Genetic Prion Disease: Insight from the Features and Experience of China National Surveillance for Creutzfeldt-Jakob Disease / 神经科学通报·英文版

Human genetic prion diseases (gPrDs) are directly associated with mutations and insertions in the PRNP (Prion Protein) gene. We collected and analyzed the data of 218 Chinese gPrD patients identified between Jan 2006 and June 2020. Nineteen different subtypes were identified and gPrDs accounted for 10.9% of all diagnosed PrDs within the same period. Some subtypes of gPrDs showed a degree of geographic association. The age at onset of Chinese gPrDs peaked in the 50–59 year group. Gerstmann–Sträussler–Scheinker syndrome (GSS) and fatal familial insomnia (FFI) cases usually displayed clinical symptoms earlier than genetic Creutzfeldt–Jakob disease (gCJD) patients with point mutations. A family history was more frequently recalled in P105L GSS and D178N FFI patients than T188K and E200K patients. None of the E196A gCJD patients reported a family history. The gCJD cases with point mutations always developed clinical manifestations typical of sporadic CJD (sCJD). EEG examination was not sensitive for gPrDs. sCJD-associated abnormalities on MRI were found in high proportions of GSS and gCJD patients. CSF 14-3-3 positivity was frequently detected in gCJD patients. Increased CSF tau was found in more than half of FFI and T188K gCJD cases, and an even higher proportion of E196A and E200K gCJD patients. 63.6% of P105L GSS cases showed a positive reaction in cerebrospinal fluid RT-QuIC. GSS and FFI cases had longer durations than most subtypes of gCJD. This is one of the largest studies of gPrDs in East Asians, and the illness profile of Chinese gPrDs is clearly distinct. Extremely high proportions of T188K and E196A occur among Chinese gPrDs; these mutations are rarely reported in Caucasians and Japanese.

Genetic Prion Disease: Insight from the Features and Experience of China National Surveillance for Creutzfeldt-Jakob Disease / 神经科学通报·英文版

Human genetic prion diseases (gPrDs) are directly associated with mutations and insertions in the PRNP (Prion Protein) gene. We collected and analyzed the data of 218 Chinese gPrD patients identified between Jan 2006 and June 2020. Nineteen different subtypes were identified and gPrDs accounted for 10.9% of all diagnosed PrDs within the same period. Some subtypes of gPrDs showed a degree of geographic association. The age at onset of Chinese gPrDs peaked in the 50-59 year group. Gerstmann-Sträussler-Scheinker syndrome (GSS) and fatal familial insomnia (FFI) cases usually displayed clinical symptoms earlier than genetic Creutzfeldt-Jakob disease (gCJD) patients with point mutations. A family history was more frequently recalled in P105L GSS and D178N FFI patients than T188K and E200K patients. None of the E196A gCJD patients reported a family history. The gCJD cases with point mutations always developed clinical manifestations typical of sporadic CJD (sCJD). EEG examination was not sensitive for gPrDs. sCJD-associated abnormalities on MRI were found in high proportions of GSS and gCJD patients. CSF 14-3-3 positivity was frequently detected in gCJD patients. Increased CSF tau was found in more than half of FFI and T188K gCJD cases, and an even higher proportion of E196A and E200K gCJD patients. 63.6% of P105L GSS cases showed a positive reaction in cerebrospinal fluid RT-QuIC. GSS and FFI cases had longer durations than most subtypes of gCJD. This is one of the largest studies of gPrDs in East Asians, and the illness profile of Chinese gPrDs is clearly distinct. Extremely high proportions of T188K and E196A occur among Chinese gPrDs; these mutations are rarely reported in Caucasians and Japanese.

Analysis of the Electrophoretic Profiles of Prion Protein in Carcinous and Pericarcinous Lysates of Six Different Types of Cancers / 生物医学与环境科学（英文）

Objective@#To find the different electrophoretic profiles of prion protein in carcinous and individual pericarcinous tissues in lysates of gastric, colon, liver, lung, thyroid, and laryngeal cancers.@*Methods@#Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were used to test the amounts and electrophoretic patterns of total PrP and the tolerance of PK (protease K) digestion among six various cancer tissue types.@*Results@#A mass of PrP signals with a large molecular weight were identified in the homogenates of peripheral tissues. The amounts and electrophoretic patterns of total PrP did not differ significantly between carcinous and pericarcinous tissues. PrPs in all types of the tested cancer samples were PK sensitive but showed diversity in the tolerance of PK digestion among various tissue types.@*Conclusions@#The study revealed that the included electrophoretic patterns of carcinous and pericarcinous tissues were almost similar. Unlike PrP-specific immunohistochemical assay, evaluation of PrP electrophoretic patterns in the peripheral organs and tissues by Western blot does not reflect tumor malignancy.

