Accurate and highly sensitive detection of Alzheimer's disease-related extracellular vesicles via förster resonance energy transfer.

Alzheimer's disease (AD) is the most common neurodegenerative disease in the world and poses a huge challenge to global healthcare. Early and accurate detection of amyloid-ß (1-42) (Aß42), a key biomarker of AD, is crucial for effective diagnosis and intervention of AD. Specific or overexpressed proteins on extracellular vesicles (EVs) describe a close correlation with the occurrence and development of diseases. EVs are a very promising non-invasive biomarker for the diagnosis of AD and other diseases. As a sensitive, simple and rapid analytical method, fluorescence resonance energy transfer (FRET) has been widely applied in the detection of EVs. Herein, we developed a dual labelling strategy for simultaneously detecting EV membrane proteins of Aß42 and CD63 based on FRET pair consisting of Au nanoclusters (AuNCs) and polydopamine nanospheres (PDANSs). The constructed nanoprobe, termed EVMPFAP assay, could specifically measure the Aß42 and CD63 on EVs with excellent sensitivity, high specificity and satisfactory accuracy. The limit of detection of EVMPFAP assay was 1.4 × 103 particles mL-1 and the linear range was from 104 to 108 particles mL-1. EVMPFAP assay was successfully used to analyze plasma EVs to distinguish AD and healthy mice. We expect that EVMPFAP assay can be routinely applied for early diagnosis and development-monitoring of AD, thus facilitating the fight against AD.

Intestinal Carnitine Status and Fatty Acid Oxidation in Response to Clofibrate and Medium-Chain Triglyceride Supplementation in Newborn Pigs.

To investigate the role of peroxisome proliferator-activated receptor alpha (PPARα) in carnitine status and intestinal fatty acid oxidation in neonates, a total of 72 suckled newborn piglets were assigned into 8 dietary treatments following a 2 (±0.35% clofibrate) × 4 (diets with: succinate+glycerol (Succ), tri-valerate (TC5), tri-hexanoate (TC6), or tri-2-methylpentanoate (TMPA)) factorial design. All pigs received experimental milk diets with isocaloric energy for 5 days. Carnitine statuses were evaluated, and fatty acid oxidation was measured in vitro using [1-14C]-palmitic acid (1 mM) as a substrate in absence or presence of L659699 (1.6 µM), iodoacetamide (50 µM), and carnitine (1 mM). Clofibrate increased concentrations of free (41%) and/or acyl-carnitine (44% and 15%) in liver and plasma but had no effects in the intestine. The effects on carnitine status were associated with the expression of genes involved in carnitine biosynthesis, absorption, and transportation. TC5 and TMPA stimulated the increased fatty acid oxidation rate induced by clofibrate, while TC6 had no effect on the increased fatty acid oxidation induced by clofibrate (p > 0.05). These results suggest that dietary clofibrate improved carnitine status and increased fatty acid oxidation. Propionyl-CoA, generated from TC5 and TMPA, could stimulate the increased fatty acid oxidation rate induced by clofibrate as anaplerotic carbon sources.

Effects of medium chain triglycerides on hepatic fatty acid oxidation in clofibrate-fed newborn piglets.

