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1.  Bipolar loop-like non-volatile strain in the (001)-oriented Pb(Mg1/3Nb2/3)O3-PbTiO3 single crystals 
Scientific Reports  2014;4:4591.
Strain has been widely used to manipulate the properties of various kinds of materials, such as ferroelectrics, semiconductors, superconductors, magnetic materials, and “strain engineering” has become a very active field. For strain-based information storage, the non-volatile strain is very useful and highly desired. However, in most cases, the strain induced by converse piezoelectric effect is volatile. In this work, we report a non-volatile strain in the (001)-oriented Pb(Mg1/3Nb2/3)O3-PbTiO3 single crystals and demonstrate an approach to measure the non-volatile strain. A bipolar loop-like S-E curve is revealed and a mechanism involving 109° ferroelastic domain switching is proposed. The non-volatile high and low strain states should be significant for applications in information storage.
doi:10.1038/srep04591
PMCID: PMC3975321  PMID: 24699506
2.  A Small Peptide with Potential Ability to Promote Wound Healing 
PLoS ONE  2014;9(3):e92082.
Wound-healing represents a major health burden, such as diabetes-induced skin ulcers and burning. Many works are being tried to find ideal clinical wound-healing biomaterials. Especially, small molecules with low cost and function to promote production of endogenous wound healing agents (i.e. transforming growth factor beta, TGF-β) are excellent candidates. In this study, a small peptide (tiger17, c[WCKPKPKPRCH-NH2]) containing only 11 amino acid residues was designed and proved to be a potent wound healer. It showed strong wound healing-promoting activity in a murine model of full thickness dermal wound. Tiger17 exerted significant effects on three stages of wound healing progresses including (1) the induction of macrophages recruitment to wound site at inflammatory reaction stage; (2) the promotion of the migration and proliferation both keratinocytes and fibroblasts, leading to reepithelialization and granulation tissue formation; and (3) tissue remodeling phase, by promoting the release of transforming TGF-β1 and interleukin 6 (IL-6) in murine macrophages and activating mitogen-activated protein kinases (MAPK) signaling pathways. Considering its easy production, store and transfer and function to promote production of endogenous wound healing agents (TGF-β), tiger17 might be an exciting biomaterial or template for the development of novel wound-healing agents.
doi:10.1371/journal.pone.0092082
PMCID: PMC3960170  PMID: 24647450
3.  Single Domain SmCo5@Co Exchange-coupled Magnets Prepared from Core/shell Sm[Co(CN)6]·4H2O@GO Particles: A Novel Chemical Approach 
Scientific Reports  2013;3:3542.
SmCo5 based magnets with smaller size and larger maximum energy product have been long desired in various fields such as renewable energy technology, electronic industry and aerospace science. However, conventional relatively rough synthetic strategies will lead to either diminished magnetic properties or irregular morphology, which hindered their wide applications. In this article, we present a facile chemical approach to prepare 200 nm single domain SmCo5@Co core/shell magnets with coercivity of 20.7 kOe and saturation magnetization of 82 emu/g. We found that the incorporation of GO sheets is responsible for the generation of the unique structure. The single domain SmCo5 core contributes to the large coercivity of the magnets and the exchange-coupled Co shell enhances the magnetization. This method can be further utilized in the synthesis other Sm-Co based exchange-coupled magnets.
doi:10.1038/srep03542
PMCID: PMC3868969  PMID: 24356309
4.  Nek9 regulates spindle organization and cell cycle progression during mouse oocyte meiosis and its location in early embryo mitosis 
Cell Cycle  2012;11(23):4366-4377.
Nek9 (also known as Nercc1), a member of the NIMA (never in mitosis A) family of protein kinases, regulates spindle formation, chromosome alignment and segregation in mitosis. Here, we showed that Nek9 protein was expressed from germinal vesicle (GV) to metaphase II (MII) stages in mouse oocytes with no detectable changes. Confocal microscopy identified that Nek9 was localized to the spindle poles at the metaphase stages and associated with the midbody at anaphase or telophase stage in both meiotic oocytes and the first mitotic embyros. Depletion of Nek9 by specific morpholino injection resulted in severely defective spindles and misaligned chromosomes with significant pro-MI/MI arrest and failure of first polar body (PB1) extrusion. Knockdown of Nek9 also impaired the spindle-pole localization of γ-tubulin and resulted in retention of the spindle assembly checkpoint protein Bub3 at the kinetochores even after 10 h of culture. Live-cell imaging analysis also confirmed that knockdown of Nek9 resulted in oocyte arrest at the pro-MI/MI stage with abnormal spindles, misaligned chromosomes and failed polar body emission. Taken together, our results suggest that Nek9 may act as a MTOC-associated protein regulating microtubule nucleation, spindle organization and, thus, cell cycle progression during mouse oocyte meiotic maturation, fertilization and early embryo cleavage.
doi:10.4161/cc.22690
PMCID: PMC3552919  PMID: 23159858
meiosis; microtubule-organizing center (MTOC) spindle; oocyte; γ-tubulin
5.  Biomonitoring of Non-Dioxin-Like Polychlorinated Biphenyls in Transgenic Arabidopsis Using the Mammalian Pregnane X Receptor System: A Role of Pectin in Pollutant Uptake 
PLoS ONE  2013;8(11):e79428.
