Identification of Genes Related to Resistance to Ichthyophthirius multifiliis Based on Co-expression Network Analysis in Grass Carp.

The ciliate protozoan Ichthyophthirius multifiliis is an essential parasite causing white spot disease in grass carp, leading to significant economic losses. Understanding the molecular basis of grass carp's response to I. multifiliis has important scientific and environmental values. The transcriptional network analysis offers a valuable strategy to decipher the changes in gene expression in grass carp infected with I. multifiliis. Our goal was to screen the genes and pathways involved in resistance to I. multifiliis in grass carp. The different traits exhibited by grass carp infected with I. multifiliis may be caused by the differences in gene expression among grass carp individuals. Herein, to reveal those resistance-associated genes against I. multifiliis infection, we performed RNA sequencing using weighted gene co-expression network analysis (WGCNA). The biological function analysis and hub gene annotation for highly relevant modules revealed that different pathogen recognition and clearance responses resulted in different resistance to I. multifiliis infection. Furthermore, gene enrichment analysis revealed that I. multifiliis invasion in the disease-resistant group mainly activated immune pathways, including scavenger receptor activity and kappa B kinase/NF-kappa B signaling. By the annotation of the highly correlated module of the hub gene, we revealed that the apoptosis and ribosome biogenesis-related genes were enriched in the disease-resistant grass carp. The results of the dark grey module showed that several genes were mainly enriched in the two-component system (ko02020) and steroid biosynthesis (ko00100), suggesting that they are resistance-associated and energy metabolism-associated genes. In the disease resistance group, hub genes mainly included Nlrc3, fos, AAP8, HAP2, HAX, cho2, and zgc:113,036. This study revealed the gene network associated with disease resistance after I. multifiliis infection. The disease resistance-related pathways and central genes identified in this study are candidate references for breeders breeding disease-resistant. The results of this study may also provide some references for the development of drugs to antagonize I. multifiliis infection.

Transcriptome Analysis of the Spleen Provides Insight into the Immune Regulation of GCRV Resistance in Grass Carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is one of the most economically important fish in China, and its production is commonly lost due to GCRV infection. To understand the molecular mechanism of GCRV resistance in grass carp, we compared the spleen transcriptome of the GCRV-resistant and susceptible individuals under GCRV infection (Res-Sus) and the GCRV-resistant individuals under different conditions of injection with GCRV and PBS (Res-Ctl). A total of 87.56 GB of clean data were obtained from 12 transcriptomic libraries of spleen tissues. A total of 379 DEGs (156 upregulated genes and 223 downregulated genes) were identified in the comparison group Res-Ctl. A total of 1207 DEGs (633 upregulated genes and 574 downregulated genes) were identified in the comparison group Res-Sus. And 54 DEGs were shared including immune-related genes of stc2 (stanniocalcin 2), plxna1 (plexin A1), ifnα (interferon alpha), cxcl 11 (C-X-C motif chemokine ligand 11), ngfr (nerve growth factor receptor), mx (MX dynamin-like GTPase), crim1 (cysteine-rich transmembrane BMP regulator 1), plxnb2 (plexin B2), and slit2 (slit guidance ligand 2). KEGG pathway analysis revealed significant differences in the expression of genes mainly involved in immune system and signal transduction, including antigen processing and presentation, Toll-like receptor signaling pathway, natural killer cell-mediated cytotoxicity, and Hippo signaling pathway. This study investigates the immune mechanism of the resistance to GCRV infection in grass carp and provides useful information for the development of methods to control the spread of the GCRV infection.

Anti-inflammatory and antioxidant properties of rice bran oil extract in copper sulfate-induced inflammation in zebrafish (Danio rerio).

