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HS3ST1 is a genetic risk gene associated with Alzheimer’s disease (AD) and overexpressed in patients, but how it contributes to the disease progression is unknown. We report the analysis of brain heparan sulfate (HS) from AD and other tauopathies using a LC-MS/MS method. A specific 3- O -sulfated HS displayed sevenfold increase in the AD group ( n = 14, P < 0.0005). Analysis of the HS modified by recombinant sulfotransferases and HS from genetic knockout mice revealed that the specific 3- O -sulfated HS is made by 3- O -sulfotransferase isoform 1 (3-OST-1), which is encoded by the HS3ST1 gene. A synthetic tetradecasaccharide (14-mer) carrying the specific 3- O -sulfated domain displayed stronger inhibition for tau internalization than a 14-mer without the domain, suggesting that the 3- O -sulfated HS is used in tau cellular uptake. Our findings suggest that the overexpression of HS3ST1 gene may enhance the spread of tau pathology, uncovering a previously unidentified therapeutic target for AD.

Heparan sulfate (HS) is a linear polysaccharide with complex structures and modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Recently, we generated a HS mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell individually or in their combination. Here, we describe the experimental procedure using the mutant cell library to determine the structure-function relationship of HS in the regulation of FGF2-FGFR1 signaling at the levels of cell surface FGF2 binding and the downstream intracellular signaling activation. Our results demonstrated that strictly defined fine structure is required for HS to act as a co-receptor for FGF2-FGFR1 signaling.

Heparan sulfate (HS) is a linear polysaccharide with complex structures and modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Recently, we generated a HS mutant mouse lung endothelial cell library by systematic deletion of HS genes expressed in the cell individually or in their combination. Here, we describe the experimental procedure using the mutant cell library to determine the structure-function relationship of HS in the regulation of FGF2-FGFR1 signaling at the levels of cell surface FGF2 binding and the downstream intracellular signaling activation. Our results demonstrated that strictly defined fine structure is required for HS to act as a co-receptor for FGF2-FGFR1 signaling.

Diabetic cardiomyopathy is a common complication in patients with diabetes and is associatedwith impaired responsiveness of ischemic myocardium to proangiogenic factors, subsequentlyleading to heart failure. STK35, a novel kinase that binds to nuclear actin, has been shown toregulate important cellular functions such as cell migration, proliferation, survival, andangiogenesis. Currently, the contribution of altered STK35 expression in human diseases remainsunexplored. In initial studies, we observed that human cardiac biopsies from diabetic patientsshowed a significant decrease in STK35 expression as compared to non-diabetic control hearts.Intriguingly, in a STZ-induced mouse model of diabetes, i.v . injection of rAAV9-STK35 to expressconstitutive STK35 in heart in FVB/N male mice promoted neovascularization and lowered cardiacfibrosis, leading to improved cardiac function of diabetic heart. Our in vitro studies observed highglucose decreased STK35 expression in mouse cardiac endothelial cells (MCEC), whereasSTK35 overexpression increased MCEC migration and vascular tube formation, and upregulatedMCEC to expression of multiple pro-angiogenic proteins. Taken together, our results demonstratethat cardiac-targeted STK35 gene therapy exerts a marked beneficial action by attenuating bothcardiac remodeling and cardiac function in a mouse model of diabetes mellitus. Mechanistically,the beneficial effect may be attributed, at least partially, to enhanced neovascularization in heart.

Oxidative stress plays a crucial role in ammonia nitrogen toxicity. In this study, the beneficial effects of dietary nano cerium oxide (nano CeO2) as a potent antioxidant were examined in the Chinese mitten crab (Eriocheir sinensis). Crabs were fed a diet supplemented with 0, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, or 12.8 mg/kg nano CeO2 for 60 d. The optimum supplementation level of nano CeO2 that significantly increased weight gain rate and decreased feed coefficient was 0.8 mg/kg. This level also offered immune protection when crabs were kept under ammonia nitrogen stress and/or exposed to pathogen infection (Aeromonas hydrophila). Supplementation with 0.8 mg/kg of CeO2 (i) relieved pathological damage to the hepatopancreas; (ii) increased hemocyte counts, including total number of hemocytes, granulocytes, and hyalinocytes; (iii) decreased malondialdehyde content and increased antioxidant enzyme activities of superoxide dismutase and catalase in the hemolymph; (iv) increased the activities of lysozyme, acid phosphatase, and alkaline phosphatase in the hemolymph; and (v) increased gene and protein expression of cathepsin L in the hepatopancreas. Mortality increased when crabs were injected with bacteria under ammonia nitrogen stress, but dietary supplementation with 0.8 mg/kg nano CeO2 decreased the mortality rate. Thus, the results of this study suggested that dietary supplementation with nano CeO2 in crabs promoted growth and up-regulated immunity to bacterial infection under ammonia nitrogen stress.