Epidemiological Characteristics of Notifiable Infectious Diseases among Foreign Cases in China, 2004-2017 / 生物医学与环境科学（英文）

Objective@#We aimed to assess the features of notifiable infectious diseases found commonly in foreign nationals in China between 2004 and 2017 to improve public health policy and responses for infectious diseases.@*Methods@#We performed a descriptive study of notifiable infectious diseases among foreigners reported from 2004 to 2017 in China using data from the Chinese National Notifiable Infectious Disease Reporting System (NNIDRIS). Demographic, temporal-spatial distribution were described and analyzed.@*Results@#A total of 67,939 cases of 33 different infectious diseases were reported among foreigners. These diseases were seen in 31 provinces of China and originated from 146 countries of the world. The infectious diseases with the highest incidence number were human immunodeficiency virus (HIV) of 18,713 cases, hepatitis B (6,461 cases), hand, foot, and mouth disease (6,327 cases). Yunnan province had the highest number of notifiable infectious diseases in foreigners. There were different trends of the major infectious diseases among foreign cases seen in China and varied among provinces.@*Conclusions@#This is the first description of the epidemiological characteristic of notifiable infectious diseases among foreigners in China from 2004 to 2017. These data can be used to better inform policymakers about national health priorities for future research and control strategies.

Preparation of PrP-specific Polyclonal Antibody Immunization of -knockout Mice with Recombinant Human PrP Protein / 生物医学与环境科学（英文）

Objective@#The definite diagnosis of human and animal prion diseases depends on the examination of special pathological changes and/or detection of PrP in the brain tissues of suspected cases. Thus, developing methods to obtain PrP antibody with good specificity and sensitivity is fundamental for prion identification.@*Methods@#We prepared a PrP-specific polyclonal antibody (pAb P54) in a -knockout mouse model immunization with recombinant full-length human PrP protein residues 23-231. Thereafter, we verified that pAb in Western blot, immunohistochemistry (IHC), and immunofluorescent (IFA) assays.@*Results@#Western blot illustrated that the newly prepared pAb P54 could react with recombinant PrP protein, normal brain PrP from healthy rodents and humans, and pathological PrP in the brains of experimental rodents infected with scrapie and humans infected with different types of prion diseases. The electrophoretic patterns of brain PrP and PrP observed after their reaction with pAb P54 were nearly identical to those produced by commercial PrP monoclonal antibodies. Three glycosylated PrP molecules in the brain homogenates were clearly demonstrated in the reactions of these molecules with pAb P54. IHC assay revealed apparent PrP deposits in the GdnCl-treated brain slices of 139A-infected mice and 263K-infected hamsters. IFA tests with pAb P54 also showed clear green signals surrounding blue-stained cell nuclei.@*Conclusion@#The newly prepared pAb P54 demonstrated reliable specificity and sensitivity and, thus, may have potential applications not only in studies of prion biology but also in the diagnosis of human and experimental rodent prion diseases.

Assessment of the Sensitivity and Specificity of the Established Real-time Quaking-induced Conversion (RT-QuIC) Technique in Chinese CJD Surveillance / 生物医学与环境科学（英文）

Real-time quaking-induced conversion (RT-QuIC) assay is a newly established PrP -detecting method. The development of RT-QuIC improves the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD), showing good sensitivity and specificity in many countries when the method was used in cerebrospinal fluid (CSF) samples. However, in China, the sensitivity and specificity of RT-QuIC has yet to be determined due to the lack of definitive diagnosis samples. Recently, 30 definitive sCJD and 30 non-CJD diagnoses were evaluated by RT-QuIC assay. In the 30 sCJD CSF samples, 29 showed positive results. By contrast, all the non-CJD samples were negative. The sensitivity and specificity of our RT-QuIC assay were 96.67% and 100%, respectively, and are comparable to other published data. Results can provide a fundamental basis for the usage of RT-QuIC assay in CJD surveillance in China.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes.</p><p><b>METHODS</b>In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing.</p><p><b>RESULTS</b>Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run.</p><p><b>CONCLUSION</b>MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.</p>

Studies on chemical constituents of Valeriana plants and their biological activities / 中国中药杂志

The recent progresses on chemical components and pharmacological activities of the genus Valerianawere summarized.Besides-essential oil, the chemical composition of Valerianais mainly focused on monoterpenoids,sesquiterpenoids,lignans, flavonoids, alkaloids, etc. Iridoids are the main chemical components ofmonoterpenoids. There are two types ofiridoidson the basis of the cyclopentane open or not. The Valerianahas been drawmuch attention for their significant sedation,spasmolysis,antidepression,antitumor, against adenosine A1 receptors and cytotoxicityactivity,and had certain function for cardiovascular disease treatment. Given to the fact of the lack of systematic review and summary of studies on the Valeriana, we summarized and analyze the study literatures on the pharmacological activity of Valerianain recent years, and providedsome basisfor further study.