To investigate whether increasing tricarboxylic acid (TCA) cycle activity and ketogenic capacity would augment fatty acid (FA) oxidation induced by the peroxisome proliferator-activated receptor-alpha (PPARα) agonist clofibrate, suckling newborn piglets (n = 54) were assigned to 8 groups following a 2 ( ± clofibrate) × 4 (glycerol succinate [SUC], triglycerides of 2-methylpentanoic acid [T2M], valeric acid [TC5] and hexanoic acid [TC6]) factorial design. Each group was fed an isocaloric milk formula containing either 0% or 0.35% clofibrate (wt/wt, dry matter basis) with 5% SUC, T2M, TC5 or TC6 for 5 d. Another 6 pigs served as newborn controls. Fatty acid oxidation was examined in fresh homogenates of liver collected on d 6 using [1-14C] palmitic acid (1 mM) as a substrate (0.265 µCi/µmol). Measurements were performed in the absence or presence of L-carnitine (1 mM) or inhibitors of 3-hydroxy-3-methylglutaryl-CoA synthase (L659699, 1.6 µM) or acetoacetate-CoA deacylase (iodoacetamide, 50 µM). Without clofibrate stimulation, 14C accumulation in CO2 was higher from piglets fed diets containing T2M and TC5 than SUC, but similar to those fed TC6. Under clofibrate stimulation, accumulation also was higher in homogenates from piglets fed TC5 than all other dietary treatments. Interactions between clofibrate and carnitine or the inhibitors were observed (P = 0.0004) for acid soluble products (ASP). In vitro addition of carnitine increased 14C-ASP (P < 0.0001) above all other treatments, regardless of clofibrate treatment. The percentage of 14C in CO2 was higher (P = 0.0023) in TC5 than in the control group. From these results we suggest that dietary supplementation of anaplerotic and ketogenic FA could impact FA oxidation and modify the metabolism of acetyl-CoA (product of ß-oxidation) via alteration of TCA cycle activity, but the modification has no significant impact on the hepatic FA oxidative capacity induced by PPARα. In addition, the availability of carnitine is a critical element to maintain FA oxidation during the neonatal period.

Crystal Structure and Noncovalent Interactions of Heterocyclic Energetic Molecules.

Nitrogen-rich heterocyclic compounds are important heterocyclic substances with extensive future applications for energetic materials due to their outstanding density and excellent physicochemical properties. However, the weak intermolecular interactions of these compounds are not clear, which severely limits their widespread application. Three nitrogen-rich heterocyclic compounds were chosen to detect their molecular geometry, stacking mode and intermolecular interactions by crystal structure, Hirshfeld surface, RDG and ESP. The results show that all atoms in each molecule are coplanar and that the stacking mode of the three crystals is a planar layer style. A large amount of inter- and intramolecular interaction exists in the three crystals. All principal types of intermolecular contacts in the three crystals are N···H interactions and they account for 40.9%, 38.9% and 32.9%, respectively. Hydrogen bonding, vdW interactions and steric effects in Crystal c are stronger than in Crystals a and b. The negative ESPs all concentrate on the nitrogen atoms in the three molecules. This work is expected to benefit the crystal engineering of heterocyclic energetic materials.

Amlexanox-modified platinum(IV) complex triggers apoptotic and autophagic bimodal death of cancer cells.

Platinum(IV) prodrugs c,c,t-[PtCl2(NH3)2(OH)(amlexanox)] (MAP) and c,c,t-[PtCl2(NH3)2(amlexanox)2] (DAP) were synthesized by reacting amlexanox with oxoplatin and characterized by NMR, HR-MS, HPLC, and elemental analysis. The complexes could be reduced to platinum(II) species and amlexanox to exert antitumor activity. Generally, MAP was more potent than DAP and cisplatin towards various human cancer cell lines; particularly, it was active in cisplatin-resistant Caov-3 ovarian cancer and A549/DDP lung cancer cells. MAP induced serious damage to DNA, remarkable change in mitochondrial morphology, decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria, and up-regulation of pro-apoptotic protein Bax in Caov-3 cells, thereby leading to evident apoptosis. Meanwhile, MAP markedly promoted the autophagic flux, including affecting the expression of microtubule-associated protein light chain 3 (LC3) and autophagy adaptor protein p62 in Caov-3 cells, with an increase in the ratio of LC3-II/LC3-I and a decrease in p62, thus trigging the occurrence of autophagy. The MAP-induced bimodal cell death mode is uncommon for platinum complexes, which presents a new possibility to invent anticancer drugs with unique mechanism of action.

Thiosemicarbazone N-Heterocyclic Cu(II) complexes inducing nuclei DNA and mitochondria damage in hepatocellular carcinoma cells.