Tocotrienols have strong antioxidant properties; however, tocotrienol has not been investigated in detail in aquatic products. In this study, the anti-inflammatory and antioxidant activities of the tocotrienol-rich fraction from rice bran oil and its potential mechanism were verified in a zebrafish CuSO4 inflammation model. The in vitro antioxidant activity was evaluated using the 2,2-Diphenyl-1-picrylhydrazyl (DPPH) stable radical method. The copper chelating activity was determined using the pyrocatechol violet method. Intracellular reactive oxygen species in zebrafish were detected using a fluorescent ROS probe. Transgenic Tg (lyz: DsRed2) zebrafish were used for neutrophil transmigration assays. The mRNA expression levels of antioxidant and pro-inflammatory factor genes were measured using quantitative real-time reverse transcription PCR. In the concentration range tested, 100 µg/mL TRF had the highest copper chelating activity (10%). TRF showed DPPH-free radical scavenging ability, which was 53% at 100 µg/mL TRF. TRF effectively repressed ROS generation and inhibited neutrophil migration to the inflamed site. Moreover, TRF upregulated the expression of antioxidant genes sod and gpx4b, inhibited the expression of pro-inflammatory factors tnfa and il8, and suppressed CuSO4-induced inflammation. In conclusion, TRF has significant anti-inflammatory and antioxidant properties, which supports the use of TRF as an aquatic feed additive to improve the anti-inflammatory and antioxidant capacity of aquatic products.

LIFGO: A modular laser-induced fluorescence detection system based on plug-in blocks.

In this work, a laser-induced fluorescence (LIF) detection system built in a modular assembling mode was developed based on commercial LEGO blocks and 3D printed blocks. We designed and fabricated a variety of 3D printed building blocks fixed with optical components, including laser light source, filters, lens, dichroic mirror, photodiode detector, and control circuits. Utilizing the relatively high positioning precision of the plug-in blocks, a modular construction strategy was adopted using the flexible plug-in combination of the blocks to build a highly sensitive laser-induced fluorescence detection system, LIFGO. The LIFGO system has a simple structure which could be constructed by inexperienced users within 3 h. We optimized the structure and tested the performance of the LIFGO system, and its detection limits for sodium fluorescein solution in 100 µm i.d. and 250 µm i.d. capillaries were 7 nM and 0.9 nM, respectively. Based on the LIFGO system, we also built a simple capillary electrophoresis (CE) system and applied it to the analysis of DNA fragments to demonstrate its application possibility in biochemical analysis. The separation of 7 fragments in DL500 DNA markers were completed in 600 s. Because of the features of low cost (less than $100) and easy-to-build construction, we introduced the LIFGO system to the experimental teaching of instrumental analysis for undergraduate students. The modular construction form of the LIF detection system greatly reduces the threshold of instrument construction, which is conducive to the popularization of the LIF detection technique in routine laboratories as well as the reform of experimental teaching mode.

Gene expression profiling of DNA methyltransferase genes in Siniperca chuatsi based on transcriptome sequencing.

The mandarin fish (Siniperca chuatsi) DNA methyltransferase gene 1 (dnmt1) was highly expressed in the mesonephros, head kidney and gonad, whereas dnmt2 was expressed in most tissues. dnmt3a was highly expressed in the brain and spleen, but dnmt3b was mainly expressed in the brain and head kidney. The genes dnmt1 and dnmt2 were highly expressed in the early stages of embryonic development, and dnmt3a and dnmt3b were expressed later. These genes also showed certain changes after artificial diet acclimation, salinity adaptation and immune stress.

Identification, Virulence, and Molecular Characterization of a Recombinant Isolate of Grass Carp Reovirus Genotype I.

The hemorrhagic disease of grass carp (HDGC) caused by grass carp reovirus (GCRV) still poses a great threat to the grass carp industry. Isolation and identification of the GCRV genotype I (GCRV-I) has been rarely reported in the past decade. In this study, a new GCRV was isolated from diseased fish with severe symptoms of enteritis and mild hemorrhages on the body surface. The isolate was further identified by cell culture, transmission electron, indirect immunofluorescence, and SDS-PAGE electrophoretic pattern analysis of genomic RNA. The results were consistent with the new isolate as a GCRV-I member and tentatively named GCRV-GZ1208. Both grass carp and rare minnow infected by the GCRV-GZ1208 have no obvious hemorrhagic symptoms, and the final mortality rate was ≤10%, indicating that it may be a low virulent isolate. GZ1208 possessed highest genomic homology to 873/GCHV (GCRV-I) and golden shiner reovirus (GSRV). Additionally, it was found a 90.7-98.3% nucleotide identity, a 96.4-100% amino acid identity, and <50% identity with GCRV-II and III genotypes. Interestingly, the sequences of some segments of GZ1208 were similar to GCRV-8733/GCHV, whereas the remaining segments were more closely related to GSRV, suggesting that a recombination event had occurred. Bootscan analysis of the complete genomic sequence confirmed this hypothesis, and recombination events between 873/GCHV and other GSRV-like viruses were also accompanied by gene mutations.