Dietary lipid oxidation is a lasting concern to aquafeed manufacturers and aquaculture farmers. Here, we determined if there was an association between oxidized fish oil (OFO) and hepatopancreatic injury in Chinese mitten crab, and evaluated the alleviating effects of a combination of vitamins C and E on hepatopancreatic injury. There were significant reductions in the body weight gain, hepatopancreatic index, and feed efficiency in crabs that received OFO. Diets with OFO also resulted in reduced crude fat contents in the hepatopancreas and muscle tissues, and alternations in the hepatopancreatic fatty acid compositions, especially a reduction in polyunsaturated fatty acids. Moreover, dietary OFO exposure reduced the antioxidant defense and non‐specific immune response, caused hepatopancreatic injury, and even apparent whitening. An ultrastructural examination of the whitened hepatopancreas revealed that hepatopancreatic cells were injured at varying degrees following OFO exposure. The combination of vitamins C and E could efficiently maintain a constant fatty acid profile in the hepatopancreas exposed to OFO, thus alleviating OFO‐induced hepatopancreatic injury. In addition, a biochemical analysis indicated that a combination of vitamins C and E not only improved the antioxidant status by mitigating the harmful effects of oxidized oil, but also modulated non‐specific immune responses in the crab that received OFO. Overall, OFO‐induced hepatopancreatic injury may be alleviated by a dietary combination of vitamins C and E, and the feeding of rancid forage fish or diets that contain highly oxidized oil is likely a cause of hepatopancreatic necrosis that is frequently observed in farmed crabs.

Intestinal microbiota has increasingly been recognized as a major determinant of host health due to its critical role in the maintenance of immune homeostasis. An experimental fish model with disrupted intestinal microbiota allows us to identify potential strategies for restoring or modulating microbiota composition. The aim of this study was to develop a reliable grass carp model with disrupted intestinal microbiota by treatment with an antibiotic cocktail consisting of vancomycin, enrofloxacin, florfenicol, and metronidazole at different concentrations. To identify the appropriate concentration and exposure time of antibiotic treatment required to disrupt intestinal microbiota, five antibiotic regimens were compared in terms of their effects on intestinal microbiota composition, growth and health status, and histological changes in the intestine, hepatopancreas, and spleen. Antibiotic-induced changes in the expression of inflammation- and apoptosis-related genes in grass carp intestines were also examined. The results revealed that the highest antibiotic concentration over a 28 day period was required to disrupt both luminal and mucosal microbiota through significant expansion of Proteobacteria and suppression of Fusobacteria. Treatment with this regimen induced significant down-regulation of caspase-8, MLCK, ZO-1, IL-17N, and IL-23R, and up-regulation of IL-1β and IL-6 in the intestine, suggesting possible effects on intestinal epithelial tight junction and inflammatory conditions. This treatment did not produce substantial histopathological changes, and was not directly detrimental to growth and health. Therefore, this antibiotic regimen may provide a suitable option to disrupt intestinal microbiota in grass carp. In addition, our study also implies that fish may serve as a promising model for investigating growth-promoting effects of antibiotics, when treated with antibiotics in low doses.

International audience; Prion-like transcellular spreading of tau in Alz-heimersD isease (AD) is mediated by tau binding to cell surface heparan sulfate (HS). However,t he structural determinants for tau-HS interaction are not well understood. Microarraya nd SPR assays of structurally defined HS oligosaccharides showt hat ar are 3-O-sulfation (3-O-S) of HS significantly enhances tau binding. In Hs3st1 À/À (HS 3-O-sulfotransferase-1 knockout) cells,reduced 3-O-S levels of HS diminished both cell surface binding and internalization of tau. In ac ell culture,t he addition of a3-O-S HS 12-mer reduced both tau cell surface binding and cellular uptake.N MR titrations mapped 3-O-S binding sites to the microtubule binding repeat 2(R2) and proline-richregion 2(PRR2) of tau. Taui so nly the seventh protein currently knownt or ecognize HS 3-O-sulfation. Our work demonstrates that this rare 3-O-sulfation enhances tau-HS binding and likely the transcellular spread of tau, providing an ovel target for disease-modifying treatment of AD and other tauopathies.

The grass carp accounts for a large proportion of aquacultural production in China, but the hemorrhagic disease caused by grass carp reovirus (GCRV) infection often causes huge economic losses to the industry. Interleukin 17 (IL-17) is an important cytokine that plays a critical role in the inflammatory and immune responses. Although IL-17 family members have been extensively studied in mammals, our knowledge of the activity of IL-17 proteins in teleosts in response to viral infection is still limited. In this study, the role of IL-17 in GCRV infection and its mechanism were investigated. The expression levels of IL-17AF1, IL-17AF2, and IL-17AF3 in Ctenopharyngodon idella kidney (CIK) cells gradually increased from 6 h after infection with GCRV. The nuclear translocation of p65, which acts in the NF-κB signaling pathway, was also increased by GCRV infection. The overexpression of IL-17AF1, IL-17AF2, or IL-17AF3 also promoted the nuclear translocation of p65 and the levels of phospho-IκBα in CIK cells, and reduced the expression of the viral structural protein VP7. An NF-κB signal inhibitor abolished the inhibition of GCRV infection by IL-17 proteins. These results suggested that the NF-κB signaling pathway was activated by the overexpression of IL-17 proteins, resulting in the inhibition of viral infection. In conclusion, in this study, we demonstrated that IL-17AF1, IL-17AF2, and IL-17AF3 acted as immune cytokines, exerting an antiviral effect by activating the NF-κB signaling pathway., Highlights • IL-17AF1, IL-17AF2, and IL-17AF3 exerted an antiviral effect by activating the NF-κB signaling pathway. • The expression levels of IL-17AF1, IL-17AF2, IL-17AF3 gradually increased after GCRV infection. • The nuclear translocation of p65 in CIK cells gradually increased after GCRV infection. • Overexpression of IL-17AF1, IL-17AF2, or IL-17AF3 promoted the nuclear translocation of p65 and p-IκBα level in CIK cells. • Overexpression of IL-17AF1, IL-17AF2, or IL-17AF3 reduced the expression of the viral structural proteinVP7.