Uniformity of Coptidis Rhizoma decoction pieces dispensing based on effective constituent equivalence / 中国中药杂志

Traditional Chinese medicine (TCM) dispensing is the final step of TCM used for clinical treatment, the stability of TCM dispensing is the guarantee of good clinical effect. Establishment of effect-constituent equivalence for Chinese herbal pieces based on clinical efficacy, can not only guarantee the stability of TCM dispensing, but also relate to the precision of clinical effect. This study chose Coptidis Rhizoma as the model, established effect-constituent equivalence of Coptidis Rhizoma, based on the effect-constituent index already established by our research group, and taking into consideration of homogeneity of clinical dosage and compliance of decoction, the uniformity of dispensing for different specification of Coptidis Rhizoma decoction pieces was studied. This research model was then applied to guide the specification-optimization of Coptidis Rhizoma and its clinical dispensing. The result indicated, effective constituent equivalence could reflect the fluctuation of specification, dosage and decoction to the fluctuation of efficacy; Optimized Coptidis Rhizoma decoction pieces had the characteristic of high homogeneity as for clinical dispensing, good compliance as for decoction, and high effective constituent equivalence. In conclusion, effective constituent equivalence could improve relevance of methods of TCM dispensing control to clinical effect. Preparated Superior-standard Decoction Pieces based on effective constituent equivalence was featured by good quality and a good practice of adjustable dosage, which could promote the development of TCM decoction pieces toward precision medicine.

Dynamic monitoring of the cytotoxic effects of protoberberine alkaloids from Rhizoma Coptidis on HepG2 cells using the xCELLigence system / 中国天然药物

AIM@#To investigate the cytotoxic effects of the six protoberberine alkaloids (PAs) from Rhizoma Coptidis on HepG2 cells.@*METHOD@#A systematic screening was conducted to investigate the dynamic response of HepG2 cells to the PAs using the impedance-based xCELLigence system. Cisplatin was selected as the positive control. The real time, concentration-response curves and the 50% inhibitory concentrations (IC50) were acquired to evaluate the anticancer activity of the PAs.@*RESULTS@#All of the six PAs inhibited cell growth and induce death in HepG2 cells in a time- and concentration-dependent manner. The IC50 values of cisplatin, berberine, columbamine, coptisine, epiberberine, jatrorrhizine, and palmatine were 5.13, 42.33, 226.54, 36.90, 302.72, 383.54, and 456.96 μg·mL(-1), respectively. The results obtained using the xCELLigence system corresponded well with those of the conventional methods.@*CONCLUSION@#The xCELLigence system is a reliable and efficient tool for real-time screening of the cytotoxic effect of compounds in cell-based in vitro assays. Coptisine and berberine, with methylenedioxy group at C2 and C3 on the phenyl ring showed stronger effect.than the other four PAs. However, compared with cisplatin, the six PAs didn't show obvious cytotoxic effect on HepG2 cells. These results provided some useful data for the evaluation of the anticancer compounds, and the clinical application of traditional Chinese medicine.

Analysis of the expressions of alphaB-crystallin in the brain tissues of agent 263K-infected hamsters at terminal stage / 病毒学报

alphaB-crystallin is a member of the sHSP (Small heat shock protein) family, which plays an impor tant role in multiple neurodegeneration diseases. To give insight into the possible alternation and the role of aB-crystallin in prion disease, the alphaB-crystallin levels in the brain tissues of agent 263K-infected hamsters were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, the levels of alphaB-crystallin were increased up to 3-fold in the brain tissues of scrapie infected 263K hamsters compared with normal controls. Immunofluorescent assays revealed that the up-regulated alphaB-crystallin was mainly observed in astrocytes, but not in neurons. The co-localization between alphaB-crystallin and abnormal deposition of PrPsc in the brain tissues of the scrapie infected hamsters was not observed. The study may provide a foundation for further revealing the potential role of alphaB-crystallin in prion disease.

