hep1) was found to be down-regulated upon EE2 exposure and was associated with ERα. Unlike the revealed signaling pathways for hepcidin regulation in mammals, it was revealed by promoter activity analysis that the OM-hep1 transcription was not associated with canonical immune-associated and hormone-associated regulatory elements, known as the nuclear factor κB (NF-κB), signal transducer and activator of transcription 3 (STAT3), ERE and estrogen-related receptor responsive element (ERRE). Further analysis through a series of base mutations revealed a short fragment from -315 to -289 bp on the OM-hep1 promoter with high activity. This fragment was composed of a putative ERE-like element (23 bases) plus an adjacent down-streamed four bases motif GTGT. Replacement of either of the core bases (GGTCA) of ERE-like or GTGT motif showed non-activity and non-response to EE2 exposure, thus a new hepcidin-associated element named as HepERE was revealed. Evidences from electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) assay demonstrated that the EE2-mediated down-regulation of OM-hep1 expression was associated with ERα binding to HepERE but not classical ERE. Taken together, a novel signaling pathway was revealed and the regulatory mechanism associated with the ERα and HepERE element on immunomodulation of OM-hep1 expression upon EE2 exposure was first reported here.
Subject(s)
Endocrine Disruptors/toxicity , Ethinyl Estradiol/toxicity , Fish Proteins/genetics , Gene Expression Regulation , Hepcidins/genetics , Oryzias/genetics , Water Pollutants, Chemical/toxicity , Animals , Base Sequence , Fish Proteins/metabolism , Hepcidins/metabolism , Immunity, Innate , Male , Oryzias/immunology , Oryzias/metabolism , Regulatory Elements, TranscriptionalABSTRACT
A new gene homologous to the reported antimicrobial peptide (AMP) hyastatin from Hyas araneus was screened in the SSH library constructed from the hemocytes of Scylla paramamosain, and named SpHyastatin. In vivo study showed that SpHyastatin was predominantly expressed in hemocytes of S. paramamosain. With the challenge of either Vibrio parahaemolyticus or lipopolysaccharide (LPS), SpHyastatin showed a positive response, meaning that it was probably involved in the immune reaction against bacterial infection in vivo. A distinctive feature of SpHyastatin in comparison with six other known AMPs tested was that SpHyastatin could maintain a higher transcription level from megalopas to the adult crab, indicating a potential consistent resistance against pathogens conferred by this peptide existing in the blood circulation of crabs. RNA interference assay was performed to inhibit SpHyastatin transcription in vivo and the result demonstrated that silencing SpHyastatin mRNA transcripts could decrease the survival rate of crabs challenged with V. parahaemolyticus. To further understand the molecular mechanisms that regulate SpHyastatin expression, a 576 bp 5'-flanking sequence of SpHyastatin was obtained using genome walking. Here, we focused our experiments on investigating the roles of the putative NF-κB binding site in LPS-mediated transcriptional regulation of the SpHyastatin gene using endothelial progenitor cells and Hela cells. Luciferase reporter analyses demonstrated that the putative NF-κB element acted as a positive regulatory element and was essential for the induction of SpHyastatin promoter by LPS. These results should shed light on the in vivo functional property and the molecular mechanism of regulation for the crab AMP SpHyastatin.
Subject(s)
Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/immunology , Brachyura/genetics , Brachyura/immunology , Gene Expression Regulation , Vibrio parahaemolyticus/physiology , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/chemistry , Arthropod Proteins/chemistry , Arthropod Proteins/genetics , Arthropod Proteins/immunology , Brachyura/microbiology , Cloning, Molecular , Escherichia coli/chemistry , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/immunology , Lipopolysaccharides/pharmacology , Male , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
In this study, we reported a significant difference in the supramolecular hydrogelation of newly discovered NI-GFF (NI-Gly-l-Phe-l-Phe) and NI-FFG (NI-l-Phe-l-Phe-Gly) on the basis of their phase diagrams. With a small difference in the peptide chain between NI-GFF and NI-FFG, we observed a significant difference in their self-assembly properties; NI-GFF formed a stable gel at neutral pH, whereas NI-FFG did not, under the same conditions. From spectroscopic and computational studies, intermolecular π-π interactions and extended hydrogen bonding interactions might reinforce the intermolecular interactions of NI-GFF, which may facilitate the formation of the self-assembled nanostructures and the hydrogel. In addition, the aggregation-induced emission (AIE)-active NI-GFF reveals relatively good biocompatibility compared with that of NI-FFG for two commonly used cell lines, suggesting that it is a promising candidate for use as a supramolecular material in biomedical applications. Our results highlight the importance of tripeptide sequences in a self-assembling hydrogel system.
