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1.  Rare V203I mutation in the PRNP gene of a Chinese patient with Creutzfeldt–Jakob disease 
Prion  2013;7(3):259-262.
Here, we report a Chinese case of Creutzfeldt–Jakob disease (CJD) with a rare mutation in the prion protein gene (PRNP) leading to an exchange of amino acid from valine (Val) to isoleucine (I) at codon 203 (V203I). The 80-y-old male presented with sudden memory loss, rapid loss of vocabulary, inattention and slow responses, accompanied by dizziness, blurred vision and ataxia. Two weeks after admission, he exhibited tremor, myoclonus and bilateral Babinski signs. At the end of the clinical course, he developed severe akinetic mutism. The cerebrospinal fluid (CSF) was positive for 14-3-3 protein. Increased bilateral signal intensity in the frontal and parietal lobes was seen on diffusion-weighted imaging (DWI); periodic activity was recorded on an electroencephalogram (EEG). There was no family history of similar symptoms. The total clinical course was approximately two months.
PMCID: PMC3783113  PMID: 23764840
14-3-3 protein; PRNP; Creutzfeldt–Jakob disease; V203I; mutation
2.  Activation of the macroautophagic system in scrapie-infected experimental animals and human genetic prion diseases 
Autophagy  2012;8(11):1604-1620.
Macroautophagy is an important process for removing misfolded and aggregated protein in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of macroautophagy in prion diseases remain obscure. Here we demonstrated that in the terminal stages of scrapie strain 263K-infected hamsters and human genetic prion diseases, the microtubule-associated protein 1 light chain 3 (LC3) was converted from the cytosolic form to the autophagosome-bound membrane form. Macroautophagy substrate sequestosome 1 (SQSTM1) and polyubiquitinated proteins were downregulated in the brains of sick individuals, indicating enhanced macroautophagic protein degradation. The levels of mechanistic target of rapamycin (MTOR) and phosphorylated MTOR (p-MTOR) were significantly decreased, which implies that this enhancement of the macroautophagic response is likely through the MTOR pathway which is a negative regulator for the initiation of macroautophagy. Dynamic assays of the autophagic system in the brains of scrapie experimental hamsters after inoculation showed that alterations of the autophagic system appeared along with the deposits of PrPSc in the infected brains. Immunofluorescent assays revealed specific staining of autophagosomes in neurons that were not colocalized with deposits of PrPSc in the brains of scrapie infected hamsters, however, autophagosome did colocalize with PrPSc in a prion-infected cell line after treatment with bafilomycin A1. These results suggest that activation of macroautophagy in brains is a disease-correlative phenomenon in prion diseases.
PMCID: PMC3494590  PMID: 22874564
transmissible spongiform encephalopathies; autophagy; neurodegenerative diseases
3.  Dysfunction of microtubule-associated proteins of MAP2/tau family in Prion disease 
Prion  2012;6(4):334-338.
The aggregation of PrPSc is thought to be crucial for the neuropathology of prion diseases. A growing body of evidence demonstrates that the perturbation of the microtubule network contributes to PrPSc-mediated neurodegeneration. Microtubules are a component of the cytoskeleton and play a central role in organelle transport, axonal elongation and cellular architecture in neurons. The polymerization, stabilization, arrangement of microtubules can be modulated by interactions with a series of microtubule-associated proteins (MAPs). Recent studies have proposed the abnormal alterations of two major microtubule-associated proteins, tau and MAP2, in the brain tissues of naturally occurred and experimental human and animal prion diseases. Increased total tau protein and hyperphosphorylation of tau at multiple residues are observed at the terminal stage of prion disease. The abnormal aggregation of tau protein disturbs its binding ability to microtubules and affects the microtubule dynamic. Significantly downregulated MAP2 is detected in the brain tissues of scrapie-infected hamsters and PrP106–126 treated cells, which corresponds well with the remarkably low levels of tubulin. In conclusion, dysfunction of MAP2/tau family leads to disruption of microtubule structure and impairment of axonal transport, and eventually triggers apoptosis in neurons, which becomes an essential pathway for prion to induce the neuropathology.
PMCID: PMC3609059  PMID: 22874672
prion; microtubule-associated proteins; MAP2; tau; dysfunction
4.  Comparative peptidome analyses of the profiles of the peptides ranging from 1–10 KD in CSF samples pooled from probable sporadic CJD and non-CJD patients 
Prion  2012;6(1):46-51.