Polychlorinated biphenyls (PCBs) are persistent organic pollutants damaging to human health and the environment. Techniques to indicate PCB contamination in planta are of great interest to phytoremediation. Monitoring of dioxin-like PCBs in transgenic plants carrying the mammalian aryl hydrocarbon receptor (AHR) has been reported previously. Herein, we report the biomonitoring of non-dioxin-like PCBs (NDL-PCBs) using the mammalian pregnane X receptor (PXR). In the transgenic Arabidopsis designated NDL-PCB Reporter, the EGFP-GUS reporter gene was driven by a promoter containing 18 repeats of the xenobiotic response elements, while PXR and its binding partner retinoid X receptor (RXR) were coexpressed. Results showed that, in live cells, the expression of reporter gene was insensitive to endogenous lignans, carotenoids and flavonoids, but responded to all tested NDL-PCBs in a dose- and time- dependent manner. Two types of putative PCB metabolites, hydroxy- PCBs and methoxy- PCBs, displayed different activation properties. The vascular tissues seemed unable to transport NDL-PCBs, whereas mutation in QUASIMODO1 encoding a 1,4-galacturonosyltransferase led to reduced PCB accumulation in Arabidopsis, revealing a role for pectin in the control of PCB translocation. Taken together, the reporter system may serve as a useful tool to biomonitor the uptake and metabolism of NDL-PCBs in plants.
doi:10.1371/journal.pone.0079428
PMCID: PMC3827382  PMID: 24236133
6.  Analyses of the Survival Time and the Influencing Factors of Chinese Patients with Prion Diseases Based on the Surveillance Data from 2008–2011 
PLoS ONE  2013;8(5):e62553.
Background
Prion diseases are kinds of progressive, incurable neurodegenerative disorders. So far, survival time of the patients with these diseases in China is unclear.
Methods
Based upon the surveillance data from Chinese Creutzfeldt-Jakob disease (CJD) surveillance network from January 2008 to December 2011, a retrospective follow-up survey was performed. The survival times of Chinese patients with prion diseases and the possible influencing factors were analyzed.
Results
Median survival time of 121 deceased patients was 7.1 months, while those for sporadic CJD (sCJD), familial CJD (fCJD) and fatal familial insomnia (FFI) cases were 6.1, 3.1 and 8.2 months, respectively. 74.0% of sCJD patients, 100% of fCJD cases and 91.7% FFI cases died within one year. The general socio-demographic factors, abnormalities in clinical examinations, clinical manifestations, and social factors did not significantly influence the survival times of Chinese prion patients.
Conclusions
Survival time of Chinese patients with prion diseases was comparable with that of many Western countries, but obviously shorter than that of Japan. Patients with acute onset and rapid progression had significantly short survival times.
doi:10.1371/journal.pone.0062553
PMCID: PMC3645993  PMID: 23671608
7.  Analysis of Transcriptome Complexity via RNA-Seq in Normal and Failing Murine Hearts 
Circulation research  2011;109(12):1332-1341.
Rationale
Accurate and comprehensive de novo transcriptome profiling in heart is a central issue to better understand cardiac physiology and diseases. Although significant progress has been made in genome-wide profiling for quantitative changes in cardiac gene expression, current knowledge offers limited insights to the total complexity in cardiac transcriptome at individual exon level.
Objective
To develop more robust bioinformatic approaches to analyze high-throughput RNA sequencing (RNA-Seq) data, with the focus on the investigation of transcriptome complexity at individual exon and transcript levels.
Methods and Results
In addition to overall gene expression analysis, the methods developed in this study were used to analyze RNA-Seq data with respect to individual transcript isoforms, novel spliced exons, novel alternative terminal exons, novel transcript clusters (i.e., novel genes) and long non-coding RNA genes. We applied these approaches to RNA-Seq data obtained from mouse hearts following pressure-overload induced by trans-aortic constriction. Based on experimental validations, analyses of the features of the identified exons/transcripts, and expression analyses including previously published RNASeq data, we demonstrate that the methods are highly effective in detecting and quantifying individual exons and transcripts. Novel insights inferred from the examined aspects of the cardiac transcriptome open ways to further experimental investigations.