The genomic and coding sequences of four members of grass carp interleukin (IL)-17 cytokine family, IL-17A/F2, IL-17A/F3, IL-17C and IL-17N, were isolated using rapid amplification of cDNA ends and conventional PCR techniques. A phylogenetic analysis showed that grass carp IL-17 family genes, including previously identified IL-17A/F1 and IL-17D, clustered with their counterparts from other fish species. IL-17D had two exons and one intron, while all the other members were composed of three exons and two introns. A multiple sequence alignment revealed that grass carp IL-17 family cytokines shared a cystine-knot motif that was highly conserved in mammals. A quantitative real-time PCR analysis revealed that these genes were expressed in the different examined tissues at varying levels. Moreover, they displayed different tissue expression patterns in fish at 24 h after intraperitoneal injection (IP) or anal intubation (AI) of Aeromonas hydrophila, implying roles in inflammatory responses. Next, we compared disease activity status and histopathological changes in intestinal tissues over a 7 day period after IP or AI, and then examine the potential differential effects of different infection routes on the expression profiles of IL-17 family genes in inflamed intestinal tissues. The intraperitoneal route resulted in more pronounced pathological symptoms than the anal route in terms of the disease activity index (DAI). Changes in the DAI highly coincided with the severity of intestinal inflammation. The two infection routes resulted in differential IL-17 family gene expression patterns over time in grass carp intestine, which suggests that IL-17 family members may play differential roles in mucosal and systemic immune responses and that IL-17N and IL-17A/F1 might possibly function as pro-inflammatory cytokines that provide defense against bacterial infection in grass carp intestine.

Interleukin-12 receptor β2 (IL-12Rβ2) is a signaling subunit of heterodimeric receptors for IL-12 and IL-35. It plays important regulatory functions in the development of Th1 cells and in the expression of inflammatory cytokines in mammals and other higher vertebrates. However, little is known about IL-12Rβ2 in teleost fish. In this work, we have cloned and characterized IL-12Rβ2 from grass carp (Ctenopharyngodon idella). The full-length cDNA of grass carp IL-12Rβ2 is 2875 bp, which encodes a mature protein with 741 amino acids. This mature protein contains three fibronectin type III domains, a transmembrane helix, and CXW and WSXWS-like motifs that are characteristic of the type I cytokine receptor family. Phylogenetic analysis revealed that cyprinid fish IL-12Rβ2 formed a single branch, clearly separated from those of other vertebrates. We expressed and purified a recombinant grass carp IL-12Rβ2 protein containing major antigenic regions, which was used to raise a polyclonal antibody. The specificity of the antibody was assessed by Western blotting analysis of whole cell lysates from Escherichia coli cells expressing the recombinant IL-12Rβ2, grass carp intestinal intraepithelial lymphocytes, and cultured C. idella kidney cells. To explore the potential regulatory role of IL-12Rβ2 in inflammation, we generated an intestinal inflammation model by anal intubation of fish with Aeromonas hydrophila. Immunohistochemical staining of the inflamed intestines revealed that IL-12Rβ2 expression is consistent with inflammatory cell recruitment during intestinal inflammation. Real-time quantitative PCR revealed that IL-12Rβ2 is widely expressed in normal tissues and is up-regulated in most tissues after infecting with A. hydrophila. We found that IL-12Rβ2, IL-12p35, and interferon-γ were expressed in similar patterns in the intestines during inflammation. Taken together, our results suggest that IL-12Rβ2 is involved in the regulation of intestinal inflammation.