Subject(s)
Hydrogels/chemistry , Imides/chemistry , Naphthalenes/chemistry , Oligopeptides/chemistry , Amino Acid Sequence , Animals , Astrocytes/cytology , Astrocytes/drug effects , Cell Line , Cell Survival/drug effects , Humans , Hydrogels/pharmacology , Hydrogen Bonding , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , MCF-7 Cells , Naphthalenes/pharmacology , Oligopeptides/pharmacology , RatsABSTRACT
A new antimicrobial peptide named SCY2 with 65.08% identity in amino acid sequence to the known scygonadin (SCY1) was first characterized in Scylla paramamosain based on its cloned full-length cDNA and genomic DNA sequences. The SCY2 gene was dominantly expressed in the ejaculatory duct of male crabs and its mRNA transcripts were discerned mainly in the glandular epithelium of the inner wall and the secretion inside the ejaculatory duct. Although the SCY2 gene could not be induced with the challenge of the bacteria and fungi tested, its induction reached the highest level at the peak period of mating in mature male crabs either in June or November, suggesting its induction was likely related to seasonal reproduction changes. Moreover, it was interesting to note that, from analysis of its transcripts and protein, SCY2 was significantly expressed only in the ejaculatory duct of pre-copulatory males before mating, however it was clearly detected in the spermatheca of post-copulatory females after mating accompanied by the decreased level of SCY2 expression in the ejaculatory duct. These results suggested that the SCY2 was probably transferred from the male during mating action with the female for the purpose of protecting fertilization. The recombinant SCY2 was more active against the Gram-positive than the Gram-negative bacteria tested. It was further observed that the SCY2 transcripts were significantly increased with addition of exogenous progesterone in tissue cultures whereas the several other hormones tested had no any effect on SCY2 expression, indicating that there might be a relationship between the SCY2 expression and the induction of hormones in vivo. In summary, this study demonstrated that one role of SCY2 was likely to be involved in crab reproduction and it exerted its reproductive immune function through the mating action and the maintenance of inner sterility in the spermatheca of the female, thus leading to successful fertilization of S. paramamosain.
Subject(s)
Antimicrobial Cationic Peptides , Brachyura/immunology , Reproduction/immunology , Animals , Antimicrobial Cationic Peptides/genetics , Antimicrobial Cationic Peptides/immunology , Antimicrobial Cationic Peptides/metabolism , Antimicrobial Cationic Peptides/pharmacology , Brachyura/genetics , Brachyura/metabolism , Ejaculatory Ducts/metabolism , Female , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/growth & development , Gram-Positive Bacteria/drug effects , Gram-Positive Bacteria/growth & development , Immunity , Male , RNA, Messenger/metabolismABSTRACT
Histone H2A is known to participate in host immune defense through generating special antimicrobial peptides (AMPs), for which it has been an interesting research focus to characterize this kind of peptides in vertebrates and invertebrates. Although thousands of AMPs have been reported in variety of life species, only several AMPs are known in crabs and in particular no H2A-derived AMP has yet been reported. In the present study, a 38-amino acid peptide with antimicrobial activity was determined based on the sequence analysis of a histone H2A identified from the mud crab Scylla paramamosain. The histone H2A derived peptide was an AMP-like molecule and designated as Sphistin. Sphistin showed typical features of AMPs such as amphiphilic α-helical second structrue and positive charge net. The synthetic Sphistin exerted high antimicrobial activity against Gram-positive, Gram-negative bacteria and yeast, among which Aeromonas hydrophila, Pseudomonas fluorescens and Pseudomonas stutzeri are important aquatic pathogens. Leakage of the cell content and disruption of the cell surface were observed in bacterial cells treated with Sphistin using scanning electron microscopy. It was proved that the increasing cytoplasmic membrane permeability of Escherichia coli was caused by Sphistin. Further observation under confocal microscopy showed that Sphistin could combine onto the membrane of Staphylococcus aureus, E. coli MC1061 and Pichia pastoris but not translocate into the cytoplasm. Moreover, the affinity of Sphistin with either LPS or LTA was also testified that there was an interaction between Sphistin and cell membrane. Thus, the antimicrobial mechanism of this peptide likely exerted via adsorption and subsequently permeabilization of the bacterial cell membranes other than penetrating cell membrane. In addition, synthetic Sphistin exhibited no cytotoxicity to primary cultured crab haemolymphs and mammalian cells even at a high concentration of 100 µg/mL for 24 h. This is the first report of a histone-derived Sphistin identified from S. paramamosain with a specific antimicrobial activity and mechanism, which could be a new candidate for future application in aquaculture and veterinary medicine.