Analysis of the human papillomavirus in laryngeal squamous cell carcinoma patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the infection of human papillomavirus in laryngeal squamous cell carcinoma patients.</p><p><b>METHODS</b>The pathological samples of 64 clinical diagnosed laryngeal squamous cell carcinoma patients were collected. Lunimex and PCR techniques were used to detect the HPV gene infection and immunohistochemistry method was used to analyze the HPV protein expression in the samples.</p><p><b>RESULTS</b>In the 64 cases, 7 were positive for HPV infection by Luminex and PCR tests. 18 were positive for HPV16/18 E6 protein expression. The total positive rate was about 39. 1%.</p><p><b>CONCLUSION</b>The high HPV infection rate in laryngeal squamous cell carcinoma patients in the study indicated indirectly that the importance of the HPV infection in pathogenesis of the laryngeal squamous cell carcinoma.</p>

Chemical constituents from leaves of Rhododendron rubiginosum var rubiginosum / 中国中药杂志

Thirteen compounds were isolated from the leaves of Rhododendron rubiginosum var. rubiginosum by various chromatographic techniques. On the basis of spectroscopic data, their structures were elucidated as 3,9-dihydroxy-megastigma-5-ene (1), 3 beta-hydroxy-5alpha ,6 alpha-epoxy-7-megastigmen-9-one (2), loliolide (3), ursolic acid(4), 2 alpha, 3 beta-dihydroxy-urs-12-en-28-oic acid (5), 2 alpha, 3 beta,23-trihydroxy-urs-12-en-28-oic acid (6), 7,9-dimethoxyrhododendrol (7), 7-methoxyrhododendrol (8), zingerone (9), isofraxidin (10), scopoletin (11), (+)-pinoresinol (12) and 3'-O-demethylepipinorisenol (13). All compounds were isolated from this plant for the first time, and compounds 1-3, 7-9, and 11-13 were isolated from the genus Rhododendron for the first time.

Contamination of live virus during tissue homogenizing by ultrasonic processor and tissue disperser / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To quantitatively evaluate the contamination area and risk of a live pathogen during tissue homogenization by either ultrasonic processor or tissue disperser.</p><p><b>METHODS</b>A recombinant Herpes Simplex Virus (rHSV) containing GFP gene was used as the index virus, and fresh liver tissue from healthy mice was used as simulated specimen. After 10% liver homogenate was mixed with rHSV (100 TCID50/0.1 mL) in a 5 mL tube, the stability of rHSV in liver homogenate and influences of an ultrasonic processor and a tissue disperser on viral infectivity were determined by GFP expressions in cell cultures. The contaminating areas of live viruses during homogenization were evaluated by a cell culture-based sedimentary. The contamination radii were counted by measurement of the distance between the operator and the farthest GFP positive well.</p><p><b>RESULTS</b>The infectivity of rHSV in 10% liver homogenate maintained almost unchanged after it was incubated at room temperature for 30 min. Treatment with an ultrasonic processor clearly dropped down the virus infectivity, while a disperser not. Obvious spills and slashes of live viruses were observed in processes of homogenization with those two apparatuses. The contamination radii are positively related with sample volume, output energy of operator and handling time.</p><p><b>CONCLUSION</b>Homogenizing infectious samples with an ultrasonic processor and a tissue disperser at commonly used conditions caused obvious spills and splashes of live viruses, which possesses high risk to induce Laboratory acquired infections (LAIs).</p>

Clinical and familial characteristics of ten chinese patients with fatal family insomnia / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Fatal familial insomnia (FFI) is an autosomal dominant prion disease characterized clinically by inattention, sleep loss, dysautonomia, and motor signs. This study is aimed to investigate clinical and familial characteristics of ten Chinese Patients with FFI.</p><p><b>METHODS</b>We identified ten FFI cases from the surveillance network for Creutafeldt-Jakob disease (CJD) in China. Final diagnosis of FFI cases was made in accordance with the WHO criteria for CJD. The main clinical features and family histories of these ten FFI cases were analyzed.</p><p><b>RESULTS</b>The median age of ten cases at onset was 38 years (from 19 to 55). The foremost symptoms seemed to be various, including sleep disturbances, vision disorder, dizziness and anorexia. Sleep disturbances appeared in all cases and lasted in the whole clinical courses. Progressive sympathetic symptoms, memory loss, movement disturbances, myoclonus and hypertension were also frequently observed. The median duration of the disease was 9.5 months. EEG and MRI did not figure out special abnormality. 14-3-3 protein in CSF was positive in five out of eight tested patients. Clear family histories were identified in 8 patients.</p><p><b>CONCLUSION</b>The data from our study confirm that the Chinese FFI cases have similar clinical characteristics as that of the Caucasian cases. Compared with other genetic CJD associated mutations, the genetic frequencies of D178N in PRNP are apparently high among the Chinese cases.</p>