The α-N-Heterocyclic thiosemicarbazones and their metal complexes have been widely investigated as anticancer and antibacterial agents for their broad spectrum of pharmacological properties. Thus, two thiosemicarbazone-based Cu(II) complexes, [Cu2(ptpc)I2] (1) and [Cu(qtpc)I] (2) with thiosemicarbazone ligand (ptpc = 2-(di(pyridin-2-yl)methylene)-N-(2-(trifluoromethyl)phenyl)-hydrazine-1-carbothioamide, qtpc = 2-(quinolin-8-ylmethylene)-N-(2-(trifluoromethyl)phenyl)hydrazine-1-carbothioamide) were synthesized and evaluated for their biological activities. Complexes 1 and 2 are superior to cisplatin in vitro antiproliferative activities toward hepatocellular carcinoma cell line with the half maximal inhibitory concentration value of 0.2 and 2 µM, respectively. A series of spectroscopic assays and the DNA cleavage experiments showed that both complexes can change and distort the conformation of DNA. Molecular docking experiment further demonstrated that complex 1 binds to DNA mainly in groove mode. Meanwhile, benefiting from their good liposolubility, complexes 1 and 2 could easily enter cells, which further triggers cell cycle arrest and apoptosis. Moreover, complexes 1 and 2 caused serious mitochondrial damage, associating with increased the level of reactive oxygen species (ROS) and Ca2+, decreased adenosine triphosphate (ATP) content and mitochondrial membrane potential (Δψm), and transformed mitochondrial morphology. These findings indicated that complexes 1 and 2 might exert their anticancer activity by inducing DNA and mitochondrial damage simultaneously.

Theoretical investigation into the cooperativity effect of 1,4-dimethoxy-D-glucosamine complex with Na+ and H2O.

In order to explore the essence of the hydration process of chitin or chitosan in the presence of cation, the cooperativity effects between the H-bonding and Na+···molecule interactions in the 1,4-dimethoxy-D-glucosamine (DMGA) complexes with H2O and Na+ were investigated at the B3LYP/6-311++G(d,p), M06-2X/6-311++G(2df,2p), and ωB97X-D/6-311++G(2df,2p) levels. The result shows that the complexes in which Na+ or H2O is bonded simultaneously to the -NH and -OH groups connected to the C3 atom of DMGA are the most stable. The cooperativity and anti-cooperativity effects occur in DMGA···H2O···DMGA and DMGA···Na+···H2O, while only the cooperativities are confirmed in DMGA···Na+···DMGA. The cooperativity occurs in the DMGA···Na+···H2O complexes without the hydration, while the anti-cooperativity occurs in those with the hydration. Furthermore, the cooperativity and anti-cooperativity in DMGA···Na+···H2O are far stronger than those in DMGA···Na+···DMGA or DMGA···H2O···DMGA. Therefore, a deduction is given that the cooperativity and anti-cooperativity effects play an important role in the hydration of chitin or chitosan in the presence of Na+. When only Na+ is linked with -OH and -NH groups of chitosan or chitin, due to the cooperativity effect, the hydration does not occur. When both Na+ and H2O are linked with -OH and -NH groups, the anti-cooperativities are dominant in controlling of the aggregation process of Na+, H2O, chitosan, and chitin, leading to the possible hydration. Atoms in molecules (AIM) analysis confirms the cooperativity and anti-cooperativity effects. Graphical abstract.

Influence of the introduction of a triphenylphosphine group on the anticancer activity of a copper complex.