Novel GLP-1 analog supaglutide improves glucose homeostasis in diabetic monkeys.

Glucagon-like peptide 1 (GLP-1) is an insulinotropic hormone and plays an important role in regulating glucose homeostasis. GLP-1 has a short half-life (t1/2 < 2 min) due to degrading enzyme dipeptidyl peptidase-IV and rapid kidney clearance, which limits its clinical application as a therapeutic reagent. We demonstrated recently that supaglutide, a novel GLP-1 mimetic generated by recombinant fusion protein techniques, exerted hypoglycemic and ß-cell trophic effects in type 2 diabetes db/db mice. In the present study, we examined supaglutide's therapeutic efficacy and pharmacokinetics in diabetic rhesus monkeys. We found that a single subcutaneous injection of supaglutide of tested doses transiently and significantly reduced blood glucose levels in a dose-dependent fashion in the diabetic monkeys. During a 4-week intervention period, treatment of supaglutide of weekly dosing dose-dependently decreased fasting and random blood glucose levels. This was associated with significantly declined plasma fructosamine levels. The repeated administration of supaglutide remarkably also decreased body weight in a dose-dependent fashion accompanied by decreased food intake. Intravenous glucose tolerance test results showed that supaglutide improved glucose tolerance. The intervention also showed enhanced glucose-stimulated insulin secretion and improved lipid profile in diabetic rhesus monkeys. These results reveal that supaglutide exerts beneficial effects in regulating blood glucose and lipid homeostasis in diabetic rhesus monkeys.

Studies on the clinical symptoms, virus distribution, and mRNA expression of several antiviral immunity-related genes in grass carp after infection with genotype II grass carp reovirus.

The viral hemorrhage disease caused by grass carp reovirus (GCRV) is a serious contagious disease of grass carp that mainly infects fingerlings and yearlings. Epidemiological studies have shown that GCRV genotype II is currently the prominent genotype. However, little is known about the histopathological characteristics, virus distribution, and expression of immunity-related genes in grass carp infected by GCRV genotype II. In this study, we found that grass carp infected by GCRV genotype II lost appetite, swam alone, and rolled, and their fins, eyes, operculum, oral cavity, abdomen, intestine, and muscles showed pronounced punctate hemorrhage. Congestion, swelling, deformation, thinning of membranes, dilatation and darkened color of nucleoli, cathepsis, erythrocyte infiltration, and vacuole formation were observed in some infected tissues. A qRT-PCR test showed that the 11 genome segments of GCRV had similar expression patterns in different tissues. The S8 segment, with unknown function and no homologous sequences, had the highest expression level, while the most conserved segment, L2, had the lowest expression level. GCRV particles were distributed in different tissues, especially in the intestine. In the infected intestine, the expression of various receptors and adaptor molecules was modulated at different levels. Pro-inflammatory cytokine interleukin-1ß (IL-1ß) expression was 2160.9 times higher than that in the control group. The upregulation of immunity-related genes activated the antiviral immunity pathways. Therefore, the intestine might play a dual role in mediating GCRV infection and the antiviral immune response. This study provides detailed information about the pathogenicity of GCRV and expression of immunity-related genes, laying the foundation for further research on virus control and treatment.

Pigmentation formation and expression analysis of tyrosinase in Siniperca chuatsi.