The shotgun strategy applying tandem mass spectrometry has been widely used to identify the proteins that are differentially distributed among diseases for its high reliability and efficiency. To find out the potential difference of protein profiles in cerebrospinal fluids (CSF) between Creutzfeldt-Jakob disease (CJD) and non-CJD patients, especially in the fraction ranging from 1–10 KD, the CSF samples of 40 probable sporadic CJD (sCJD) patients, 32 non-CJD cases with dementia and 17 non-CJD cases without dementia were separately pooled and enriched by the magnetic beads based weak cation exchange chromatography (MB-WCX). After trypsin digestion, each enriched CSF was separated and identified by RP-HPLC-ESI-QTOF MS/MS. In total, 42, 53 and 47 signals of proteins were identified in the pooled CSF fraction less than 10 KD of probable sCJD, non-CJD with dementia and non-CJD without dementia, respectively. Compared with that of probable sCJD, the similarity of CSF protein profiles of non-CJD with dementia (76.2%) were higher than that of non-CJD without dementia (57.1%). Nine CSF proteins were found to be specially observed in probable sCJD group. Those data may help to select the potential biomarkers for diagnosis of CJD. Additionally, further studies of the small segments of cellular proteins in CSF of CJD patients may also provide scientific clues for understanding the neuropathogenesis of TSEs.
PMCID: PMC3338965  PMID: 22453178
Creutzfeldt-Jakob disease; CSF; MB-HPLC-ESI-QTOF; 1–10 KD; comparative peptidome
5.  CtmPrP and ER stress 
Prion  2011;5(3):123-125.
The pathogenic agent is hypothesized to be PrPSc in prion diseases. However, little accumulation of PrPSc is repeatedly observed in some kinds of natural and experimental prion diseases, including some special genetic human prion diseases. One of the specific topology forms of PrP, CtmPrP, representing a key neurotoxic intermediate in prion disorders, has been testified in cell-free translation systems and transgenic mice models. Recently, some studies have showed that point-mutations within the hydrophobic transmembrane region increase the amount of CtmPrP in cells, such as human homologue A117V which is associated with GSS and G114V associated with gCJD, while the mutations outsides transmembrane region do not. The retention of the CtmPrP in ER subsequently is able to induce ER stress and apoptosis, which is supported by upregulation of ER chaperone synthesis, such as Grp78, Grp58, Grp94, Bip and the transcription factor CHOP/GADD153. In conclusion, some kinds of intermediate forms of PrPSc, including CtmPrP, may work as the ultimate cause of neurodegeneration.
PMCID: PMC3226035  PMID: 21795854
prion; CtmPrP; ER stress; transmembrane region; mutant
6.  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.
PMCID: PMC3226051  PMID: 21791975
creutzfeldt-Jakob disease; PRNP; R208H
7.  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.
PMCID: PMC3166511  PMID: 21597335
Creutzfeldt-Jakob disease; PRNP; E196K
8.  Biotransformation of aesculin by human gut bacteria and identification of its metabolites in rat urine 
AIM: To observe the biotransformation process of a Chinese compound, aesculin, by human gut bacteria, and to identify its metabolites in rat urine.
METHODS: Representative human gut bacteria were collected from 20 healthy volunteers, and then utilized in vitro to biotransform aesculin under anaerobic conditions. At 0, 2, 4, 8, 12, 16, 24, 48 and 72 h post-incubation, 10 mL of culture medium was collected. Metabolites of aesculin were extracted 3 × from rat urine with methanol and analyzed by HPLC. For in vivo metabolite analysis, aesculetin (100 mg/kg) was administered to rats via stomach gavage, rat urine was collected from 6 to 48 h post-administration, and metabolite analysis was performed by LC/ESI-MS and MS/MS in the positive and negative modes.
