Identification of Three Antiviral Inhibitors against Japanese Encephalitis Virus from Library of Pharmacologically Active Compounds 1280

Japanese encephalitis virus (JEV) can cause severe central nervous disease with a high mortality rate. There is no antiviral drug available for JEV-specific treatment. In this study, a cytopathic-effect-based, high-throughput screening assay was developed and applied to screen JEV inhibitors from Library of Pharmacologically Active Compounds 1280. The antiviral effects of three hit compounds including FGIN-1-27, cilnidipine, and niclosamide were evaluated in cells by western blotting, indirect immunofluorescence assay, and plaque reduction assay. A time-of-addition assay proved that all three compounds inhibited JEV at the stage of replication. The EC50s of FGIN-1-27, cilnidipine, and niclosamide were 3.21, 6.52, and 5.80 µM, respectively, while the selectivity indexes were 38.79, 30.67, and 7.49. FGIN-1-27 and cilnidipine have high efficiency and selectivity against JEV. This study provided two JEV antiviral inhibitors as candidates for treatment of JEV infection.     

A virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses

How viruses evolve to select their receptor proteins for host cell entry is puzzling. We recently determined the crystal structures of NL63 coronavirus (NL63-CoV) and SARS coronavirus (SARS-CoV) receptor-binding domains (RBDs), each complexed with their common receptor, human angiotensin-converting enzyme 2 (hACE2), and proposed the existence of a virus-binding hot spot on hACE2. Here we investigated the function of this hypothetical hot spot using structure-guided biochemical and functional assays. The hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. Mutations that disturb the hot spot structure have significant effects on virus/receptor interactions, revealing critical energy contributions from the hot spot structure. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-CoV/hACE2 interface, and hence RBD/hACE2 binding affinities are decreased either by NL63-CoV mutations decreasing the tunnel space or by SARS-CoV mutations increasing the tunnel space. Furthermore, NL63-CoV RBD inhibits hACE2-dependent transduction by SARS-CoV spike protein, a successful application of the hot spot theory that has the potential to become a new antiviral strategy against SARS-CoV infections. These results suggest that the structural features of the hot spot on hACE2 were among the driving forces for the convergent evolution of NL63-CoV and SARS-CoV.     

A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses

Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains.     

A cluster randomised controlled trial of an occupational therapy intervention for residents with stroke living in UK care homes (OTCH): study protocol

ABSTRACT: BACKGROUND: The occupational therapy (OT) in care homes study (OTCH) aims to investigate the effect of a targeted course of individual OT (with task training, provision of adaptive equipment, minor environmental adaptations and staff education) for stroke survivors living in care homes, compared to usual care. METHODS/DESIGN: A cluster randomised controlled trial of United Kingdom (UK) care homes (n?=?90) with residents (n?=?900) who have suffered a stroke or transient ischaemic attack (TIA), and who are not receiving end-of-life care. Homes will be stratified by centre and by type of care provided and randomised (50:50) using computer generated blocked randomisation within strata to receive either the OT intervention (3 months intervention from an occupational therapist) or control (usual care). Staff training on facilitating independence and mobility and the use of adaptive equipment, will be delivered to every home, with control homes receiving this after the 12 month follow-up.Allocation will be concealed from the independent assessors, but the treating therapists, and residents will not be masked to the intervention. Measurements are taken at baseline prior to randomisation and at 3, 6 and 12 months post randomisation. The primary outcome measure is independence in self-care activities of daily living (Barthel Activities of Daily Living Index). Secondary outcome measures are mobility (Rivermead Mobility Index), mood (Geriatric Depression Scale), preference based quality of life measured from EQ-5D and costs associated with each intervention group. Quality adjusted life years (QALYs) will be derived based on the EQ-5D scores. Cost effectiveness analysis will be estimated and measured by incremental cost effectiveness ratio. Adverse events will be recorded. DISCUSSION: This study will be the largest cluster randomised controlled trial of OT in care homes to date and will clarify the currently inconclusive literature on the efficacy of OT for stroke and TIA survivors residing in care homes. TRIAL REGISTRATION: ISRCTN00757750.     

