Persistence of Japanese Encephalitis Virus Is Associated with Abnormal Expression of the Nonstructural Protein NS1 in Host Cells

AbstractPersistent infection with Japanese encephalitis virus (JEV) was established in murine neuroblastoma N18 cells, and the persistency has been maintained in cell culture for over 6 months. From the persistently infected cells, a clone named C2-2 was selected and expanded to form a stable cell line. The vast majority of C2-2 cells showed viral protein staining by immunofluorescence and continuously produced low levels of virus (103to 104PFU/ml) without marked cytopathic effects or cyclic variations. In addition to the wild-type viral proteins, truncated forms of the viral nonstructural protein 1 (NS1) as well as its derivative NS1′ were produced in C2-2 cells. Both truncated NS1 and NS1′ contain deletions at their N-termini; however, the analyses by RT–PCR and direct sequencing of the viral RNA failed to detect any truncations or mutations within the NS1 region, suggesting that NS1 truncation was a result of a unique posttranslational proteolytic cleavage of NS1 in the persistently infected cells. Similar but not identical truncation of NS1 was also observed in two other persistently infected cell lines established in Vero and DBT (murine astrocytoma) cells. However, viruses released from C2-2 cells did not produce truncated NS1 upon infection of N18 cells, suggesting that NS1 truncations were the result of virus–cell interaction in persistently infected cells. These data indicate a strong association between abnormal NS1 expression and JEV persistency. A probable involvement of dysfunctional NS1 in the establishment and/or maintenance of JEV persistency in tissue culture is discussed

Phenotypes and Genotypes of Patients with Pantothenate Kinase-Associated Neurodegeneration in Asian and Caucasian Populations: 2 Cases and Literature Review

Objectives. Pantothenate kinase-associated neurodegeneration (PKAN) is a rare disease caused by pantothenate kinase 2 (PANK2, OMIM 606157) mutations. This study is aimed to investigate clinical presentations, pathologies, and genetics in patients with PKAN. Methods. Two patients with PKAN were reported. We reviewed the literature to include additional 19 patients with PKAN in Eastern Asia. These patients were divided into classic and atypical groups by the age of onset. We compared the data on PKAN patients of Asian and Caucasian populations. Results. We found iron deposits in the globus pallidus in our Patient 1 and a heterozygous truncating mutation (c.1408insT) in Patient 2. Literature review shows that generalized dystonia and bulbar signs are more common in classic PKAN patients, whereas segmental dystonia and tremors are more specific to atypical ones. Asian patients have less complex presentations-lower prevalence of pyramidal signs, mental impairment, and parkinsonism-than Caucasians. D378G in exon 3 is the most frequent mutation (28%) in Asians. Conclusions. Our study demonstrates that the distribution of dystonia is the major distinction between subgroups of PKAN. Caucasian patients have more complex presentations than Asians. Exon 3 and 4 are hot spots for screening PANK2 mutations in Asian patients

Trypsin-induced proteome alteration during cell subculture in mammalian cells

<p>Abstract</p> <p>Background</p> <p>It is essential to subculture the cells once cultured cells reach confluence. For this, trypsin is frequently applied to dissociate adhesive cells from the substratum. However, due to the proteolytic activity of trypsin, cell surface proteins are often cleaved, which leads to dysregulation of the cell functions.</p> <p>Methods</p> <p>In this study, a triplicate 2D-DIGE strategy has been performed to monitor trypsin-induced proteome alterations. The differentially expressed spots were identified by MALDI-TOF MS and validated by immunoblotting.</p> <p>Results</p> <p>36 proteins are found to be differentially expressed in cells treated with trypsin, and proteins that are known to regulate cell metabolism, growth regulation, mitochondrial electron transportation and cell adhesion are down-regulated and proteins that regulate cell apoptosis are up-regulated after trypsin treatment. Further study shows that bcl-2 is down-regulated, p53 and p21 are both up-regulated after trypsinization.</p> <p>Conclusions</p> <p>In summary, this is the first report that uses the proteomic approach to thoroughly study trypsin-induced cell physiological changes and provides researchers in carrying out their experimental design.</p

Highly reliable GIGA-sized synthetic human therapeutic antibody library construction