Animal pigmentation primarily depends on the presence and mixing ratio of chromatophores, functioning in animal survival and communication. For the benthic and carnivorous Siniperca chuatsi, pigmentation pattern is key to concealment and predation. In this study, the formation, distribution, and main pattern of chromatophores were observed in the embryos, larvae, skins, and visceral tissues from S. chuatsi. Melanophores were firstly visualized in the yolk sac at segmentation stage, and then they were migrated to the whole body and further clustered into the black stripes, bands, and patches. In adult S. chuatsi, the head, black band, and body side skins mainly contained melanophores, showing as deep or light black. The abdomen skin mainly contained iridophores, showing as silvery. In the eye, the pigment layers were located in the epithelial layers of iris and retina and shown as black. Then, the pigmentation-related gene, tyrosinase gene from S. chuatsi (Sc-tyr) was analyzed by bioinformatics and quantitative methods. The Sc-tyr gene encoded a protein with 540 amino acids (Sc-TYR). The Sc-TYR contained two copper ion binding sites, which were coordinated by six conserved histidines (H182, H205, H214, H366, H370, H393) and necessary for catalytic activity. The Sc-TYR was well conserved compared with TYR of various species with higher degree of sequence similarity with other fishes (77.6-98.3%). The qRT-PCR test showed that the Sc-tyr mRNA reached the peak value at segmentation stage in the embryo development, the black skins displayed a higher expression level than that in silvery skin, and the eye had the highest expression level compared with other tissues. Further research on enzyme activity showed that the expression patterns of tyrosinase activity were similar to that of the Sc-tyr mRNA. Comparing with the results of molecular and phenotype, it was found that the temporal and spatial distributions of tyrosinase corresponded well with changes in pigmentation patterns and the intensity of skin melanization. This study initially explored the pigmentation formation and tyrosinase expression, which served as a foundation for further insight into the genetics mechanism of body color formation in S. chuatsi.

Expression and Functional Analysis of Hepcidin from Mandarin Fish (Siniperca chuatsi).

Hepcidin is a liver-derived peptide hormone that is related to iron balance and immunity in humans. However, its function in Siniperca chuatsi has not been well elucidated. In this study, we analyzed the expression and function of the S. chuatsi hepcidin (Sc-hep) gene. Sc-hep was specifically expressed in the liver and appeared to be one of the most highly expressed genes in the liver. After spleen and kidney necrosis virus (ISKNV) infection and lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly I:C) stimulation, the expression of Sc-hep in the liver increased by approximately 110, 6500, and 225 times, respectively. After ferrous sulfate (FS) injection, the expression of Sc-hep in the liver increased approximately 520-fold. We found that miR-19c-5p could inhibit Sc-hep expression. Five CpG dinucleotides distributed in the promoter region showed no differential methylation between the liver and the stomach, both presenting high methylation rates. After FS or LPS injection, the expression of three iron balance-related genes (FPN1, TFR1, and FTN) and five immune-related cytokine genes (IL-1ß, IL8, TNF-α, TLR22, and SOCS3) significantly changed. These results indicate that Sc-hep participates in the regulation of iron balance and plays an important role in the immune system. Sc-hep increased approximately 52-fold when mandarin fish were domesticated with artificial diets. Sc-hep might be used as a real-time biomarker of mandarin fish liver because its expression markedly varies under different physiological conditions.

Integrative cytological analysis of the effects of Ca2+ and vitamin D3 on extracellular Ca2+ flux and intracellular Ca2+ reserves in the mantle of the pearl oyster (Hyriopsis cumingii Lea).