Calf thymus DNA binding studies of the new neodymium-naproxen complex

Fluorescence spectroscopy in combination with UV absorption spectroscopy was carried out to investigate the interaction between the neodymium-naproxen complex (Nd-NAP) and calf thymus DNA (ctDNA). The experimental results showed that Nd-NAP intercalated with the ctDNA base pairs. Analysis of fluorescence quenching data of Nd-NAP by ctDNA at different temperatures using a Stern-Volmer equation revealed that dynamic and static quenching occurred simultaneously. The binding constants and the number of binding sites at 293 and 310 K were obtained as 2.904 × 10(4) L mol(-1), 1.172 and 2.432 × 10(4) L mol(-1), 1.143, respectively. The thermodynamic parameters ΔG, ΔH, and ΔS calculated at different temperatures indicated that hydrogen bonding and van der Waals force were the main binding forces.     

Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, a real-time quantitative PCR (qPCR) protocol targeting the Borrelia burgdorferi-specific recA gene was adapted for use with a Lightcycler for rapid detection and quantification of the Lyme disease spirochete, B. burgdorferi, in field-collected Ixodes scapularis ticks. The sensitivity of qPCR for detection of B. burgdorferi DNA in infected ticks was comparable to that of a well-established nested PCR targeting the 16S-23S rRNA spacer. Of the 498 I. scapularis ticks collected from four northeastern states (Rhode Island, Connecticut, New York, and New Jersey), 91 of 438 (20.7%) nymphal ticks and 15 of 60 (25.0%) adult ticks were positive by qPCR assay. The number of spirochetes in individual ticks varied from 25 to 197,200 with a mean of 1,964 spirochetes per nymphal tick and a mean of 5,351 spirochetes per adult tick. No significant differences were found in the mean numbers of spirochetes counted either in nymphal ticks collected at different locations in these four states (P = 0.23 by one-way analysis of variance test) or in ticks infected with the three distinct ribosomal spacer restriction fragment length polymorphism types of B. burgdorferi (P = 0.39). A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k = 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.     

Synthesis and biological evaluation of novel 4-azaindolyl-indolyl-maleimides as glycogen synthase kinase-3β (GSK-3β) inhibitors

A series of novel 4-azaindolyl-indolyl-maleimides were synthesized and evaluated for their GSK-3β inhibitory activity. Most compounds exhibited high potency to GSK-3β. Among them, compound 7c was the most promising GSK-3β inhibitor. Preliminary structure–activity relationships were discussed based on the experimental data obtained and showed that different substituents on the indole ring and side chains at 1-position of indole had varying degrees of influence on the GSK-3β inhibitory potency. In a cell-based functional assay, compounds 7c and 15a significantly reduced Aβ-induced Tau hyperphosphorylation by inhibiting GSK-3β.     

物种多样性及生物量与地下水位的关系——以海流兔河流域为例

以3条样带上117块植被群落调查样方为基础资料,研究了海流兔河流域植被物种多样性及生物量与地下水位之间的关系。结果表明:1)地下水位高低及地貌类型均会影响草本层植物群落组成及优势种构成。滩地样地中,随地下水位降低,优势草本的更替方向为寸草,芨芨草,马蔺,狗尾草,碱茅;沙坡样地中,优势草本的更替方向为大针茅,沙鞭,沙蓬,沙打旺。2)地下水位为1.5 m时是草本植物群落生长发育最适宜区域,物种多样性及丰富度达到最大,而灌木层物种多样性及丰富度随地下水位下降呈现波动变化的特征;当地下水位埋深小于5.0 m时,草本层物种多样性及丰富度明显高于灌木层,在地下水位埋深大于5.0 m时,草本层物种多样性指数开始出现低于灌木层的现象。3)草本植物多样性及丰富度和生物量之间关联性不强,滩地样地中,草本层地上生物量及地下生物量在地下水位为1.8 m时具有最大值,但植物群落结构较为单一;沙坡样地中,地上生物量最大值出现在地下水位为5.0 m的区域内,而地下生物量最大值出现在地下水位为3.5 m时。综上,物种多样性、地上及地下生物量与地下水位都不是简单的线性关系,而是有一个最适水位;高于或低于这个最适水位,多样性和生物量都会下降。