BackgroundMonoclonal antibodies (mAbs) and their derivatives are the fastest expanding category of pharmaceuticals. Efficient screening and generation of appropriate therapeutic human antibodies are important and urgent issues in the field of medicine. The successful in vitro biopanning method for antibody screening largely depends on the highly diverse, reliable and humanized CDR library. To rapidly obtain potent human antibodies, we designed and constructed a highly diverse synthetic human single-chain variable fragment (scFv) antibody library greater than a giga in size by phage display. Herein, the novel TIM-3-neutralizing antibodies with immunomodulatory functions derived from this library serve as an example to demonstrate the library’s potential for biomedical applications.MethodsThe library was designed with high stability scaffolds and six complementarity determining regions (CDRs) tailored to mimic human composition. The engineered antibody sequences were optimized for codon usage and subjected to synthesis. The six CDRs with variable length CDR-H3s were individually subjected to β-lactamase selection and then recombined for library construction. Five therapeutic target antigens were used for human antibody generation via phage library biopanning. TIM-3 antibody activity was verified by immunoactivity assays.ResultsWe have designed and constructed a highly diverse synthetic human scFv library named DSyn-1 (DCB Synthetic-1) containing 2.5 × 1010 phage clones. Three selected TIM-3-recognizing antibodies DCBT3-4, DCBT3-19, and DCBT3-22 showed significant inhibition activity by TIM-3 reporter assays at nanomolar ranges and binding affinities in sub-nanomolar ranges. Furthermore, clone DCBT3-22 was exceptionally superior with good physicochemical property and a purity of more than 98% without aggregation.ConclusionThe promising results illustrate not only the potential of the DSyn-1 library for biomedical research applications, but also the therapeutic potential of the three novel fully human TIM-3-neutralizing antibodies

The LRRK2 Arg1628Pro variant is a risk factor for Parkinson's disease in the Chinese population

The c.G4883C variant in the leucine-rich repeat kinase 2 (LRRK2) gene (protein effect: Arg1628Pro) has been recently proposed as a second risk factor for sporadic Parkinson's disease in the Han Chinese population (after the Gly2385Arg variant). In this paper, we analyze the Arg1628Pro variant and the associated haplotype in a large sample of 1,337 Han subjects (834 patients and 543 controls) ascertained from a single referral center in Taiwan. In our sample, the Arg1628Pro allele was more frequent among patients (3.8%) than among controls (1.8%; p = 0.004, OR 2.13, 95% CI 1.29-3.52). Sixty heterozygous and two homozygous carriers of the Arg1628Pro variant were identified among the patients, of which only one was also a carrier of the LRRK2 Gly2385Arg variant. We also show that carriers of the Arg1628Pro variant share a common, extended haplotype, suggesting a founder effect. Parkinson's disease onset age was similar in patients who carried the Arg1628Pro variant and in those who did not carry it. Our data support the contention that the Arg1628Pro variant is a second risk factor for Parkinson's disease in the Han Chinese population. Adding the estimated effects of Arg1628Pro (population attributable risk [PAR] ∼4%) and Gly2385Arg variants (PAR ∼6%) yields a total PAR of ∼10%

Identifying Farm Pond Habitat Suitability for the Common Moorhen (Gallinula chloropus): A Conservation-Perspective Approach

The purpose of this study was to establish a habitat-suitability assessment model for Gallinula chloropus, or the Common Moorhen, to be applied to the selection of the most suitable farm pond for habitat conservation in Chiayi County, Taiwan. First, the fuzzy Delphi method was employed to evaluate habitat selection factors and calculate the weights of these factors. The results showed that the eight crucial factors, by importance, in descending order, were (1) area ratio of farmlands within 200 m of the farm pond; (2) pond area; (3) pond perimeter; (4) aquatic plant coverage of the pond surface; (5) drought period; (6) coverage of high and low shrubs around the pond bank; (7) bank type; and (8) water-surface-to-bank distance. Subsequently, field evaluations of 75 farm ponds in Chiayi County were performed. The results indicated that 15 farm ponds had highly-suitable habitats and were inhabited by unusually high numbers of Common Moorhens; these habitats were most in need of conservation. A total of two farm ponds were found to require habitat-environment improvements, and Common Moorhens with typical reproductive capacity could be appropriately introduced into 22 farm ponds to restore the ecosystem of the species. Additionally, the habitat suitability and number of Common Moorhens in 36 farm ponds were lower than average; these ponds could be used for agricultural irrigation, detention basins, or for recreational use by community residents. Finally, the total habitat suitability scores and occurrence of Common Moorhens in each farm pond were used to verify the accuracy of the habitat-suitability assessment model for the Common Moorhen. The overall accuracy was 0.8, and the Kappa value was 0.60, which indicates that the model established in this study exhibited high credibility. To sum up, this is an applicable framework not only to assess the habitat suitability of farm ponds for Common Moorhens, but also to determine whether a particular location may require the implementation of conservation practices. Furthermore, the findings in this research can provide useful information to all relevant stakeholders involved in the implementation of wildlife-habitat conservation and restoration at farm ponds