Subject(s)
Antimicrobial Cationic Peptides/genetics , Arthropod Proteins/genetics , Brachyura/genetics , Gene Expression Regulation , White spot syndrome virus 1/physiology , Amino Acid Sequence , Animals , Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/metabolism , Antimicrobial Cationic Peptides/pharmacology , Arthropod Proteins/chemistry , Arthropod Proteins/metabolism , Arthropod Proteins/pharmacology , Bacteria/drug effects , Base Sequence , Brachyura/growth & development , Brachyura/metabolism , Brachyura/virology , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/metabolism , Histones/chemistry , Histones/genetics , Histones/metabolism , Molecular Sequence Data , Organ Specificity , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Yeasts/drug effectsABSTRACT
Innexins are a class of transmembrane proteins that are important for embryonic development, morphogenesis and electrical synapse formation. In the present study, a novel innexin2 gene from Scylla paramamosain was named Sp-inx2 and characterized. The complete cDNA and genomic DNA sequences of Sp-inx2 were revealed. Sp-inx2 mRNA transcripts were distributed in various tissues of S. paramamosain and were most abundant in the hemocytes. The Sp-inx2 was significantly upregulated in hemocyte, gill and hepatopancreas tissues with the challenge of either Vibrio alginolyticus, Vibrio parahaemolyticus or lipopolysaccharides (LPSs) when analyzed at 3 and 6 h using quantitative real-time PCR, suggesting that it could activate an immune response against the challenge of LPSs or Vibrio species. Using the chemical inhibitors carbenoxolone and probenecid, the absorption of the fluorescent dye Lucifer yellow decreased in the primary cultured hemocytes of crabs, thus confirming that hemichannels composed of Sp-inx2 existed in the crab hemocytes. With LPS stimulation, the level of mRNA transcripts and protein expression of Sp-inx2 in the same cultured hemocytes gradually increased from 6 to 48 h, while the activity of hemichannels was down-regulated at 6 and 12 h, demonstrating that LPSs could modulate the absorption activity of hemichannels in addition to its upregulation of Sp-inx2 gene expression. Furthermore, the dye uptake rate in HeLa cells in which Sp-inx2 was ectopically expressed increased dramatically but the increase was significantly down-regulated with the addition of 50 µg mL(-1) LPS, suggesting that the LPS stimulation could effectively reduce the activity of hemichannels. Interestingly, with the ectopic expression of Sp-inx2 in HeLa and EPC cells, apoptosis spontaneously occurred in both cultured cell lines when detected using TUNEL assay. In summary, a new Sp-inx2 gene was first characterized in a marine animal S. paramamosain and it had a function associated with immune response and cell apoptosis.
Subject(s)
Apoptosis , Arthropod Proteins/genetics , Brachyura/genetics , Brachyura/immunology , Connexins/genetics , Immunity, Innate , Signal Transduction , Amino Acid Sequence , Animals , Arthropod Proteins/chemistry , Arthropod Proteins/metabolism , Base Sequence , Brachyura/metabolism , Brachyura/microbiology , Connexins/chemistry , Connexins/metabolism , Gills/immunology , Hemocytes/immunology , Hepatopancreas/immunology , Lipopolysaccharides/pharmacology , Phylogeny , Sequence Alignment , Up-Regulation , Vibrio alginolyticus/physiology , Vibrio parahaemolyticus/physiologyABSTRACT
In animals, biotransformation and the immune system interact with each other, however, knowledge of the toxic mechanism of benzo[a]pyrene (BaP) on these two systems is not well known. The present study investigated the toxic effects of BaP on the biotransformation system, cortisol level and DNA integrity of red sea bream (Pagrus major). The results showed that cortisol level was induced under the challenge of lipopolysaccharide (LPS). Short-term exposure (96 h) of BaP at environmental concentration significantly increased the cortisol level, hepatic EROD activity and CYP1A1 mRNA expression. When P. major was exposed to BaP for 14 d followed by LPS challenge this increased the cortisol level, EROD activity and hepatic DNA damage except CYP1A1 mRNA expression. Combined with our previous data, which showed that BaP exposure can modulate the immunologic response in P. major challenged with LPS, a hypothetical adverse outcome pathway of BaP on fish was suggested.