Aiming at obtaining new copper complexes with good cytotoxicity against cancer cells, triphenylphosphine (TPP) was introduced to obtain insight into the influence of the co-ligands. In this paper, two copper complexes, Cu(2-pbmq)(CH3OH)Br2 (1) and [Cu(2-pbmq)(TPP)Br]2 (2) were designed, synthesized, and characterized by X-ray crystallography, 2-((2-(pyrazin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl))quinolone (2-pbmq), to investigate the influence of the TPP group on the anticancer activity of the metal complex. Although the presence of the TPP group diminished the intensity of the interaction properties of the complex with DNA, the in vitro anticancer activity and cellular uptake of the TPP-containing complex were markedly superior to those of its TPP-lacking counterpart. Detailed studies on the more potently cytotoxic complex 2 revealed that it accumulated in nucleus, arrested the cell cycle at the G0-G1 phase, causing mitochondrial dysfunction, involving the potential simultaneous mitochondrial membrane collapse, cellular ATP level depletion, and Ca2+ leakage, eventually inducing cell apoptosis. In summary, the introduction of a TPP group enhances the biological activity and cytotoxicity of the complex.

Effects of Dietary Anaplerotic and Ketogenic Energy Sources on Renal Fatty Acid Oxidation Induced by Clofibrate in Suckling Neonatal Pigs.

Maintaining an active fatty acid metabolism is important for renal growth, development, and health. We evaluated the effects of anaplerotic and ketogenic energy sources on fatty acid oxidation during stimulation with clofibrate, a pharmacologic peroxisome proliferator-activated receptor α (PPARα) agonist. Suckling newborn pigs (n = 72) were assigned into 8 dietary treatments following a 2 × 4 factorial design: ± clofibrate (0.35%) and diets containing 5% of either (1) glycerol-succinate (GlySuc), (2) tri-valerate (TriC5), (3) tri-hexanoate (TriC6), or (4) tri-2-methylpentanoate (Tri2MPA). Pigs were housed individually and fed the iso-caloric milk replacer diets for 5 d. Renal fatty acid oxidation was measured in vitro in fresh tissue homogenates using [1-14C]-labeled palmitic acid. The oxidation was 30% greater in pig received clofibrate and 25% greater (p < 0.05) in pigs fed the TriC6 diet compared to those fed diets with GlySuc, TriC5, and Tri2MPA. Addition of carnitine also stimulated the oxidation by twofold (p < 0.05). The effects of TriC6 and carnitine on palmitic acid oxidation were not altered by clofibrate stimulation. However, renal fatty acid composition was altered by clofibrate and Tri2MPA. In conclusion, modification of anaplerosis or ketogenesis via dietary substrates had no influence on in vitro renal palmitic acid oxidation induced by PPARα activation.

Chemical biology suggests pleiotropic effects for a novel hexanuclear copper(ii) complex inducing apoptosis in hepatocellular carcinoma cells.

A novel hexanuclear copper(ii)-based complex, [Cu6(tpbb)2(NO3)12] (1), was synthesized, which shows potent cytotoxicity to hepatoma carcinoma cells by inducing apoptosis and apoptosis-related processes. Furthermore, mechanistic investigations based on proteomes revealed that the induced apoptosis was mediated by acting on several targets and multiple pathways in a pleiotropic way.

Synthesis and anticancer evaluation of benzo-N-heterocycles transition metal complexes against esophageal cancer cell lines.

Three novel transition metal complexes, Cu(p-2-bmq)Cl2 (1), Zn(p-2-bmq)Cl2 (2) and [Co(p-2-bmq)Cl2]2 (3) (where p-2-bmq = 2-((1-(pyridin-2-yl)-1H-benzoimidazol-2-yl)methyl) quinolone, have been synthesized. The complexes were detected for their cytotoxicity in vitro against four human esophageal cancer cell lines (SMMC7721, BGC823, HCT116 and HT29) by MTT assay. The results showed that they all have anti-tumor cell proliferation activity. E specially, complex 1 exhibited significant cytotoxicity with IC50 value of 15.89 µM against SMMC7721 cells for 72 h. The morphological changes of nuclei by fluorescence staining methods proved that complex 1 could induce intracellular DNA damage. The flow cytometry analysis revealed that the treatment of SMMC7721 cells with complex 1 induced intracellular ROS increased, mitochondrial potential collapse, G2/M-phase arrest, and even apoptosis. These studies should highly valuable for the development of transition metal-based compounds to the potential anticancer medicinal applications.