Bacterial enteritis is a widespread inflammatory disease in most farmed fish, yet its pathogenesis is largely unknown, primarily because of the paucity of reproducible experimental models of intestinal inflammation in fish. The aim of the present study was to develop a model of intestinal inflammation in the grass carp by challenging it with a strain of Aeromonas hydrophila . To induce intestinal inflammation, the fish were challenged with A . hydrophila via anal intubation at 2.7 × 10 6 cfu/fish, an appropriate dose based on the disease activity index for inflammatory signs. The pathological changes occurring in the intestines over the 21 day experimental period were examined with light microscopy and scanning electron microscopy. Histopathological examinations showed that A . hydrophila infection caused severe intestinal lesions, including intestinal villus fusion and shedding, and induced heavy inflammatory cell infiltration on day 3 after challenge. Ultrastructural observations also revealed that bacterial infection caused microvillus effacement. The injured intestinal villi were gradually repaired during the subsequent 18 days of the experimental period. Our results also indicated that the inflammatory responses induced by A . hydrophila infection were closely associated with the expression of pro-inflammatory cytokines interleukin 1β (IL-1β), IL-8, and tumor necrosis factor α (TNF-α) and myeloperoxidase (MPO) activity. The highest mRNA levels of these cytokines and highest MPO activity in the intestine were observed on day 3 after challenge. These results show that the intestinal lesions caused by A . hydrophila infection and their repair were completely consistent with the time course of inflammation-related genes or biomarkers. Thus, we have developed an experimental model of intestinal inflammation in an intensively farmed fish. This model is expected to be useful in clarifying the pathogenesis of bacterial enteritis in the grass carp and other farmed fish, and in testing antimicrobial drugs for potential use in aquaculture.

This paper is to explore the biogas production potential of wetland aquatic biomass plants. 7 species of wetland aquatic biomass plants are used in the study, which include 4 plants with more fiber carbohydrate, Acorus calamus Linn, Typha orientalis Presl, Pontederia cordata and Canna indica , and 3 plants with more starch carbohydrate, Colocasia tonoimo Nakai, Thalia dealbata and Hydrocotyle vulgaris. In the experiment, these plants were treated by anaerobic fermentation in batch mode at 37 °C. The results show that the anaerobic biogas production potential (ABP, mL·g −1 VS) of aquatic biomass plants is different for different components content (%TS). The correlation between ABP and hemicellulose content is significant and negative ( R = −0.784, 0.01 p R = 0.767, 0.01 p y = 238.62 + 2.60 x 1 + 28.55 x 2 −2.08 x 2 x 3 + 12.67 x 3 , (Adj- R 2 = 0.962, p (intercept) = 0.034, p ( x 1 ) = 0.101, p ( x 2 ) = 0.036, p ( x 2 x 3 ) = 0.066, p ( x 3 ) = 0.031, p = 0.025, SD = 9.95), y represents ABP (mL g −1 VS), x 1 , x 2 and x 3 represents the cellulose, lignin and starch carbohydrate content (%TS) respectively.

A dielectric barrier discharge system with a discharging zone apart from a degrading zone is developed to remove 4-chlorophenol from water. Oxygen is fed through the discharging zone where it dissociates to form a plasma stream. The plasma stream is introduced into the degrading zone to make 4-chlorophenol degraded effectively. The removal of 4-chlorophenol was influenced by the processing parameters such as gas flow rate, pH and applied voltage. Increasing the peak voltage or gas flow rate among a certain range, the removal efficiency was enhanced. More than 80% of 4-chlorophenol was removed in 6 min at pH 9.00, resulting in a high energy efficiency of 46.34 × 10 −9 mol J −1 (21.46 g/kW h). Discharging in initial 4-chlorophenol solution, the removal percent was about 90% at 30 min with peak voltage of 14.64 kV and oxygen flow rate of 200 L h −1 . The corresponding energy efficiency was 12.35 × 10 −9 mol J −1 (5.72 g/kW h). During the degradation, aromatic products and organic acids were produced. The main aromatic intermediates were different at different pH, suggesting different pathways of the target contaminant. After the benzene ring was opened, organic acid such as formic acid, oxalic acid, d -(+)-malic acid and maleic acid were formed.

Interleukin-8 (IL-8) is a CXC chemokine that plays key regulatory roles in the immune and inflammatory responses implicated in many human diseases. In this study, we identified and characterized an IL-8 homologue from the grass carp, Ctenopharyngodon idellus . A sequence alignment of the full-length cDNA and genomic DNA showed that the exon/intron organization of grass carp IL-8 (gcIL-8) is identical to those of other known CXC chemokine genes. A multiple alignment analysis showed that gcIL-8 is an ELR – CXC chemokine, and its deduced amino acid sequence shares 81% and 36% identity with common carp IL-8s L1 (GenBank ID: ABE47600 ) and L2 (GenBank ID: AB470924 ), respectively, suggesting that it belongs to the lineage 1 group of fish IL-8 proteins. On a phylogenetic tree, gcIL-8 clustered with other teleost IL-8 proteins to form a fish-specific clade, clearly distinct from those of bird, mammal, and amphibian proteins. Real-time quantitative PCR analysis indicated that gcIL-8 is differentially expressed in various tissues under normal conditions and that the expression of gcIL-8 mRNA in immune-related tissues is clearly upregulated by Aeromonas hydrophila infection. To explore the biological effects of gcIL-8, we produced a recombinant protein, rgcIL-8, in a prokaryotic expression system. Purified rgcIL-8 was confirmed to be chemoattractive for head kidney neutrophils and mononuclear leukocytes in vitro . Our histopathological study also revealed that rgcIL-8 exerts proinflammatory effects by inducing neutrophil infiltration and erythrocyte extravasation. Overall, these results suggest that IL-8 is crucially involved in the inflammatory responses of fish.