Subject(s)
Benzo(a)pyrene/toxicity , DNA Damage , Hydrocortisone/metabolism , Sea Bream/metabolism , Animals , Biotransformation , Cytochrome P-450 CYP1A1/metabolism , Fishes/metabolism , Lipopolysaccharides , Liver/metabolismABSTRACT
The effects of polycyclic aromatic hydrocarbons (PAHs) have been reported to modulate the immune response in aquatic animals, but the collected information of their effects on fish immunity is so far ambiguous. This study demonstrated that Benzo[a]pyrene (BaP) exposure altered the expression pattern of an antimicrobial peptide hepcidin (PM-hepc) gene and the activities of some immune-associated parameters in the lipopolysaccharide (LPS)-challenged red sea bream (Pagrus major). It was observed that LPS could increase respiratory burst, lysozyme and antibacterial activity in P. major. However when the P. major was exposed to different concentrations of BaP (1, 4, or 8 µg L(-1) ) for 14 days and then challenged with LPS there was no significant change in the lysozyme and antibacterial activity. It was further observed that LPS could induce the PM-hepc mRNA expression at 3, 6, and 12-h post-LPS challenge. However, when P. major was exposed first to BaP for 14 days and then challenged with LPS, the expression of PM-hepc mRNA was delayed in the liver until 24 h and not significantly induced until 48 and 96 h. The mRNA expression pattern was completely different from that only with LPS challenge, showing that BaP exposure changed the PM-hepc mRNA expression pattern of fish with LPS challenge. This study demonstrated that BaP exposure can weaken or inhibit the induction of lysozyme and antibacterial activity in the LPS-challenged P. major; conversely BaP exposure could enhance the mRNA expression of PM-hepc gene, indicating that the effect of BaP has different modulatory mechanism on hepcidin genes and immune-associated parameters.
Subject(s)
Benzo(a)pyrene/pharmacology , Lipopolysaccharides/pharmacology , Sea Bream/immunology , Animals , Blood Bactericidal Activity , Hepcidins/metabolism , Liver/drug effects , Liver/metabolism , Muramidase/blood , RNA, Messenger/metabolism , Respiratory Burst , Toxicity Tests, Acute , Toxicity Tests, SubacuteABSTRACT
Heat shock proteins (Hsps) are an evolutionarily conserved group of molecules present in all eukaryotic and prokaryotic organisms. Hsp10 and Hsp60 were originally described as the essential mitochondrial proteins involved in protein folding. Recent studies demonstrate that Hsp10 has additional roles including immune modulation. In our study, an homologous Hsp10 (Sp-Hsp10) was identified in the mud crab Scylla paramemosain, and its genomic DNA organization was determined. The cDNA sequence of Sp-Hsp10 contains an open reading frame of 309 bp, encoding a putative protein of 102 amino acid residues with approximately 10 kDa. The Sp-Hsp10 gene is located next to the Sp-Hsp60 gene and shares a 1916-bp intergenic region. The promoter activity of the Sp-Hsp10 flanking gene was analyzed using luciferase reporter assays in transfected endothelial progenitor cells. The upregulation of Sp-Hsp10 expression was detected after exposure of hemocytes to a heat shock of 1 h at 37 °C compared with unstressed hemocytes raised at 20 °C. To our knowledge, this is the first report characterizing the genomic organization of a new Hsp10 in a crustacean.