Novel binuclear and trinuclear metal (II) complexes: DNA interactions and in vitro anticancer activity through apoptosis.

A water soluble trinuclear copper(II) complex and a binuclear cobalt(II) complex, namely Cu3(ppbm)2(SO4)3 (1) and Co2(ppbm)2(NO3)4 (2) (ppbm = 2-(pyridin-2-yl)-1-(pyridin-3-ylmethyl)- 1H-benzo[d]imidazole), have been successfully synthesized and characterized by elemental analysis, IR Spectroscopy, electrospray ionization mass spectra (ESI-MS). The interaction of the new complexes with DNA has been explored using spectroscopy methods, indicating that the complexes 1 and 2 bind to DNA via noncovalent interactions. DNA cleavage experiment suggested that the complex 1 exhibits efficient DNA cleavage activities in the presence of ascorbate (Asc), hydrogen peroxide may serve as the major cleavage active species. The cytotoxicity assay showed that complex 1 exhibited significant inhibitory activity toward the proliferation of several tumor cell lines, with a lower IC50 value than cisplatin and complex 2, indicating that it had the potential to act as effective anticancer agent. The morphological staining assays showed that 1 apparently induced the TFK-1 cells apoptosis. Besides, cellular uptake experiment on TFK-1 cells revealed that complex 1 accumulates primarily inside the nucleus. The apoptosis was attributable to the metal-assisted generation of reactive oxygen species (ROS).

DNA interactions and in vitro anticancer evaluations of pyridine-benzimidazole-based Cu complexes.

Copper is an essential element and has redox potential, thus copper complexes have been developed rapidly with the hope of curing cancer. To further develop anticancer agents and investigate their anticancer mechanisms, two Cu complexes, [Cu(bpbb)0.5·Cl·SCN]·(CH3OH) (1) and [Cu2(bpbb)·Br3·(OH)] n (2), were synthesized and characterized using 4,4'-bis((2-(pyridin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)biphenyl (bpbb), with associated Cu(ii) salts. Complex 1 is a binuclear structure, whereas 2 is a one-dimensional complex. Compared with 2, complex 1 exhibited potent in vitro cytotoxicity toward four cell lines (HCT116, BGC823, HT29, and SMMC7721), and was most effective against HCT116 cells. Therefore, further in-depth investigation was carried out using complex 1. Absorption spectral titration experiments, ethidium bromide displacement assays, and circular dichroism spectroscopic studies suggested that complex 1 binds strongly to DNA by intercalation. Complex 1 exhibited a clear concentration-dependent pBR322 DNA cleavage activity. Inductively coupled plasma mass spectrometry testing implied that complex 1 could enter cells and that DNA was one important target. Cellular level assays suggested that complex 1 activates the generation of intracellular reactive oxygen species, causing DNA damage, promoting cell cycle arrest and mitochondria dysfunction, and inducing cellular apoptosis.

Cytotoxicity, dual-targeting apoptosis induction evaluation of multinuclear cu complexes based on pyrazine-benzimidazole derivative.

To investigate the cytotoxicity and mechanism of action of multinuclear Cu complexes against tumor cell lines, two complexes, Cu6(bpbib)4Br8 (1) and Cu2(bpbib)2(BF4)2Cl2 (2) (bpbib = 1,4-bis((2-(pyrazin-2-yl)-1H-benzo[d]imidazol-1-yl)methyl)benzene) were synthesized and characterized. Both Cu complexes showed high selectivity toward cancer and not normal cells, and the SMMC7721 cell line showed most sensitivity toward both complexes. Complex 1 exhibited more potent cytotoxicity and enhanced cellular uptake, and therefore, was comprehensively investigated. Complex 1 exhibited dual effects in the inhibition of tumor cell proliferation of SMMC7721 cells, causing nuclear DNA damage and mitochondrial dysfunction involving simultaneous reactive oxygen species (ROS) generation and Ca2+ increase. DNA binding studies suggest that intercalation might be the most probable binding mode. Fluorescence spectrometry also detected a medium affinity of complex 1 to bovine serum albumin (BSA) at distinct temperatures and resulted in BSA fluorescence static quenching.