Detecting Bakanae disease in rice seedlings by machine vision

Bakanae disease, or "foolish seedling", is a seed-borne disease of rice (Oryza sativa L.). Infected plants can yield empty panicles or perish, resulting in a loss of grain yield. The disease occurs most frequently when contaminated seeds are used. Once the seeds are contaminated, the pathogen Fusarium fujikuroi spreads in the field. Therefore, infected plants must be screened at early developmental stages. This work proposes an approach to nondestructively distinguish infected and healthy seedlings at the age of 3 weeks using machine vision. Seeds of the rice cultivars Tainan 11 and Toyonishiki were inoculated with a conidial suspension of F. fujikuroi. The seedling were cultivated in an incubator for 3 weeks. The images of infected and control seedlings were acquired using flatbed scanners to quantify their morphological and color traits. Support vector machine (SVM) classifiers were developed for distinguishing the infected and healthy seedlings. A genetic algorithm was used for selecting essential traits and optimal model parameters for the SVM classifiers. The proposed approach distinguished infected and healthy seedlings with an accuracy of 87.9% and a positive predictive value of 91.8%. (C) 2016 Elsevier B.V. All rights reserved

Effects of swimming training on myocardial protection in rats

Swimming is important for promoting and maintaining health, as it can increase the efficiency of the cardiovascular system and decrease the occurrence of cardiovascular diseases. The objective of the present study was to examine whether swimming training could decrease myocardial injury in rats caused by myocardial ischemia/reperfusion (I/R). Sprague-Dawley rats were randomized into four groups, namely the Sham, coronary artery occlusion, swimming training and ischemic preconditioning (IPC) groups. Myocardial I/R was induced in anesthetized male Sprague-Dawley rats by a 40-min occlusion followed by a 3-h reperfusion of the left anterior descending coronary artery. The rats were sacrificed after surgery and their hearts were examined. The results demonstrated that the number of TUNEL-positive nuclei and degree of caspase-3 activation were both significantly increased in the myocardium following myocardial I/R in rats, indicating increased cardiomyocyte apoptosis. On the other hand, swimming training decreased the serum levels of creatine phosphokinase, lactate dehydrogenase and cardiac troponin I, and was associated with reduced histological damage and myocardial infarct size. Furthermore, swimming training also reduced TNF-α levels, caspase-3 activation and enhanced Bcl-2 activation, which decreased the number of apoptotic cells in the myocardium. The findings of the present study showed that swimming training and IPC could similarly decrease myocardial injury following myocardial I/R, and may therefore be used as exercise training to effectively prevent myocardial injury.補正完畢GR

Genome-wide association mapping of gene loci affecting disease resistance in the rice-Fusarium fujikuroi pathosystem

Background Rice bakanae disease has emerged as a new threat to rice production. In recent years, an increase in the occurrence and severity of bakanae disease has been reported in several areas in Asia. Although bakanae disease affects rice yield and quality, little is known about the genetics of bakanae resistance in rice. The lack of large-scale screens for bakanae resistance in rice germplasm has also limited the development and deployment of resistant varieties. Results A genome-wide association study (GWAS) was conducted to identify genes/loci conferring bakanae resistance in rice. A total of 231 diverse accessions from Rice Diversity Panel 1 (RDP1) were inoculated with a highly virulent Taiwanese Fusarium fujikuroi isolate and assessed for resistance using two parameters: (1) disease severity index based on visual rating and (2) colonization rate determined by reisolation of F. fujikuroi from the basal stems of infected rice seedlings. We identified 14 quantitative trait loci (QTLs) (10 for disease severity and 4 for colonization rate), including 1 mapped for both parameters. A total of 206 candidate genes were identified within the 14 QTLs, including genes encoding leucine-rich repeat (LRR)-containing and NB-ARC (nucleotide-binding adaptor shared by APAF-1, R proteins, and CED-4) proteins, hormone-related genes, transcription factor genes, ubiquitination-related genes, and oxidase/oxidoreductase genes. In addition, a candidate QTL (qBK1.7) that co-localized with the previously identified QTLs qBK1 and qFfR1, was verified by linkage analysis using a population of 132 recombinant inbred lines derived from IR64 x Nipponbare. GWAS delineated qBK1.7 to a region of 8239 bp containing three genes. Full-length sequencing across qBK1.7 in 20 rice accessions revealed that the coding regions of two LRR-containing genes Os01g0601625 and Os01g0601675 may be associated with bakanae resistance. Conclusions This study facilitates the exploitation of bakanae resistance resources in RDP1. The information on the resistance performance of 231 rice accessions and 14 candidate QTLs will aid efforts to breed resistance to bakanae and uncover resistance mechanisms. Quantification of the level of F. fujikuroi colonization in addition to the conventional rating of visual symptoms offers new insights into the genetics of bakanae resistance