To examine Ca2+ absorption and transportation in the freshwater pearl oyster, Hyriopsis cumingii Lea, we studied the effects of different levels of either extracellular Ca2+ or 1,25(OH)2D3 on extracellular Ca2+ flux and intracellular Ca2+ concentrations in mantle cells using the non-invasive micro-test technique and laser scanning confocal microscopy. The inner and outer mantle (IM and OM) cells from mussels were cultured and then treated with different concentrations of Ca2+ and 1,25(OH)2D3. Extracellular Ca2+ flux and intracellular Ca2+ reserves were analyzed. The results showed that both extracellular Ca2+ and 1,25(OH)2D3 had significant effects on Ca2+ flux and reserves in mantle cells, especially in IM cells (P < .05). The increase in extracellular Ca2+ concentrations resulted in the conversion of extracellular Ca2+ flux into influx with an increase in flow rate (P < .05). The calcium ion fluorescence intensity of OM cells was higher than that of IM cells (P < .05). 1,25(OH)2D3 addition also significantly increased the influx rate of extracellular Ca2+, especially in IM cells, which were more sensitive to 1,25(OH)2D3 addition and had significantly higher Ca2+ influx rates than did OM cells (P < .05). Fluorescence intensities of intracellular Ca2+ first increased and then decreased with increasing 1,25(OH)2D3 levels. The study showed that IM cells play an important role in absorbing Ca2+ from the environment, while OM cells mainly function in the temporary storage and transportation of Ca2+ in the body. The current results suggested that high levels of extracellular Ca2+ (1.25 mM) or 1,25(OH)2D3 (over 100 IU/L) were favorable for Ca2+ uptake and maintenance in the body.

Irf3 from mandarin fish thymus initiates interferon transcription.

Interferon regulatory factors (IRFs) are transcription factors of the interferon (IFN)-inducible signaling pathway essential for host immunity against antimicrobial infection by virus and bacteria. Interferon regulatory factor 3 (IRF3) regulates the expression of IFNs and IFN-stimulated genes by binding to the IFN stimulatory response element (ISRE). In this study, we analyze the thymus transcriptome of the mandarin fish Siniperca chuatsi and report the functional analysis of Irf3 from the thymus as an emerging model of antiviral approaches. The predicted S. chuatsi IRF3 (Sc-Irf3) protein has 465 amino acid residues and evolutionarily conserved domains and is clustered in the IRF3 subfamily on a phylogenetic tree. Sc-Irf3 upon transgenic expression was mainly found in the cytoplasm through Western blot analysis and microscopy, but it translocated to the nucleus after polyinosinic:polycytidylic acid (ploly I:C) treatment. Endogenous Sc-irf3 RNA expression was detected in all eight adult organs examined. Importantly, Sc-irf3 RNA expression was significantly upregulated by ploly(I:C) treatment in the adult organs. Concurrently, reporter assays revealed that Sc-Irf3 increased the transcriptional activity of the ifnß promoter, a minimal ISRE-containing promoter, and ifn promoter of mandarin fish. Therefore, Sc-Irf3 plays a major role in the IFN immune defense system against virus infection.

Structure and function of S9 segment of grass carp reovirus Anhui strain.

A highly virulent grass carp reovirus (GCRV) strain, named GCRV-AH528, was recently purified from a diseased grass carp with hemorrhage disease in Anhui, China. GCRV-AH528 S9 segment was 1320 nucleotides in length and encoded a 418 amino acid VP6 protein. BLAST search showed that the VP6 protein owned a conserved domain belonging to the reoviral σ2 family. Phylogenetic analysis of VP6 presented that GCRV-AH528 belonged to GCRV genotype II, which was more closely related to Orthoreovirus than GCRV genotype I and genotype III. Further analysis revealed that GCRV-AH528 S9 and mammalian orthoreovirus S8 might have evolved from a common ancestral precursor and have identical mechanism in virus assembly. The expression level of vp6 gene was detected by quantitative real-time PCR (qRT-PCR). Over time, the expression level of vp6 gradually increased in Ctenopharyngodon idellus kidney cells. However, the level of vp6 expression in blood sharply increased at 4-6 days, and then decreased to a low level after GCRV-AH528 challenge (P < 0.05). The vp6 gene was detected in all tissues examined, whereas at relatively higher levels in blood, kidney, and liver (P < 0.05). The yeast two-hybrid (Y2H) system was used to identify VP6 self-interaction, while no interaction was detected in VP6-VP6. This study not only revealed the S9 segment structure and expression pattern but also analyzed the VP6 mechanism by yeast hybridization method. The present study provides valuable informations for further experimental design and investigation of VP6 functions.

Sequence and functional analysis of intestinal alkaline phosphatase from Lateolabrax maculatus.