In vitro antitumor activity of novel benzimidazole-based Cu(II) complexes.

Four benzimidazole-based Cu(II) complexes: Cu2(p-2-bmp)2Br4 (1), Cu2(p-2-bmp)2Cl4 (2), Cu2(p-2-bmb)2(DMF)2Br4·(CHCl3) (3), and Cu(p-2-bmb)(NO3)2·(CHCl3) (4) were isolated and characterized, where p-2-bmp is 1-((2-(pyridine-2-yl)-1H-benzoimidazol-1-yl)methyl)-1H-pyridine and p-2-bmb is 1-((2-(pyridin-2-yl)-1-benzoimidazol-1-yl)methyl)-1H-benzotriazole. Complexes 1 and 2 have binuclear configurations, 3 has a mononuclear structure, and 4has a one-dimensional (1-D) chain skeleton. To evaluate their potential anticancer effects on human carcinoma cells, anti-proliferation, DNA binding and cleavage, and apoptosis elicitation were examined. Compared with complexes 2, 3, and 4, complex 1 exhibited potent in vitro cytotoxicity toward four cell lines (MCF7, EC109, SH-SY5Y and QBC939), with SH-SY5Y cells demonstrating the most sensitivity. Therefore, further in-depth investigations were performed using complex 1. Absorption titration experiments, circular dichroism spectroscopic studies, and ethidium bromide displacement assays suggested that complex 1 binds to DNA through intercalation, significantly cleaves supercoiled pBR322 DNA, and inhibits DNA transcription. Cell cycle analysis revealed that SH-SY5Y cells were arrested in the G2/M phase after treatment with complex 1. Membrane permeability analysis and nuclear staining of SH-SY5Y cells showed that complex 1 could induce apoptosis.

miR­140­5p inhibits human glioma cell growth and invasion by targeting JAG1.

miR­140­5p has been reported to be a tumor suppressor in several types of human cancer, however, little is known about its expression and function in human gliomas. The present study aimed to detect the expression of miR­140­5p in human glioma tissues and cell lines, and to investigate the effect of miR­140­5p on glioma cell growth, invasion and adhesion using in vitro gain­of­function and loss­of­function experiments. Furthermore, the hypothesis that Jagged1 (JAG1) may be a target gene of miR­140­5p was tested. Reverse transcription­quantitative polymerase chain reaction analysis revealed that miR­140­5p was significantly downregulated in human glioma tissues and cell lines compared with normal tissues, and that its expression was correlated with the grade of gliomas. Transfection of a miR­140­5p mimic into SW1783 glioma cells promoted cell growth, invasion and adhesion, as determined by MTT, Transwell and cell adhesion assays respectively. By contrast, transfection of a miR­140­5p inhibitor had the opposite effect. A dual­luciferase reporter assay confirmed that JAG1 was a target gene of miR­140­5p, and miR­140­5p inhibited JAG1 expression both at the mRNA and protein level. In addition, JAG1 overexpression reversed the effect of miR­140­5p on glioma cell growth, invasion and adhesion. In conclusion, the present study is the first to reveal that miR­140­5p acts as a tumor suppressor in human gliomas. JAG1 was demonstrated to be a novel target of miR­140­5p, and miR­140­5p exerted its inhibitory effect on human glioma growth and invasion, partly by suppressing JAG1. The present study may provide useful information toward novel targets for the treatment of gliomas.