Subject(s)
Brachyura/genetics , Chaperonin 10/genetics , Promoter Regions, Genetic , Amino Acid Sequence , Animals , Base Sequence , Chaperonin 10/metabolism , Chaperonin 60/genetics , Molecular Sequence Data , Open Reading Frames , Phylogeny , Sequence Analysis, DNA , Transcription, GeneticABSTRACT
Organic and inorganic contaminants can suppress immune function in molluscs and crustaceans. It was postulated that metals could modulate immune function in marine crabs. To test this hypothesis, sublethal effects of mercury (Hg) on cellular immune and biochemical responses of crabs were determined. When crabs were exposed for 14 d to environmentally-relevant concentrations of Hg, changes in immune-associated parameters including, total haemocyte count, lysosomal membrane stability, phenoloxidase, super oxide generation and phagocytosis were observed. Oxidative stress, as measured by lipid peroxidation, antioxidant responses, including superoxide dismutase and catalase activities and glutathione-mediated antioxidant enzymes in serum, haemocyte lysate, gills, hepatopancreas and muscle were assessed in crabs exposed to Hg. Exposure to Hg resulted in significantly lesser immune-associated parameters in haemolymph and antioxidants in all tissues studied. Conversely, GST and phenoloxidase activity, were greater in crabs exposed to Hg. Responses of antioxidant parameters (SOD, CAT and GP(x)) were positively correlated with immune responses, including THC, superoxide and phagocytosis. These results were postulated to be due to an immediate response of antioxidant defense to oxygen radicals generated. Overall, the results suggest that 14 d exposure to environmentally realistic concentrations of Hg causes immunomodulation and potentially harmful lessened antioxidant defenses of crabs.
Subject(s)
Brachyura/immunology , Mercury/immunology , Water Pollutants, Chemical/immunology , Animals , Arthropod Proteins/immunology , Brachyura/drug effects , Catalase/immunology , Glutathione Peroxidase/immunology , Hemolymph/drug effects , Hemolymph/immunology , Immunologic Factors/immunology , Immunologic Factors/toxicity , Mercury/toxicity , Oxidative Stress/drug effects , Phagocytosis/drug effects , Superoxide Dismutase/immunology , Superoxides/immunology , Water Pollutants, Chemical/toxicityABSTRACT
Invertebrates are dependent on cellular and humoral immune defences against microbial infection. Scylla paramamosain is an important commercial species, but the fundamental knowledge on its immune defense related to the antioxidant and immune-associated reactions is still lacking. The study was to differentiate the responses of immune-associated parameters of haemolymph components in S. paramamosain when challenged with bacterial lipopolysaccharides (LPSs). The immunostimulating effects of LPS in crab by triggering various immune parameters (phagocytosis, lysozyme, antibacterial activity, phenoloxidase, and the generation of superoxide and nitric oxide) were investigated. Results showed that the generation of free radicals, phenoloxidase, lysozyme and antibacterial activities was significantly increased through the exposure periods. Conversely, total hemocyte count and lysosomal membrane stability decreased significantly as the exposure period extended to 96 h. The relationship between the antioxidant enzymes and immune reactions due to LPS was highly significant. In addition, ROS production was positively correlated with antioxidant showing immediate response of antioxidant defense to the oxyradicals generated. Overall, the study indicated that nonspecific immune components in hemocytes of crab showed active response to the LPS stimulation, and their responses suggested that many immune-associated parameters could be modulated and interrelated with the influence of antioxidants in crustaceans.
ABSTRACT
Scygonadin (Scy) is an important antimicrobial peptide which was first isolated from the seminal plasma of Scylla serrata (now renamed as Scylla paramamosain). Elucidation of the Scy expression pattern in tissues will help in understanding its potential function associated with the reproductive immunity. In our study, Scy mRNA transcripts and its protein were found widely distributed in mature male and female crabs. Scy mRNA transcripts were significantly demonstrated in the ejaculatory duct and hemocytes of males but were much less expressed in the other tissues tested. In addition, Scy mRNA transcripts were discerned in a number of cells in the glandular epithelium of the inner wall and in the secretion inside the ejaculatory duct using the in situ hybridization method. In females, Scy mRNA transcripts were obviously demonstrated in the hemocytes and gills but weakly detected in other tissues tested. The copy number of scygonadin mRNA transcripts in the ejaculatory duct of males was greatly higher than those in other tissues, in particular, was over 60,000 fold that in the hemocytes of females. Using immunohistochemistry, the Scy protein was found at higher levels in male tissues than in female ones, particularly in the reproductive duct of males. It was also interesting to note that Scy gene expression was not significantly induced with lipopolysaccharide challenge. However, it was highly expressed in the ejaculatory duct and the seminal vesicle of pre-copulatory males and in the spermathecae of post-copulatory females under mating conditions. The results suggested that Scy, as an important antimicrobial component, probably performed more functions in males, and was likely to be involved in a function associated with crab fertilization and reproduction in both males and females during mating.