Introduction Atrophic vaginitis is a common occurrence, particularly among postmenopausal women; however, few seek or receive treatment. One therapeutic solution is topically applied products. Estrogen‐based treatments have been shown to be effective; however, many patients are reluctant to use such formulations due to health concerns, hence the need to assess the efficacy of acceptable alternatives. Aim This multicenter, randomized, controlled, open‐label, parallel‐group clinical trial set out to evaluate the efficacy and safety of hyaluronic acid vaginal gel to treat vaginal dryness compared with estriol cream in postmenopausal women. Methods One hundred forty‐four subjects were randomized, 72 to the test group treated with hyaluronic acid vaginal gel (Hyalofemme) and 72 to the control group treated with estriol cream (Ovestin). Treatment in both groups was applied by means of a device once every 3 days for a total of 10 applications over 30 days. Main Outcome Measures Efficacy was measured by grading vaginal dryness and three other vaginal symptoms on a visual analog scale. Safety assessments included vital signs, laboratory examinations of the vaginal microecosystem, vaginal pH value, vaginal B ultrasound, and incidence of adverse events. Assessments were performed at baseline, by telephone after the third application, and at the final visit. Results Both hyaluronic acid vaginal gel and estriol cream can significantly improve the clinical symptoms of vaginal dryness in postmenopausal women, with improvement rate of 84.44% and 89.42%, respectively, after 10 applications, without statistically significant difference between them. Conclusion Both hyaluronic acid vaginal gel and estriol cream are effective in the treatment of vaginal dryness. Hyaluronic acid vaginal gel may be considered as a valid alternative to estrogen‐based treatments in relieving the symptoms of vaginal dryness.

Air is often used as bubbling gas with the purpose of improving the liquid discharge performance in some pulsed high-voltage discharge systems. Nitrogen in air could dissociate into active species that might participate in a series of liquid chemical reactions. In the discharge system with 4-chlorophenol as target contaminant, some more toxic intermediates were formed and the energy efficiency was decreased by bubbling air and nitrogen. The G50 (energy cost at 50% conversion) is about 0.91 g (KW h)−1, 0.70 g (KW h)−1 and 0.49 g (KW h)−1 respectively by bubbling oxygen, air and nitrogen. Nitro- compounds such as 4-chloro-2-nitrophenol and 4-nitrocatechol were identified, providing evidence for the formation of nitro group. Chloride ion (Cl−) dropped in the degradation process could be activated and react with non-degradable 4-chlorophenol to form 2,6-dichlorophenol. Phenol was also identified for higher yield of reductive radicals. Comparing 4-chlorophenol degradation by bubbling air or nitrogen with that by oxygen, the degradation rates of original contaminants and intermediate products were lower under same energy input. By discharging for 60 min with applied input energy density of 175 W L−1, almost all of aromatic substances were removed with bubbling oxygen but they still remained at about 8.83% with bubbling air and 12.79% with nitrogen. Furthermore, nitrogen in the bubbling gas was transformed into inorganic nitrogen such as NO2- and NO3- that increased linearly with discharge time and achieved a formation rate twice as the degradation rate of original target contaminant.

Inflammation is a protective response that is implicated in bacterial enteritis and other fish diseases. The inflammatory mechanisms behind Aeromonas hydrophila infections in fish remain poorly understood. In this study, we performed a de novo grass carp transcriptome assembly using Illumina’s Solexa sequencing technique. On this basis we carried out a comparative analysis of intestinal transcriptomes from A. hydrophila-challenged and physiological saline solution (PSS/mock) -challenged fish, and 315 genes were up-regulated and 234 were down-regulated in the intestines infected with A. hydrophila. The GO enrichment analysis indicated that the differentially expressed genes were enriched to 12, 4, and 8 GO terms in biological process, molecular function, and cellular component, respectively. A KEGG analysis showed that 549 DEGs were involved in 165 pathways. Moreover, 15 DEGs were selected for quantitative real-time PCR analysis to validate the RNA-seq data. The results confirmed the consistency of the expression levels between RNA-seq and qPCR data. In addition, a time-course analysis of the mRNA expression of 12 inflammatory genes further demonstrated that the intestinal inflammatory responses to A. hydrophila infection simultaneously modulated gene expression variations. The present study provides intestine-specific transcriptome data, allowing us to unravel the mechanisms of intestinal inflammation triggered by bacterial pathogens.