Alkaline phosphatases (Alps) belong to a class of phosphate transferases that dephosphorylate lipopolysaccharide (LPS), adenosine triphosphate, and nucleotides. In this study, a 1874-base pair (bp) intestinal alp cDNA sequence was cloned from Lateolabrax maculatus and designated as Lm-alpi. It contained a 1611 bp open reading frame which encoded a protein with 537 amino acids. Protein sequence alignment showed that Lm-AlpI shared 29.8-79.8% identity with its homologs. Lm-AlpI catalytic sites contained three metal ion sites (two Zn2+ and one Mg2+), referring to D73, H184, D348, H349, H352, H464, D389, and H390 residues, which are essential for enzymatic activity and conservation in different organisms. Two predicted disulfide bonds in Lm-AlpI were composed of four cysteines (C152-C214 and C499-C506), which were homologous to those of mammals. Immunohistochemical staining revealed that Lm-AlpI was mainly expressed on the mucosal surface of the gastrointestinal tract, including stomach, intestine, and gastric cecum. Lm-AlpI was mainly located on the plasma membrane of transiently transfected HeLa cells. The mRNA of Lm-alpi was mainly expressed in the intestine, and its expression levels gradually increased after LPS treatment and further increased by 1.81-fold after 48 h. After desalting culture, the relative mRNA expression level of Lm-alpi decreased at 30 and 50 days after hatching (DAH) and then returned to normal levels at 70 DAH. Further experiments demonstrated that the enzyme activity of Lm-AlpI exhibited an expression pattern similar to that of the mRNA expression of Lm-alpi after LPS treatment and desalting culture. This study provided valuable information on the Lm-AlpI functions associated with the mucosal immunity and salinity adaptation of L. maculatus.

A Novel Model-Based Driving Behavior Recognition System Using Motion Sensors.

In this article, a novel driving behavior recognition system based on a specific physical model and motion sensory data is developed to promote traffic safety. Based on the theory of rigid body kinematics, we build a specific physical model to reveal the data change rule during the vehicle moving process. In this work, we adopt a nine-axis motion sensor including a three-axis accelerometer, a three-axis gyroscope and a three-axis magnetometer, and apply a Kalman filter for noise elimination and an adaptive time window for data extraction. Based on the feature extraction guided by the built physical model, various classifiers are accomplished to recognize different driving behaviors. Leveraging the system, normal driving behaviors (such as accelerating, braking, lane changing and turning with caution) and aggressive driving behaviors (such as accelerating, braking, lane changing and turning with a sudden) can be classified with a high accuracy of 93.25%. Compared with traditional driving behavior recognition methods using machine learning only, the proposed system possesses a solid theoretical basis, performs better and has good prospects.

Identification and differential expression of microRNAs during metamorphosis of the Japanese flounder (Paralichthys olivaceus).

BACKGROUND: MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of 20-25 nucleotides that play a key role in diverse biological processes. Japanese flounder undergo dramatic metamorphosis in their early development. The metamorphosis is characterized by morphological transformation from a bilaterally symmetrical to an asymmetrical body shape concomitant with extensive morphological and physiological remodeling of organs. So far, only a few miRNAs have been identified in fish and there are very few reports about the Japanese flounder miRNA. METHODOLOGY/PRINCIPAL FINDINGS: Solexa sequencing technology was used to perform high throughput sequencing of the small RNA library from the metamorphic period of Japanese flounder. Subsequently, aligning these sequencing data with metazoan known miRNAs, we characterized 140 conserved miRNAs and 57 miRNA: miRNA* pairs from the small RNA library. Among these 57 miRNA: miRNA* pairs, twenty flounder miRNA precursors were amplified from genomic DNA. We also demonstrated evolutionary conservation of Japanese flounder miRNAs and miRNA* in the animal evolution process. Using miRNA microarrays, we identified 66 differentially expressed miRNAs at two metamorphic stages (17 and 29 days post hatching) of Japanese flounder. The results show that miRNAs might play a key role in regulating gene expression during Japanese flounder metamorphosis. CONCLUSIONS/SIGNIFICANCE: We identified a large number of miRNAs during flounder metamorphosis, some of which are differentially expressed at two different metamorphic stages. The study provides an opportunity for further understanding of miRNA function in the regulation of flounder metamorphosis and gives us clues for further studies of the mechanisms of metamorphosis in Japanese flounder.