RESULTS: Human gut bacteria could completely convert aesculin into aesculetin in vitro. The biotransformation process occurred from 8 to 24 h post-incubation, with its highest activity was seen from 8 to 12 h. The in vitro process was much slower than the in vivo process. In contrast to the in vitro model, six aesculetin metabolites were identified in rat urine, including 6-hydroxy-7-gluco-coumarin (M1), 6-hydroxy-7-sulf-coumarin (M2), 6, 7-di-gluco-coumarin (M3), 6-glc-7-gluco-coumarin (M4), 6-O-methyl-7-gluco-coumarin (M5) and 6-O-methyl-7-sulf-coumarin (M6). Of which, M2 and M6 were novel metabolites.
CONCLUSION: Aesculin can be transferred into aesculetin by human gut bacteria and is further modified by the host in vivo. The diverse metabolites of aesculin may explain its pleiotropic pharmaceutical effects.
PMCID: PMC2665149  PMID: 19322928
Aesculin; Biotransformation; Human gut bacteria; Rat urine; Sulfated derivatives; LC/ESI-MS; Aseculetin
9.  A strategy for trade monitoring and substitution of the organs of threatened animals 
Scientific Reports  2013;3:3108.
The use of threatened animals as a source of traditional medicines is accelerating the extinction of such species and imposes great challenges to animal conservation. In this study, we propose a feasible strategy for the conservation of threatened medicinal animals that combines trade monitoring and the search for substitutes. First, DNA barcoding provides a powerful technique for monitoring the trade of animal species, which helps in restricting the excessive use and illegal trade of such species. Second, pharmacological tests have been adopted to evaluate the biological equivalence of threatened and domestic animals; based on such testing, potential substitutes are recommended. Based on a review of threatened animal species and their substitutes, we find that the search for substitutes deserves special attention; however, this work is far from complete. These results may be of great value for the conservation of threatened animals and maintaining the heritage of traditional medicine.
PMCID: PMC3813934  PMID: 24173429
10.  Brain microglia were activated in sporadic CJD but almost unchanged in fatal familial insomnia and G114V genetic CJD 
Virology Journal  2013;10:216.
Microglial activations have been described in different subtypes of human prion diseases such as sporadic Creutzfeldt-Jakob disease (CJD), variant CJD, Kuru and Gerstmann-Sträussler-Scheinker disease (GSS). However, the situation of microglia in other genetic prion diseases such as fatal familial insomnia (FFI) and familial CJD remains less understood. The brain microglia was evaluated comparatively between the FFI, G114V and sCJD cases in the study.
Specific Western blots, immunohistochemical and immunofluorescent assays were used to detect the changes of microglia and ELISA tests were used for levels of inflammatory cytokines.
Western blots, immunohistochemical and immunofluorescent assays illustrated almost unchanged microglia in the temporal lobes of FFI and G114V gCJD, but obviously increased in those of sCJD. The Iba1-levels maintained comparable in six different brain regions of FFI and G114V cases, including thalamus, cingulate gyrus, frontal cortex, parietal cortex, occipital cortex and temporal cortex. ELISA tests for inflammatory cytokines revealed significantly up-regulated IL-1β, IL-6 and TNF-α in the brain homogenates from sCJD, but not in those from FFI and G114V gCJD.
Data here demonstrates silent brain microglia in FFI and G114V gCJD but obviously increased in sCJD, which reflects various pathogenesis of different human prion diseases subtypes.
PMCID: PMC3716817  PMID: 23816234
Prions; Microglia; Creutzfeldt-Jakob disease; Fatal familial insomnia; G114V; Cytokines
11.  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.
Prion diseases are kinds of progressive, incurable neurodegenerative disorders. So far, survival time of the patients with these diseases in China is unclear.
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.
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.
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.
PMCID: PMC3645993  PMID: 23671608
12.  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.
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.
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.
These data imply that Hsp70 has central role in the metabolism of cytosolic PrPs
PMCID: PMC3544727  PMID: 23216755
Hsp70; Cytosolic PrP; Apoptosis; Prion disease; Geldanamycin
13.  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.
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.
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.
PMCID: PMC3369880  PMID: 22685557
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.
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.
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.
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.
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.
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 
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.
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.
These results suggest that the mutation from Ala to Val at aa 338 is critical for E2 DNA-binding and its transcriptional regulation.
PMCID: PMC3307029  PMID: 22333459
HPV-2; E2; DNA-binding; Transcriptional regulation; Promoter
16.  Remarkable Reduction of MAP2 in the Brains of Scrapie-Infected Rodents and Human Prion Disease Possibly Correlated with the Increase of Calpain 
PLoS ONE  2012;7(1):e30163.