Conclusions
Our work provided a comprehensive set of methods to analyze mouse cardiac transcriptome complexity at individual exon and transcript levels. Applications of the methods may infer important new insights to gene regulation in normal and disease hearts in terms of exon utilization and potential involvement of novel components of cardiac transcriptome.
doi:10.1161/CIRCRESAHA.111.249433
PMCID: PMC3243366  PMID: 22034492
RNA-Seq; transcriptome profiling; hypertrophy; heart failure
8.  Heat shock protein 70 selectively mediates the degradation of cytosolic PrPs and restores the cytosolic PrP-induced cytotoxicity via a molecular interaction 
Virology Journal  2012;9:303.
Background
Although the aggregation of PrPSc is thought to be crucial for the neuropathology of prion diseases, there is evidence in cultured cells and transgenic mice that neuronal death can be triggered by the accumulation of cytosolic PrPs, leading to the hypothesis that the accumulation of PrPs in the cytosol of neurons may be a primary neurotoxic culprit. Hsp70, a molecular chaperone involved in protein folding/refolding and degradation in the cytoplasm, has a protective effect in some models of neurodegenerative diseases, e.g., Alzheimer’s and Parkinson’s diseases, but its role in prion diseases remains unclear.
Results
To study the role of Hsp70 in prion diseases, we used immunoprecipitation to first identify a molecular interaction between Hsp70 and PrPs. Using immunofluorescence, we found that Hsp70 colocalized with cytosolic PrPs in HEK293 cells transiently transfected with plasmids for Cyto-PrP and PG14-PrP but not with wild-type PG5-PrP or endoplasmic reticulum (ER)-retained PrPs (3AV-PrP and ER-PrP). Using western blot analysis and apoptosis assays of cultured cells, we found that the overexpression of Hsp70 by transfection or the activation of Hsp70 by geldanamycin selectively mediated the degradation of cytosolic PrPs and restored cytosolic PrP-induced cytotoxicity. Moreover, we found that Hsp70 levels were up-regulated in cells expressing Cyto-PrP and in hamster brains infected with the scrapie agent 263K.
Conclusion
These data imply that Hsp70 has central role in the metabolism of cytosolic PrPs
doi:10.1186/1743-422X-9-303
PMCID: PMC3544727  PMID: 23216755
Hsp70; Cytosolic PrP; Apoptosis; Prion disease; Geldanamycin
9.  Global impact of RNA splicing on transcriptome remodeling in the heart *  
In the eukaryotic transcriptome, both the numbers of genes and different RNA species produced by each gene contribute to the overall complexity. These RNA species are generated by the utilization of different transcriptional initiation or termination sites, or more commonly, from different messenger RNA (mRNA) splicing events. Among the 30 000+ genes in human genome, it is estimated that more than 95% of them can generate more than one gene product via alternative RNA splicing. The protein products generated from different RNA splicing variants can have different intracellular localization, activity, or tissue-distribution. Therefore, alternative RNA splicing is an important molecular process that contributes to the overall complexity of the genome and the functional specificity and diversity among different cell types. In this review, we will discuss current efforts to unravel the full complexity of the cardiac transcriptome using a deep-sequencing approach, and highlight the potential of this technology to uncover the global impact of RNA splicing on the transcriptome during development and diseases of the heart.
doi:10.1631/jzus.B1201006
PMCID: PMC3411092  PMID: 22843179
Alternative RNA splicing; Transcriptome; Gene regulation; Heart; RNA-seq
10.  The first Chinese case of Creutzfeldt-Jakob disease patient with R208H mutation in PRNP 
Prion  2011;5(3):232-234.
A case of Creutzfeldt-Jakob disease (CJD) with a rare mutation of the prion protein (PrP) gene (PRNP) at codon 208 (R208H), while the codon 129 was a methionine homozygous genotype is reported. The patient initial displayed hand tremor, dizziness and progressive cognitive dysfunction. Subsequently, other symptoms gradually appeared, including cerebellar ataxia and mental disorder. No periodic activity was recorded at electroencephalography (EEG) and 14-3-3 protein in cerebrospinal fluid was negative. Total clinical course was about four months. Retrospective investigation of this family across seven generations did not figure out clear family history. However, genetic analyses revealed six first-degree family members with the R208H allele.
doi:10.4161/pri.5.3.16796
PMCID: PMC3226051  PMID: 21791975
creutzfeldt-Jakob disease; PRNP; R208H
11.  Protein Disulfide Isomerase Regulates Endoplasmic Reticulum Stress and the Apoptotic Process during Prion Infection and PrP Mutant-Induced Cytotoxicity 
PLoS ONE  2012;7(6):e38221.
Background
Protein disulfide isomerase (PDI), is sorted to be enzymatic chaperone for reconstructing misfolded protein in endoplasmic reticulum lumen. Recently, PDI has been identified as a link between misfolded protein and neuron apoptosis. However, the potential for PDI to be involved in the pathogenesis of prion disease remains unknown. In this study, we propose that PDI may function as a pleiotropic regulator in the cytotoxicity induced by mutated prion proteins and in the pathogenesis of prion diseases.