Hepatopancreas necrosis disease (HPND) is a disease and serious impacts on the industry of Chinese mitten crabs (Eriocheir sinensis) culture, however the actual cause of this disease is still not known. In the present study, to explore the pathogenic changes and risk factors caused by HPND, ultrathin sections of different tissues from the diseased crabs were observed with transmission electron microscope. The hepatopancreatic cells, spermatogonium, gill tissues and muscle cells of the diseased crabs showed severe structural and morphological changes. To further investigate whether HPND was caused by pathogenic microorganism, the healthy crabs were fed/injected with diseased tissues, the symptoms of HPND were not found, suggesting that HPND was not caused by virus or microsporidian infections. In addition, the toxic effect of avermectin and high pH water were also examined in this study. 40% (p

By applying pulsed high voltage discharge to a needle-mesh reactor that using seven acupuncture needles as discharge electrode and stainless steel wire mesh as ground electrode, nitrogen from bubbling gas could be fixed into NO 2 − and NO 3 − with equivalent mol H + produced in the liquid phase and a small amount of NO and NO 2 yielded in the gas phase. The HNO 2 was originally formed and then converted into HNO 3 . The ·OH and H 2 O 2 stimulated the conversion reaction from HNO 2 to HNO 3 , which caused HNO 2 concentration increased in the first 12 min and then decreased until lower than its detection limit. The concentration of HNO 3 still increased with discharge time. After 36 min, HNO 3 was the only and ultimate product in the liquid. The total yield of HNO 2 and HNO 3 could be affected by processing parameters such as electric factors of peak voltage and frequency, mesh size of ground electrode and content of nitrogen in N 2 /O 2 bubbling. Increasing peak voltage or frequency, the total yield of HNO 2 and HNO 3 increased. Gas composition had a heavy impact on the fixation efficiency that obtained its maximum value at an oxygen content of 66.7% with bubbling O 2 /N 2 gas. At the end of the 36 min discharge, the HNO 3 concentration with bubbling air was 2.215 mmol L −1 at an applied voltage of 25 kV, pulse repetition frequency of 140 Hz and ground electrode mesh of 20 × 20. The energy yield was about 1.22 g (HNO 3 )/kWh.

Degradation of 4-chlorophenol by pulsed high voltage discharge is an intricate process involving a series of complex chemical reactions. Hydroxylation of 4-chlorophenol to form hydroquinone, 4-chlororesorcinol and 4-chlorocatechol is the first step, though a very small amount of direct cleavage products of the C1–C2 or C5–C6 bond are observed. The yield of 4-chlorocatechol is about twice as much as that of hydroquinone. Less 4-chloresorcinol is produced. The free chloride ions dropped from the 4-chlorophenol degradation can obtain reactivity again from the discharge, and react with undegraded 4-chlorophenol to form 2,4-dichlorophenol. Some ring-opened products have also been identified and their possible reaction routes are proposed. Several compounds are verified by use of authentic samples. The more stable ring-opened products are low molecular weight (LMW) acids such as formic, acetic, oxalic, malonate, maleic and malic acid. By discharging 4-chlorophenol aqueous solution for 36 min, the amount of carbons obtained from organic acids is more than 50% while that of carbons from aromatic products less than 20% in the carbons of degraded 4-chlorophenol, which is about 94% of initial carbons. After 60 min of discharge, all the 4-chlorophenol and its aromatic intermediates have been removed completely and the organic carbons are mainly presented as organic acid such as acetic and oxalate acid. At the end of the 120 min discharge, the amount of the remaining organic carbons is not more than 14% of the initial carbons.

Introduction To date, no approved medication is available for the treatment of female sexual arousal disorder (FSAD). Aim The purpose of this study was to evaluate the clinical efficacy and safety of a novel alprostadil topical cream for the treatment of FSAD. Methods This was a multicenter, randomized, double blind, placebo‐controlled, parallel design dose‐ranging study. Four hundred female patients with FSAD (22–62 years of age), after a 4‐week nontreatment baseline period, were provided with 10 blinded doses of 500, 700, or 900 mcg alprostadil or a placebo cream to be applied to the clitoris and the G‐spot in the vagina prior to vaginal intercourse. Main Outcome Measures The primary efficacy end point was the arousal success rate (equal number of the Yes responses to Question 3 of the Female Sexual Encounter Profile [FSEP] or number of the sexual encounters). Secondary endpoints included the Female Sexual Function Index (FSFI), Global Assessment Questionnaire, other FSEP question responses, and post‐treatment changes in Female Sexual Distress Scale. Results A total of 374 FSAD patients completed the study. Primary efficacy analysis of the intent‐to‐treat (ITT) population showed a significant increase in arousal success rates with dose. Arousal success rates at the end of the total evaluation period were 33.1%, 46.3% ( P = 0.0161), 43.5% ( P = 0.0400), and 53.9% ( P = 0.0002) in the placebo , 500, 700, and 900 mcg alprostadil groups, respectively. The changes of the FSFI score, relative to baseline were 14.7%, 20.7% ( P = 0.067), 21.7% ( P = 0.035), and 22.9% ( P = 0.002) for the placebo , 500, 700, and 900 mcg treatment groups, respectively. The other secondary efficacy end point values showed a consistent trend in support of the primary efficacy results. Conclusion These results demonstrated that the application of topical alprostadil prior to vaginal intercourse significantly improved the sexual arousal rate of the subjects with FSAD. Liao Q, Zhang M, Geng Li, Wang X, Song X, Xia P, Lu T, Lu M, and Liu V. Efficacy and safety of alprostadil cream for the treatment of female sexual arousal disorder: A double‐blind, placebo‐controlled study in Chinese population. J Sex Med 2008;5:1923–1931.