Microtubule-associated protein 2 (MAP2) belongs to the family of heat stable MAPs, which takes part in neuronal morphogenesis, maintenance of cellular architecture and internal organization, cell division and cellular processes. To obtain insight into the possible alteration and the role of MAP2 in transmissible spongiform encephalopathies (TSEs), the MAP2 levels in the brain tissues of agent 263K-infected hamsters and human prion diseases were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, MAP2 levels in the brain tissues of scrapie infected hamsters, a patient with genetic Creutzfeldt-Jakob disease (G114V gCJD) and a patient with fatal familial insomnia (FFI) were almost undetectable. The decline of MAP2 was closely related with prolonged incubation time. Exposure of SK-N-SH neuroblastoma cell line to cytotoxic PrP106-126 peptide significantly down-regulated the cellular MAP2 level and remarkably disrupted the microtubule structure, but did not alter the level of tubulin. Moreover, the levels of calpain, which mediated the degradation of a broad of cytoskeletal proteins, were significantly increased in both PrP106-126 treated SK-N-SH cells and brain tissues of 263K prion-infected hamsters. Our data indicate that the decline of MAP2 is a common phenomenon in TSEs, which seems to occur at an early stage of incubation period. Markedly increased calpain level might contribute to the reduction of MAP2.
PMCID: PMC3260227  PMID: 22272295
17.  Fever with Thrombocytopenia Associated with a Novel Bunyavirus in China 
The New England journal of medicine  2011;364(16):1523-1532.
Heightened surveillance of acute febrile illness in China since 2009 has led to the identification of a severe fever with thrombocytopenia syndrome (SFTS) with an unknown cause. Infection with Anaplasma phagocytophilum has been suggested as a cause, but the pathogen has not been detected in most patients on laboratory testing.
We obtained blood samples from patients with the case definition of SFTS in six provinces in China. The blood samples were used to isolate the causal pathogen by inoculation of cell culture and for detection of viral RNA on polymerase-chain-reaction assay. The pathogen was characterized on electron microscopy and nucleic acid sequencing. We used enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and neutralization testing to analyze the level of virus-specific antibody in patients’ serum samples.
We isolated a novel virus, designated SFTS bunyavirus, from patients who presented with fever, thrombocytopenia, leukocytopenia, and multiorgan dysfunction. RNA sequence analysis revealed that the virus was a newly identified member of the genus phlebovirus in the Bunyaviridae family. Electron-microscopical examination revealed virions with the morphologic characteristics of a bunyavirus. The presence of the virus was confirmed in 171 patients with SFTS from six provinces by detection of viral RNA, specific antibodies to the virus in blood, or both. Serologic assays showed a virus-specific immune response in all 35 pairs of serum samples collected from patients during the acute and convalescent phases of the illness.
A novel phlebovirus was identified in patients with a life-threatening illness associated with fever and thrombocytopenia in China. (Funded by the China Mega-Project for Infectious Diseases and others.)
PMCID: PMC3113718  PMID: 21410387
18.  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.
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.
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.
PMCID: PMC3164193  PMID: 21904617
19.  Molecular Interaction of TPPP with PrP Antagonized the CytoPrP-Induced Disruption of Microtubule Structures and Cytotoxicity 
PLoS ONE  2011;6(8):e23079.
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.
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.
PMCID: PMC3155546  PMID: 21857997
20.  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.
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.
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.
PMCID: PMC3029303  PMID: 21298055
21.  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.
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.
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.
PMCID: PMC2912377  PMID: 20686702
22.  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.
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.
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.
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.
EV71 shedding from respiratory tract may continue for nearly four weeks after onset, but its excretion through feces can persist more than five weeks.
PMCID: PMC2895604  PMID: 20565813
23.  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 
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.
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.
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.
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.
PMCID: PMC2868850  PMID: 20356412
24.  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.
PMCID: PMC2740053  PMID: 19830016
25.  Surveillance for Creutzfeldt-Jakob disease in China from 2006 to 2007 
BMC Public Health  2008;8:360.
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.
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.
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.
Chinese CJD patients possessed similar epidemiological and clinical characteristics as worldwide.
PMCID: PMC2596798  PMID: 18928564

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