Methodology/Principal Findings
To elucidate potential alterations of PDI in prion diseases, the levels of PDI and relevant apoptotic executors in 263K infected hamsters brain tissues were evaluated with the use of Western blots. Abnormal upregulation of PDI, Grp78 and Grp58 was detected. Dynamic assays of PDI alteration identified that the upregulation of PDI started at the early stage and persistently increased till later stage. Obvious increases of PDI and Grp78 levels were also observed in cultured cells transiently expressing PrP mutants, PrP-KDEL or PrP-PG15, accompanied by significant cytotoxicities. Excessive expression of PDI partially eased ER stress and cell apoptosis caused by accumulation of PrP-KDEL, but had less effect on cytotoxicity induced by PrP-PG15. Knockdown of endogenous PDI significantly amended cytotoxicity of PrP-PG15, but had little influence on that of PrP-KDEL. A series of membrane potential assays found that apoptosis induced by misfolded PrP proteins could be regulated by PDI via mitochondrial dysfunction. Moreover, biotin-switch assays demonstrated active S-nitrosylted modifications of PDI (SNO-PDI) both in the brains of scrapie-infected rodents and in the cells with misfolded PrP proteins.
Conclusion/Significance
Current data in this study highlight that PDI and its relevant executors may function as a pleiotropic regulator in the processes of different misfolded PrP proteins and at different stages during prion infection. SNO-PDI may feed as an accomplice for PDI apoptosis.
doi:10.1371/journal.pone.0038221
PMCID: PMC3369880  PMID: 22685557
12.  Multiple injuries after earthquakes: a retrospective analysis on 1,871 injured patients from the 2008 Wenchuan earthquake 
Critical Care  2012;16(3):R87.
Introduction
Multiple injuries have been highlighted as an important clinical dimension of the injury profile following earthquakes, but studies are scarce. We investigated the pattern and combination of injuries among patients with two injuries following the 2008 Wenchuan earthquake. We also described the general injury profile, causes of injury and socio-demographic characteristics of the injured patients.
Methods
A retrospective hospital-based analysis of 1,871 earthquake injured patients, totaling 3,177 injuries, admitted between 12 and 31 May 2008 to the People's Hospital of Deyang city (PHDC). An electronic, webserver-based database with International Classification of Diseases (ICD)-10-based classification of earthquake-related injury diagnoses (IDs), anatomical sites and additional background variables of the inpatients was used. We analyzed this dataset for injury profile and number of injuries per patient. We then included all patients (856) with two injuries for more in-depth analysis. Possible spatial anatomical associations were determined a priori. Cross-tabulation and more complex frequency matrices for combination analyses were used to investigate the injury profile.
Results
Out of the 1,871 injured patients, 810 (43.3%) presented with a single injury. The rest had multiple injuries; 856 (45.8%) had two, 169 (9.0%) patients had three, 32 (1.7%) presented with four injuries, while only 4 (0.2%) were diagnosed with five injuries. The injury diagnoses of patients presenting with two-injuries showed important anatomical intra-site or neighboring clustering, which explained 49.1% of the combinations. For fractures, the result was even more marked as spatial clustering explained 57.9% of the association pattern. The most frequent combination of IDs was a double-fracture, affecting 20.7% of the two-injury patients (n = 177). Another 108 patients (12.6%) presented with fractures associated with crush injury and organ-soft tissue injury. Of the 3,177 injuries, 1,476 (46.5%) were fractures. Most injuries were located in the head (22.9%) and lower extremities (30.8%).
Conclusions
Multiple injuries are put forward as an important component of the injury profile after this earthquake. A pattern of injury combinations and spatial aggregation of injuries was also found. Clinical diagnosis and treatment should be adapted to care of these patients. More studies are needed to generalize these findings.
doi:10.1186/cc11349
PMCID: PMC3580632  PMID: 22594875
13.  A Chinese Creutzfeldt-Jakob disease patient with E196K mutation in PRNP 
Prion  2011;5(2):117-120.
Genetic Creutzfeldt-Jakob disease (gCJD) is caused by a range of mutations in the prion protein gene (PRNP) and account for approximately 10–15% of overall human prion diseases worldwide. They are different with disease onset, disease duration, clinical signs and diagnostic findings. Here we reported a 71 year-old female with an E196K mutation in one PRNP allele, while the codon 129 was a methionine homozygous genotype. The patient started with non-specific symptoms, but displayed rapidly progressive disturbances of speech, memory, cognitive and physical movement. No periodic activity was recorded at electroencephalography (EEG) during the entire disease course. Retrospective investigation of her family members did not reveal similar neurological disorders. Total clinical course was about seven months.
doi:10.4161/pri.5.2.15846
PMCID: PMC3166511  PMID: 21597335
Creutzfeldt-Jakob disease; PRNP; E196K
14.  Mouse-adapted scrapie strains 139A and ME7 overcome species barrier to induce experimental scrapie in hamsters and changed their pathogenic features 
Virology Journal  2012;9:63.