Subject(s)
Brachyura/genetics , Animals , Brachyura/immunology , Brachyura/metabolism , Brachyura/physiology , Ejaculatory Ducts/metabolism , Female , Gene Expression/immunology , Gills/metabolism , Hemocytes/metabolism , Male , Peptides/genetics , Peptides/immunology , Peptides/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproduction , Seminal Vesicles/metabolismABSTRACT
The antimicrobial peptide scygonadin (Scy) was first isolated from the gonad of Scylla serrata and its gene is predominantly expressed in the ejaculatory duct of adult males. Thus, its function was predicted to be associated with reproductive immunity, but this is still unclear and needs further investigation. In our study, the expression pattern of Scy at different developmental stages of both male and female S. paramamosain was investigated, so that the potential function of this peptide could be examined. Using real-time quantitative PCR, Scy mRNA transcripts were demonstrated obviously in the vulnerable embryos and larvae-zoea I but very weakly detected in the larvae-zoea III, megalops and juveniles. The gene expression pattern showed a decreasing trend during the early developmental stages. The Scy gene had low expression in the ejaculatory duct of small and medium crabs (100g and 200g in weight) whose gonads were underdeveloped. However, the level of Scy expression was significantly increased in large crabs (300g in weight), which had normally become sexually mature at this size. It was further observed that the numbers of Scy mRNA transcripts in sexually mature crabs were significantly more abundant than in immature ones. In addition, the Scy gene was significantly expressed in the ejaculatory duct of mature male crabs during the mating period (April and May) and reached their highest expression in May. Using immunohistochemistry, the Scy protein was strongly detected in the testis and seminal vesicle of small crabs. However, in large crabs, Scy protein was intensively present in more tissues than in small crabs, including the ejaculatory duct, posterior ejaculatory duct, gill and muscle of males, and also in the spermatheca, gill and muscle of females. It is also interesting to note that Scy mRNA transcripts were detected in other crab species and showed similar expression pattern to those in S. paramamosain. This study extended our knowledge concerning the antimicrobial peptide scygonadin, which has its function principally in the ejaculatory duct of males but which may also play a role at different developmental stages of S. paramamosain from embryogenesis to maturation, and is also widely distributed in other crabs.
Subject(s)
Anti-Infective Agents/immunology , Antimicrobial Cationic Peptides/immunology , Brachyura , Immunity/genetics , Animals , Anti-Infective Agents/metabolism , Antimicrobial Cationic Peptides/metabolism , Brachyura/embryology , Brachyura/growth & development , Brachyura/immunology , Ejaculatory Ducts/immunology , Ejaculatory Ducts/metabolism , Female , Gene Expression/immunology , Larva/growth & development , Larva/immunology , Male , RNA, Messenger/immunology , RNA, Messenger/metabolism , Reproduction/immunology , Seminal Vesicles/immunology , Seminal Vesicles/metabolism , Testis/immunology , Testis/metabolismABSTRACT
BACKGROUND: Spinal muscular atrophy (SMA) is the most common neuromuscular autosomal recessive disorder. The American College of Medical Genetics has recently recommended routine carrier screening for SMA because of the high carrier frequency (1 in 25-50) as well as the severity of that genetic disease. Large studies are needed to determine the feasibility, benefits, and costs of such a program. METHODS AND FINDINGS: This is a prospective population-based cohort study of 107,611 pregnant women from 25 counties in Taiwan conducted during the period January 2005 to June 2009. A three-stage screening program was used: (1) pregnant women were tested for SMA heterozygosity; (2) if the mother was determined to be heterozygous for SMA (carrier status), the paternal partner was then tested; (3) if both partners were SMA carriers, prenatal diagnostic testing was performed. During the study period, a total of 2,262 SMA carriers with one copy of the SMN1 gene were identified among the 107,611 pregnant women that were screened. The carrier rate was approximately 1 in 48 (2.10%). The negative predictive value of DHPLC coupled with MLPA was 99.87%. The combined method could detect approximately 94% of carriers because most of the cases resulted from a common single deletion event. In addition, 2,038 spouses were determined to be SMA carriers. Among those individuals, 47 couples were determined to be at high risk for having offspring with SMA. Prenatal diagnostic testing was performed in 43 pregnant women (91.49%) and SMA was diagnosed in 12 (27.91%) fetuses. The prevalence of SMA in our population was 1 in 8,968. CONCLUSION: The main benefit of SMA carrier screening is to reduce the burden associated with giving birth to an affected child. In this study, we determined the carrier frequency and genetic risk and provided carrier couples with genetic services, knowledge, and genetic counseling.