Polyethylene glycol (PEG) coupled r-hirudin mutein is determined by biological methods-the coagulation system. In the present study, a competitive enzyme-linked immunosorbent assay (ELISA) is described that permits the measurement of PEG r-hirudin. The ELISA system adopts a rabbit IgG antibody to quantitatively detect PEG-hirudin in human plasma. A PEG-hirudin calibration curve ranged from 50 to 7000 ng/mL. The limit of detection was 87 ng/mL. The intraassay coefficients of variation (CV) ranged between 16 and 21%, and interassay CV between 8 and 22% for low and high PEG-hirudin concentrations, respectively. The recovery of the compound in plasma was between 96 and 111.5%. The interindividual differences between 100 and 5000 ng/mL PEG-hirudin were between 12 and 22%. The correlation of the concentration of PEG-hirudin determined with the ELISA and the ecarin clotting time was r = 0.902. No interactions between unfractionated heparin, low molecular-weight heparin, or phenprocoumon and PEG-hirudin were observed in the ELISA. Deficiencies of thrombin or antithrombin as well as low, normal, and high fibrinogen levels did not interfere with the assay. It is concluded that the ELISA determines the concentration of PEG-hirudin and is not influenced by other major anticoagulants or by plasma levels of some coagulation proteins.

[14C]-Serotonin release assay (14C-SRA) from platelets is considered to be the most sensitive test for laboratory confirmation of heparin-induced thrombocytopenia (HIT). This study compared 14C-SRA with an enzyme immunoassay (EIA) to determine the release of serotonin from platelets in the presence of heparin and serum from HIT patients. The normal range (median, 2.5 and 97.5 percentiles) of serotonin release from platelets in healthy subjects (n = 149) is 38 ng/ml (19 and 62) measured by EIA-SRA. Serum from HIT patients (n = 42) released 2548 ng/ml (244 and 7987) serotonin in the presence of 0.1 IU/ml heparin and 29 ng/ml (13 and 76) in the presence of 100 IU/ml heparin. One hundred per cent and 15% of HIT samples exhibited an elevated serotonin release from platelets in the presence of 0.1 IU/ml low molecular weight (LMW) heparin, 2100 ng/ml (869 and 5968), or danaparoid, 272 ng/ml (143 and 403), respectively. The sensitivity and specificity of the EIA-SRA was 100% and 97.4% and of the 14C-SRA 100% and 92.9% in HIT patients. No false-positive results were found in patients receiving heparin (n = 28), in patients with elevated levels of bilirubin (n = 5), in patients with antiphospholipid antibody syndrome (n = 10) or in non-HIT patients (n = 78) with both assays. The EIA technique to quantify serotonin release from platelets provides a reliable non-radioactive method to diagnose heparin-induced thrombocytopenia and to assess in vitro crossreactivity of low molecular weight heparins and heparinoid.

Background —Hirudin is a small protein with strong thrombin inhibition that may be antigenic. The generation and disappearance of anti-hirudin antibodies were investigated in patients with heparin-induced thrombocytopenia who were treated with recombinant hirudin (r-hirudin) for ≥5 days. Methods and Results —The IgA, IgE, IgG, and IgM isotypes of anti-hirudin antibodies were determined by ELISA before and after the start of r-hirudin therapy. A total of 56% of patients (13 of 23) developed ≥1 antibody isotype during therapy. No IgE antibodies were generated. IgA, IgG, and IgM antibodies were detected in 30% (7 of 23), 52% (12 of 23), and 17% (4 of 23) of patients, respectively. Four patients generated only IgG, 2 patients developed either IgM or IgG and IgM, 5 patients IgG and IgA, and 2 patients IgG, IgM, and IgA antibodies. IgM antibodies disappeared within 8 days of the cessation of r-hirudin. IgA and IgG antibodies disappeared within 1 year in all but 1 patient. Binding of purified IgG to r-hirudin in IgG antibody–positive patients (n=7) was demonstrated by competitive ELISA for r-hirudin. Of the 7 IgG antibody samples, 1 each neutralized or enhanced the anticoagulant activity of r-hirudin. Conclusions —R-hirudin may be antigenic in patients with heparin-induced thrombocytopenia. More comprehensive investigations will be required to determine the biological relevance of this and to establish the antibody-generation pattern in other diseases.