Background
Transmissible spongiform encephalopathy (TSE) diseases are known to be zoonotic diseases that can infect different kinds of animals. The transmissibility of TSE, like that of other infectious diseases, shows marked species barrier, either being unable to infect heterologous species or difficult to form transmission experimentally. The similarity of the amino acid sequences of PrP among species is believed to be one of the elements in controlling the transmission TSE interspecies. Other factors, such as prion strains and host's microenvironment, may also participate in the process.
Methods
Two mouse-adapted strains 139A and ME7 were cerebrally inoculated to Golden hamsters. Presences of scrapie associate fibril (SAF) and PrPSc in brains of the infected animals were tested by TEM assays and Western blots dynamically during the incubation periods. The pathogenic features of the novel prions in hamsters, including electrophoretic patterns, glycosylating profiles, immunoreactivities, proteinase K-resistances and conformational stabilities were comparatively evaluated. TSE-related neuropathological changes were assayed by histological examinations.
Results
After long incubation times, mouse-adapted agents 139A and ME7 induced experimental scrapie in hamsters, respectively, showing obvious spongiform degeneration and PrPSc deposits in brains, especially in cortex regions. SAF and PrPSc in brains were observed much earlier than the onset of clinical symptoms. The molecular characteristics of the newly-formed PrPSc in hamsters, 139A-ha and ME7-ha, were obviously distinct from the original mouse agents, however, greatly similar as that of a hamster-adapted scrapie strain 263 K. Although the incubation times and main disease signs of the hamsters of 139A-ha and ME7-ha were different, the pathogenic characteristics and neuropathological changes were highly similar.
Conclusions
This finding concludes that mouse-adapted agents 139A and ME7 change their pathogenic characteristics during the transmission to hamsters. The novel prions in hamsters' brains obtain new molecular properties with hamster-specificity.
doi:10.1186/1743-422X-9-63
PMCID: PMC3325885  PMID: 22400710
15.  A point mutation in the DNA-binding domain of HPV-2 E2 protein increases its DNA-binding capacity and reverses its transcriptional regulatory activity on the viral early promoter 
Background
The human papillomavirus (HPV) E2 protein is a multifunctional DNA-binding protein. The transcriptional activity of HPV E2 is mediated by binding to its specific binding sites in the upstream regulatory region of the HPV genomes. Previously we reported a HPV-2 variant from a verrucae vulgaris patient with huge extensive clustered cutaneous, which have five point mutations in its E2 ORF, L118S, S235P, Y287H, S293R and A338V. Under the control of HPV-2 LCR, co-expression of the mutated HPV E2 induced an increased activity on the viral early promoter. In the present study, a series of mammalian expression plasmids encoding E2 proteins with one to five amino acid (aa) substitutions for these mutations were constructed and transfected into HeLa, C33A and SiHa cells.
Results
CAT expression assays indicated that the enhanced promoter activity was due to the co-expressions of the E2 constructs containing A338V mutation within the DNA-binding domain. Western blots analysis demonstrated that the transiently transfected E2 expressing plasmids, regardless of prototype or the A338V mutant, were continuously expressed in the cells. To study the effect of E2 mutations on its DNA-binding activity, a serial of recombinant E2 proteins with various lengths were expressed and purified. Electrophoresis mobility shift assays (EMSA) showed that the binding affinity of E2 protein with A338V mutation to both an artificial probe with two E2 binding sites or HPV-2 and HPV-16 promoter-proximal LCR sequences were significantly stronger than that of the HPV-2 prototype E2. Furthermore, co-expression of the construct containing A338V mutant exhibited increased activities on heterologous HPV-16 early promoter P97 than that of prototype E2.
Conclusions
These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.
doi:10.1186/1471-2199-13-5
PMCID: PMC3307029  PMID: 22333459
HPV-2; E2; DNA-binding; Transcriptional regulation; Promoter
16.  The Epidemiological, Clinical, and Laboratory Features of Sporadic Creutzfeldt-Jakob Disease Patients in China: Surveillance Data from 2006 to 2010 
PLoS ONE  2011;6(8):e24231.
Background
Creutzfeldt-Jakob disease (CJD) is a rare, rapidly progressive fatal central nervous system disorder, which consists of three main catalogues: sporadic, familial, and iatrogenic CJD.
Methodology/Principal Findings
In China, the surveillance for CJD started in 2006, covering 12 provincial Centers for Disease Control and Prevention (CDCs) and 15 hospitals. From 2006 to 2010, 624 suspected patients were referred to China CJD surveillance. The epidemiological, clinical and laboratory features of sporadic CJD (sCJD) were analysed. Both groups of probable and possible sCJD showed highest incidences in the population of 60 to 69 year-olds. The most common presenting symptoms were progressive dementia and mental-related symptoms (neurological symptoms including sleeping turbulence, depression, anxiety and stress). Among the four main clinical manifestations, myoclonus was more frequently observed in the probable sCJD patients. About 2/3 of probable sCJD cases showed positive 14-3-3 in CSF and/or periodic sharp wave complexes (PSWC) in electroencephalography (EEG). The presence of myoclonus was significantly closely related with the appearance of PSWC in EEG. Polymorphisms of codon 129 in PRNP of the notified cases revealed a highly predominant M129M genotype in Han Chinese. Among 23 genetic human prion diseases, ten were D178N/M129M Fatal familial insomnia (FFI) and five were T188K genetic CJD (gCJD), possibly indicating a special distribution of gCJD-related mutations in Han Chinese.