Prolonged hemolysis and methemoglobinemia following organic copper fungicide ingestion.

Acute ingestion of copper sulfate has been reported to cause gastrointestinal injury, hemolysis, methemoglobinemia, hepatorenal failure, shock; or even death. The toxicity of organocopper compounds, however, remains largely unknown. A 40-y-old man attempted suicide by ingesting some 50 ml of Sesamine fungicide. He immediately developed headache, vomiting and abdominal pain, followed by progressive dyspnea, cyanosis, dark urine and diarrhea. Severe methemoglobinemia and hemolysis were documented, and treatment with ascorbic acid and hydration was commenced. He was referred to our service 3 d later for methylene blue treatment. Despite the above treatment, his symptomatology persisted and it was not until 5 d post-ingestion that the implicated fungicide was identified as copper-8-hydroxyquinolate. BAL therapy and plasma exchange were instituted, which decreased his plasma hemoglobin from 1,300 mg/dL to 29.1 mg/dL, and lowered his methemoglobin level from 20.9% to 1.1%. His serum and urine copper concentration dropped from 238 microg/dL to 96 microg/dL and from 112 microg/dL to 16 microg/dL, respectively. He was discharged uneventfully 18 d post-ingestion. Pre-existing glucose-6-phosphate dehydrogenase (G6PD) deficiency as well as copper-induced inhibition of G6PD activity was documented during hospitalization. Organocopper compounds may cause prolonged hemolysis and methemoglobinemia through oxidative stress, especially among patients with G6PD deficiency. Antidotal therapy with methylene blue is not likely to be effective in this setting: treatment with intensive supportive measures and other therapeutic options, such as plasma exchange, should be sought.

Determination of aristolochic acids in medicinal plant and herbal product by liquid chromatography-electrospray-ion trap mass spectrometry.

This paper describes a liquid chromatography-electrospray-ion trap mass spectrometry (LC-ES-ITMS) method for the determination of aristolochic acid I and II (AA-I and AA-II) in medicinal plants and Chinese herbal remedies. A reversed phase C(18) column with gradient elution was utilized. The effects of mobile phase additives, acetic acid and ammonium acetate, on LC separation and ES ionization were investigated. For both AA-I and AA-II, the [M+NH(4)](+) ion was found to be the precursor ion for target MS/MS analysis. The MS/MS product ion, [M+H-44](+), was used for the quantitative measurement of AA-I and AA-II. The linearity was good from 0.03 to 5 mug ml(-1) and good correlation (r(2)=0.999) over the range examined was determined for both AA. The detection limit based on a signal-to-noise ratio of three was 0.012 and 0.015 mug ml(-1) for AA-I and AA-II, respectively. Various Chinese herbal remedies obtained from renal failure patients and medicinal plants were examined by this newly developed method.

High-performance liquid chromatographic determination for aristolochic acid in medicinal plants and slimming products.

A HPLC procedure with a silica gel RP-18 reversed-phase column for the determination of aristolochic acids I, II in medicinal plants and slimming products was developed. The mobile system 0.3% ammonium carbonate solution-acetonitrile (75:25, v/v) with pH 7.5 was the optimal buffer to clearly separate aristolochic acids I, II within 20 min. The recovery of aristolochic acids I, II in medicinal plants and slimming products was better than 90% by extracting with methanol and purifying through a PHP-LH-20 column. The major component was aristolochic acid I in Aristolochia fangchi and the level ranged from 437 to 668 ppm. Aristolochic acid II was the major component for Aristolochia contorta and its range was <1-115 ppm. Twelve out of 16 samples of slimming pills and powders contained aristolochic acids I and/or II. The major component in most slimming products was aristolochic acid II and the level ranged from <1 to 148 ppm. It may indicate that slimming products were not mainly made of A. fangchi.