Background Heparin-induced thrombocytopenia is a relatively uncommon but severe sideeffect of heparin therapy. Heparin-induced IgG antibody has been elucidated to be the main isotype and the most pathogenic antibody in the pathophysiology. As affected patients are at high risk of developing thrombotic events, confirmation of the clinical diagnosis and avoidance of heparin re-exposure are important and desirable. Materials and methods In the present study, heparin-induced IgG was measured by the binding of neoantigens, which were prepared by incubating FITC-heparin with platelet factor 4 present in normal serum. The cross-reactivities of heparin-induced IgG with lowmolecular-weight heparin and danaparoid were analysed by competitive binding. Results A total of 81 clinically suspected heparin-induced thrombocytopenia type II patients were analysed. Thirty-seven of 38 heparin-induced thrombocytopenia type II patients, in whom thromboembolism was confirmed by objective methods, had elevated relative fluorescence intensity ratios (patient normal control) and 36 had positive heparininduced platelet activation results. The prevalence of heparin-induced IgG in heparininduced thrombocytopenia type II patients was 97·4%. Positive heparin-induced IgG results were: 0/319 healthy volunteers, 0/38 other thrombo-cytopenia and 2/56 heparin/lowmolecular-weight heparin-receiving patients without thrombocytopenia, 2/41 hyperbilirubinaemic patients and 2/50 hyperlipidaemic patients. A small amount of cross-reaction assays showed similar results as obtained in heparin-induced platelet activation. Conclusion Our results suggest that a very high frequency of heparin-induced IgG in heparin-induced thrombocytopenia type II patients can be detected using a novel antigen assay. The rapid determination of pathogenic heparin-induced IgG may be a useful tool for the rapid diagnosis of heparin-induced thrombocytopenia type II that could facilitate further management of the patients.

ZusammenfassungDie Heparin-induzierte Thrombozytopenie ist eine seltenere, jedoch schwere Nebenwirkung der Heparintherapie. Heparin-induzierter IgG-Antikörper wurde als Haupt-Isotyp und hauptsächlicher pathogener Antikörper bei der Pathophysiolgie abgeklärt. Da die Patienten ein hohes Risiko für die Entwicklung thrombotischer Ereignisse aufweisen, ist es wichtig und wünschenswert, die klinische Diagnose zu bestätigen und eine Heparin-Reexposition zu vermeiden. Zur Bestimmung von Heparin-induziertem IgG bei Patienten mit Heparin-induzierter Thrombozytopenie (HIT) Typ II wurde ein »Fluorescence-Iinked immunofiltration assay« (FLIFA) entwickelt. Anhand dieses Neoantigen-Assays läßt sich eine hohe Frequenz von Heparin-induziertem IgG bei HIT Typ II nachweisen. Die sofortige Bestimmung von pathogenem, Heparin-induziertem IgG kann ein nützliches Verfahren zur raschen Diagnose einer HIT Typ II sein, das die weitere Behandlung der Patienten erleichtern könnte.

A fluorescence-linked immunofiltration assay (FLIFA) was developed for the determination of heparin-induced IgG in heparin-induced thrombocytopenia (HIT) type II patients. Protein A was immobilized on a nitrocellulose membrane to bind heparin-induced IgG of HIT type II patients. Fluorescein-5-isothiocynate (FITC)-heparin was added to platelet factor 4 present in normal serum to form the neo-antigen which was captured by heparin-induced IgG. The heparin-induced IgG was quantified by the relative fluorescence intensity (RFI) of bound FITC-heparin. Values were expressed as a RFI ratio (RFI patient / RFI normal) and were 1.965+/-0.413 in HIT type II patients (n = 36) and 1.064+/-0.162 in healthy controls (n = 50, p

ZusammenfassungRekombinantes Hirudin ist ein potentes, spezifisches Antithrombin-Agens, das in Hefezellen exprimiert wird. Die verschiedenen rekombinanten Hirudine befinden sich nun in der klinischen Prüfung bezüglich einer Anzahl von Indikationen. Die Ergebnisse vorliegender Studien versprechen eine hohe Effektivität und Sicherheit in der Vorbeugung von tiefen Beinvenenthrombosen und Lungenembolien bei Patienten mit elektiver Hüftchirurgie, bei Patienten mit akutem Myokardinfarkt, akutem koronaren Syndrom und der instabilen Angina mit nachfolgender Angioplastie. In diesen Studien waren unter der Therapie mit rekombinantem Hirudin allergische Reaktionen nicht aufgetreten. Rekombinantes Hirudin wurde ebenfalls hinsichtlich seiner Effektivität und Sicherheit bei Patienten mit heparin-induzierter Thrombozytopenie Typ II untersucht. Hier zeigte sich eine Inzidenz von Heparin-Antikörpern in bis zu 50% aller mit Hirudin langzeitbehandelten Patienten. Eine Immunantwort gegen rekombinantes Hirudin konnte ebenfalls in Tierversuchen zur Herstellung von monoklonalen Hirudinantikörpern produziert werden. Die vorliegenden Daten zeigen, daß die Hirudinantikörper teils hirudin-neutralisierende, teils halbwertszeitverlängernde Eigenschaften aufweisen. Die biologische Relevanz der Hirudin-Antikörper bleibt weiteren Studien vorbehalten.

Patients with a history of heparin-induced thrombocytopenia (HIT) and antibodies to hirudin were re-exposed to recombinant (r)-hirudin for prophylaxis of thromboembolism. Four patients were re-exposed to 2 × 25 mg of subcutaneous r-hirudin for 8–27 d. Two patients were re-exposed once, one patient twice and one four times. Re-exposure was well tolerated in all patients and no thromboembolism occurred. Antihirudin IgG (4/4 patients), IgA and IgM (1/4 patients) antibody levels increased. Baseline ecarin clotting times showed high variability. Patients with antibodies to hirudin may be re-exposed but anticoagulant monitoring is mandatory.