Conclusion
From the period of 2006 to 2010, 261 patients were diagnosed as sCJD and 23 patients were diagnosed as genetic human prion diseases in China. The epidemiological, clinical and laboratory analysis data were consistent with the characteristics of sporadic CJD, which provide insight into the features of CJD in China.
doi:10.1371/journal.pone.0024231
PMCID: PMC3164193  PMID: 21904617
17.  Molecular Interaction of TPPP with PrP Antagonized the CytoPrP-Induced Disruption of Microtubule Structures and Cytotoxicity 
PLoS ONE  2011;6(8):e23079.
Background
Tubulin polymerization promoting protein/p25 (TPPP/p25), known as a microtubule-associated protein (MAP), is a brain-specific unstructured protein with a physiological function of stabilizing cellular microtubular ultrastructures. Whether TPPP involves in the normal functions of PrP or the pathogenesis of prion disease remains unknown. Here, we proposed the data that TPPP formed molecular complex with PrP. We also investigated its influence on the aggregation of PrP and fibrillization of PrP106–126 in vitro, its antagonization against the disruption of microtubule structures and cytotoxicity of cytosolic PrP in cells, and its alternation in the brains of scrapie-infected experimental hamsters.
Methodology/Principal Findings
Using pull-down and immunoprecipitation assays, distinct molecular interaction between TPPP and PrP were identified and the segment of TPPP spanning residues 100–219 and the segment of PrP spanning residues 106–126 were mapped as the regions responsible for protein interaction. Sedimentation experiments found that TPPP increased the aggregation of full-length recombinant PrP (PrP23–231) in vitro. Transmission electron microscopy and Thioflavin T (ThT) assays showed that TPPP enhanced fibril formation of synthetic peptide PrP106–126 in vitro. Expression of TPPP in the cultured cells did not obviously change the microtubule networks observed by a tubulin-specific immunofluorescent assay and cell growth features measured by CCK8 tests, but significantly antagonized the disruption of microtubule structures and rescued the cytotoxicity caused by the accumulation of cytosolic PrP (CytoPrP). Furthermore, Western blots identified that the levels of the endogenous TPPP in the brains of scrapie-infected experimental hamsters were significantly reduced.
Conclusion/Significance
Those data highlight TPPP may work as a protective factor for cells against the damage effects of the accumulation of abnormal forms of PrPs, besides its function as an agent for dynamic stabilization of microtubular ultrastructures.
doi:10.1371/journal.pone.0023079
PMCID: PMC3155546  PMID: 21857997
18.  Familial CJD Associated PrP Mutants within Transmembrane Region Induced Ctm-PrP Retention in ER and Triggered Apoptosis by ER Stress in SH-SY5Y Cells 
PLoS ONE  2011;6(1):e14602.
Background
Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrPC) to the pathogenic one (PrPSc). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease.
Methodology/Principal Findings
To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and capase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed.
Conclusions/Significance
The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis.
doi:10.1371/journal.pone.0014602
PMCID: PMC3029303  PMID: 21298055
19.  The Prepared Tau Exon-Specific Antibodies Revealed Distinct Profiles of Tau in CSF of the Patients with Creutzfeldt-Jakob Disease 
PLoS ONE  2010;5(7):e11886.
Background
The diagnostic value of CSF tau for Creutzfeldt-Jakob disease (CJD) has been widely evaluated, showing a markedly disease-relative manner. However, the profiles of tau isoforms in CSF of CJD patients remain unknown. Here, we prepared the exon-specific antibodies against the peptides encoded by exon-2, exon-3 and exon-10 of human tau protein and evaluated the reactive profiles of tau in CSF samples from the patients with probable CJD.
Methodology/Principal Findings
Sequences encoding exon-2, exon-3 and exon-10 of human tau protein were cloned into a prokaryotic expression vector pGEX-2T. Using recombinant fusion proteins GST-E2, GST-E3 and GST-E10, three tau exon-specific antibodies were elicited. Reliable specificities of the prepared antibodies were obtained after a serial of purification processes, not only in recognizing the tau peptides encoded by exon-2, -3 and -10, but also in distinguishing six recombinant tau isoforms by Western blot and ELISA. Three predominant tau-specific bands were observed in CSF samples with the exon-specific and the commercial tau antibodies, respectively, showing different reactive profiles between the groups of probable CJD and non-CJD. A 65 KD band was detected only in the CSF samples from probable CJD patients, especially with the antibodies against exon-2 (Anti-tE2) and exon-10 (Anit-tE10). The appearances of 65 KD band in CSF correlated well with positive 14-3-3 in CSF and typical abnormality in EEG. Such band was not observed in the CSF samples of six tested genetic CJD patients.