Genetic characterization and serological prevalence of swine hepatitis E virus in Shandong province, China.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is classified into four major genotypes (1 to 4) and swine is the main natural reservoir for genotypes 3 and 4. In this study, a total of 106 bile samples from a slaughterhouse in the Shandong province of China were tested for the partial ORF2 gene of HEV by RT-nPCR to determine the virus genotypes, and two indirect ELISA were developed for the detection of swine HEV specific IgM and IgG antibodies in 980 serum samples from 24 farms, in order to investigate the seroprevalence. Thirty-two out of 106 (30.2%) bile samples were positive for HEV and a high degree of partial ORF2 sequence similarity (86.8-100%) was observed among 20 samples. The viral sequences belonged to genotype 4, subtypes 4a and 4d. One complete genome sequence of a subtype 4d HEV was further determined and characterized. The seroprevalence of HEV IgG and IgM antibodies was 100% (24/24) and 41.7% (10/24) for herds, and 66.4% (651/980) and 1.6% (16/980) for the individual pigs, respectively. These results suggested a high prevalence of genotype 4 of swine HEV infection both in swine farms and at the slaughterhouse in Shandong province, which further raise public-health concerns for zoonosis and pork safety.

Development of a blocking ELISA for detection of antibodies against avian hepatitis E virus.

A blocking enzyme-linked immunosorbent assay (bELISA) was developed for the detection of immunoglobulin G antibodies against avian hepatitis E virus (HEV). In the bELISA, the coating antigen was a truncated protein containing C-terminal 268-amino acid region of ORF2 from an avian HEV strain isolated in China (CaHEV) and blocking antibody was a monoclonal antibody (mAb) 1H5 recognizing the epitope within amino acids 384-414 in the C-terminal 268-amino acid region. The concentration of blocking mAb 1H5 was determined as that yielded an OD450nm value of 1.0 for binding to the coating antigen and the antigen concentration and serum dilution were optimized using a checkerboard titration. A cut-off value of 20.7% at the mean percent inhibition plus 3 standard deviations was determined by testing 265 negative sera. The bELISA had a sensitivity of 98.3% by testing 116 positive sera from chickens infected experimentally with CaHEV and had no cross-reaction with other anti-avian virus antibodies. The compliance rates of the bELISA with indirect ELISA and Western blot were 83.7% and 93.3%, respectively, by testing 300 field chicken sera. These results suggested that the bELISA developed in this study can be used for detection of antibodies against avian HEV and showed high reproducibility compared with indirect ELISA and Western blot methods.

Development and application of an indirect ELISA for detection of antibodies against avian hepatitis E virus.

An indirect enzyme-linked immunosorbent assay (iELISA) that could detect immunoglobulin G antibodies against avian hepatitis E virus (HEV) was developed. This assay employs a truncated C-terminal 268-amino acid recombinant ORF2 protein from an avian HEV genotype 3 strain isolated in China (CaHEV) as the coating antigen. The antigen concentration and serum dilution were optimized using a checkerboard titration. A cut-off value of 0.368 at OD(450nm) was determined by testing 120 positive and 200 negative chicken sera for avian HEV antibodies using the two-graph receiver operating characteristic (TG-ROC) analysis. This iELISA has a sensitivity of 96.1% and a specificity of 95.8%. The overall agreement between the iELISA and a corresponding Western blot was 97%. The iELISA was used to evaluate the seroprevalence of avian HEV in poultry farms in the Shandong province. The avian HEV seropositive rate of 35.9% was determined by testing 1871 serum samples that were collected from 10 chicken flocks ranged from 10 to 60 weeks of age. The iELISA that was developed in this study can be used for detection of immunoglobulin G antibodies against avian HEV.

Bis{µ-1,3-bis-[(2-methyl-1H-benzimid-azol-1-yl)meth-yl]benzene-κN:N}bis-(diiodidocadmium).

In the title compound, [Cd(2)I(4)(C(24)H(22)N(4))(2)], the 1,3-bis-[(2-methyl-1H-benzimidazol-1-yl)meth-yl]benzene ligand bridges two CdI(2) units, forming a centrosymmetric dinuclear complex. The Cd(II) atom adopts a distorted tetra-hedral coordination geometry. In the crystal, complex mol-ecules are linked into columns parallel to [101] by π-π stacking inter-actions, with centroid-centroid distances of 3.558 (2) Å.