Conclusions/Significance
Three exon-specific polyclonal antibodies were successfully prepared. Based on these antibodies, different CSF tau profiles in Western blots were observed between the groups of probable CJD and non-CJD. A disease-specific tau band emerged in the CSF samples from probable sporadic CJD, which may supply a new biomarker for screening sporadic CJD.
doi:10.1371/journal.pone.0011886
PMCID: PMC2912377  PMID: 20686702
20.  Long persistence of EV71 specific nucleotides in respiratory and feces samples of the patients with Hand-Foot-Mouth Disease after recovery 
BMC Infectious Diseases  2010;10:178.
Background
EV71 is associated with the fatal cases of brain stem encephalitis during large HFMD outbreaks from 1998 to 2008. EV71 may continuously shed from upper respiratory tracts and feces of HFMD patients for relatively long time after recovery. However, the persistence of viruses in the patients' secretions and excretions is not clear.
Methods
Serial throat swabs and feces of 34 definitely diagnosed patients, including 30 mild cases and 4 severe cases, were traced and collected with the interval of 2 to 4 days for up to 32 and 48 days, respectively, and tested by a nested RT-PCR.
Results
The EV-71 specific sequences were identified by a Nested RT-PCR in all specimens of 0-4 days, and 5-8 days. The positive rates of EV71 in throat swabs dropped markedly to 42.86% during 9-12 days, and maintained at 20-30% during 13-24 days, while that in feces reduced to 71.43% during 9-12 days, and maintained roughly 20% till 37-40 days. EV71 nucleotide of 36.36% cases disappeared simultaneously both in throats and feces, 39.39% cases showed longer persistence of EV71 nucleotides in feces, and 21.21% were longer in throats. The longest duration of shedding observed was 24 days for throat swabs and 42 days for fecal specimens.
Conclusions
EV71 shedding from respiratory tract may continue for nearly four weeks after onset, but its excretion through feces can persist more than five weeks.
doi:10.1186/1471-2334-10-178
PMCID: PMC2895604  PMID: 20565813
21.  Cholesterol utilization in mycobacteria is controlled by two TetR-type transcriptional regulators: kstR and kstR2 
Microbiology  2010;156(Pt 5):1362-1371.
Mycobacterium tuberculosis is able to use a variety of carbon sources in vivo and current knowledge suggests that cholesterol is used as a carbon source during infection. The catabolized cholesterol is used both as an energy source (ATP generation) and as a source of precursor molecules for the synthesis of complex methyl-branched fatty acids. In previous studies, we described a TetR-type transcriptional repressor, kstR, that controls the expression of a number of genes involved in cholesterol catabolism. In this study, we describe a second TetR-type repressor, which we call kstR2. We knocked this gene out in Mycobacterium smegmatis and used microarrays and quantitative RT-PCR to examine the effects on gene expression. We identified a palindromic regulatory motif for KstR2, showed that this motif is present in three promoter regions in mycobacteria and rhodococcus, and demonstrated binding of purified KstR2 to the motif. Using a combination of motif location analysis, gene expression analysis and the examination of gene conservation, we suggest that kstR2 controls the expression of a 15 gene regulon. Like kstR, kstR2 and the kstR2 regulon are highly conserved among the actinomycetes and studies in rhodococcus suggest a role for these genes in cholesterol catabolism. The functional significance of the regulon and implications for the control of cholesterol utilization are discussed.
doi:10.1099/mic.0.034538-0
PMCID: PMC3068626  PMID: 20167624
22.  Changes of tau profiles in brains of the hamsters infected with scrapie strains 263 K or 139 A possibly associated with the alteration of phosphate kinases 
Background
Phospho-tau deposition has been described in a rare genetic human prion disease, Gerstmann-Sträussler-Scheinker syndrome, but is not common neuropathological picture for other human and animal transmissible spongiform encephalopathies (TSEs). This study investigated the possible changes of tau and phosphorylated tau (p-tau, at Ser396, Ser404, and Ser202/Thr205) in scrapie experimental animals.
Methods
The profiles of tau and p-tau (p-tau, at Ser396, Ser404, and Ser202/Thr205) in the brain tissues of agents 263K- or 139A-infected hamsters were evaluated by Western blots and real-time PCR. Meanwhile, the transcriptional and expressive levels of GSK3β and CDK5 in the brains were tested.
Results
The contents of total tau and p-tau at Ser202/Thr205 increased, but p-tau at Ser396 and Ser404 decreased at the terminal stages, regardless of scrapie strains. Transcriptional levels of two tau isoforms were also increased. Additionally, it showed higher CDK5, but lower GSK3β transcriptional and expressive levels in the brains of scrapie-infected animals. Analysis of brain samples collected from different times after inoculated with agent 263 K revealed that the changes of tau profiles and phosphate kinases were time-relative events.
Conclusion
These data suggest that changes of profiles of p-tau at Ser396, Ser404 and Ser202/Thr205 are illness-correlative phenomena in TSEs, which may arise of the alteration of phosphate kinases. Alteration of tau, p-tau (Ser396, Ser404, and Ser202/Thr205), GSK3β and CDK5 were either intermediate or consequent events in TSE pathogenesis and proposed the potential linkage of these bioactive proteins with the pathogenesis of prion diseases.
doi:10.1186/1471-2334-10-86
PMCID: PMC2868850  PMID: 20356412
23.  Human Prion disease with a T188K mutation in Chinese: a case report 
Cases Journal  2009;2:7820.
Inherited Prion diseases are characterized by mutations in the PRNP gene predispose to disease by causing the expression of abnormal PrP protein. We report a 58-year-old Chinese female with mutation in codon 188 (T188K) of the PRNP gene, while the codon 129 was a methionine homozygous genotype. The patient displayed 4-year long slowly progressive sleeping disturbance and rapid exacerbation of neurological status after other neurological manifestations appeared. Cerebral spinal fluid 14-3-3 protein was positive.
doi:10.1186/1757-1626-2-7820
PMCID: PMC2740053  PMID: 19830016
24.  Surveillance for Creutzfeldt-Jakob disease in China from 2006 to 2007 
BMC Public Health  2008;8:360.
Background
Human transmissible spongiform encephalopathies (HTSE), or Creutzfeldt-Jakob disease (CJD), is a group of rare and fatal diseases in central nervous system. Since outbreak of bovine spongiform encephalopathy (BSE) and variant CJD, a worldwide CJD surveillance network has been established under the proposition of WHO. In China, a national CJD surveillance system has started since 2002. The data of CJD surveillance from 2006 to 2007 was analyzed.
Methods
Total 12 provinces are included in CJD surveillance system. The surveillance unit in each province consists of one or two sentinel hospitals and the provincial CDC. All suspected CJD cases reported from CJD surveillance were diagnosed and subtyped based on the diagnostic criteria for CJD issued by WHO.
Results
Total 192 suspected CJD cases were reported and 5 genetic CJD, 51 probable and 30 possible sporadic CJD (sCJD) cases were diagnosed. The collected sCJD cases distribute sporadically without geographical clustering and seasonal relativity and the highest incidences in both probable and possible sCJD cases appeared in the group of 60–69 year. The most common three foremost symptoms were progressive dementia, cerebellum and mental-related symptoms. The probable sCJD patients owning both typical EEG alteration and CSF protein 14-3-3 positive have more characteristic clinical syndromes than the ones having only one positive. The polymorphisms of codon 129 of all tested reported cases shows typical patterns of Han Chinese as previous reports, that M129M are predominant whereas M129V are seldom.
Conclusion
Chinese CJD patients possessed similar epidemiological and clinical characteristics as worldwide.
doi:10.1186/1471-2458-8-360
PMCID: PMC2596798  PMID: 18928564
25.  Human prion disease with a G114V mutation and epidemiological studies in a Chinese family: a case series 
Introduction
Transmissible spongiform encephalopathies are a group of neurodegenerative diseases of humans and animals. Genetic Creutzfeldt-Jakob diseases, in which mutations in the PRNP gene predispose to disease by causing the expression of abnormal PrP protein, include familial Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome and fatal familial insomnia.
Case presentation
A 47-year-old Han-Chinese woman was hospitalized with a 2-year history of progressive dementia, tiredness, lethargy and mild difficulty in falling asleep. On neurological examination, there was severe apathy, spontaneous myoclonus of the lower limbs, generalized hyperreflexia and bilateral Babinski signs. A missense mutation (T to G) was identified at the position of nt 341 in one PRNP allele, leading to a change from glycine (Gly) to valine (Val) at codon 114. PK-resistant PrPSc was detected in brain tissues by Western blotting and immunohistochemical assays. Information on pedigree was collected notably by interviews with family members. A further four suspected patients in five consecutive generations of the family have been identified. One of them was hospitalized for progressive memory impairment at the age of 32. On examination, he had impairment of memory, calculation and comprehension, mild ataxia of the limbs, tremor and a left Babinski sign. He is still alive.
Conclusion
This family with G114V inherited prion disease is the first to be described in China and represents the second family worldwide in which this mutation has been identified. Three other suspected cases have been retrospectively identified in this family, and a further case with suggestive clinical manifestations has been shown by gene sequencing to have the causal mutation.
doi:10.1186/1752-1947-2-331
PMCID: PMC2576332  PMID: 18925969

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