The analysis of human papillomavirus infection in lip squamous cell carcinoma patients / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the infection of human papillomavirus in lip squamous cell carcinoma patients.</p><p><b>METHODS</b>The pathological samples of 9 clinical diagnosed lip cancer patients were collected. Lunimex and PCR techniques were used to detect the HPV gene infection and immunohistochemistry method was used to analyze the HPV protein expression in the samples.</p><p><b>RESULTS</b>In the 9 cases, 1 was positive for HPV16 gene and 7 were positive for HPV16/18 E6 protein expression. The total positive rate was about 8/9.</p><p><b>CONCLUSION</b>The high HPV infection rate in lip cancer patients in the study indicated indirectly that the importance of the direct contact to the infection of HPV what was the basis for pathogenesis of the lip squamous cell carcinoma.</p>

Mutant D178N prion protein converts spontaneously in RT-QuIC assay / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the conversion of mutant D178N prion protein in RT-QuIC assay.</p><p><b>METHODS</b>The D178N mutant prion PRNP was generated by the method of single site mutation. The mutant PRNP gene was inserted into plasmids of pET24. The full and N-truncated recombinant human prion proteins were expressed and purified. The fibril formations of these proteins were real-time monitored by the method of RT-QuIC. The ability to resist proteinase K (PK) of these fibrils was analyzed.</p><p><b>RESULTS</b>We succeed to construct human PrP-D178N plamids. The N-truncated human prion protein with D178N (PrP90-231-D178N) can convert spontaneously in RT-QuIC, while full length of human prion D178N protein (PrP23-231-D178N) fails to convert spontaneously. The spontaneously generated fibril has been domenstrated it is partily PK-resistant.</p><p><b>CONCLUSION</b>The N-terminal of prion protein (23-90) plays an important role for the D178N mutant protein spontaneously conversion, which provide the clues for study the pathogenesis of genetic CJD.</p>

Study on the correlation between membrane protein Flotillin-1 and PrPc endocytosis / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To explore whether the membrane-associated protein Flotillin-1 has relationship with endocytosis of PrPc.</p><p><b>METHODS</b>The expression of Flotillin-1 in different cell lines was detected with the method of Western Blot; the interaction between Flotillin-1 and PrPc in Cells which were treated with copper ions was observed using immunoprecipitation method.</p><p><b>RESULTS</b>(1) Flotillin-1 was widely expressed in many cell lines without significant difference in the amounts of expression level; (2) Only in the appearance of copper ions, the protein complexes of PrPc and Flotillin-1 can be detected with the method of IP, which were related to copper ions concentration and processing time.</p><p><b>CONCLUSION</b>The membrane-associated protein Flotillin-1 has the relationship with the endocytosis of PrPc.</p>

A redox process of ethanethiol group increases formation of beta-sheet structure and amyloid fibrils of wild type human prion protein / 病毒学报

In order to explore the potential influences of the disulfide bridge on the physical and chemical properties of PrP protein, the expressed recombinant human wild-type PrP protein was purified for using in an established redox process for the reduction and oxidation of the ethanethiol group within PrP. Sedimentation tests illustrated that redox process remarkably promoted the aggregation of recombinant PrP. Thioflavin T binding assay revealed an enhanced fibrillization of the recombinant human PrP after redox process. Far-UV circular dichroism demonstrated that the PrP treated with redox process showed a significant p-sheet rich structure. Furthermore, PrP-specific Western blot identified that the recombinant PrP after redox possessed stronger proteinase K-resistance. Those data indicates that the formation of the disulfide bridge induces the alteration of the secondary structure and enhances the progresses of aggregation and fibrillization of PrP protein.

Mutations in various functional domains of HPV2 E2 protein inhibit the transcriptional depression activities / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the potential transcriptional depression activities of HPV2 E2 proteins with mutations in different functional domains.</p><p><b>METHODS</b>The primers for constructing various E2 mutants were synthesized based on a HPV2 isolate containing several point mutations within E2 open reading frame. Different E2 mutations were generated by the method of extending PCR and inserted into plasmid pcDNA3. 1. Various recombinant mammalian expression plasmids pcDNA3. 1-E2 were co-transfected into HeLa cells together with a CAT-reporter plasmid pBLCAT-LCR containing HPV-2 prototype LCR, respectively. The transcriptional repression activities of the E2 mutants were evaluated by detection of CAT expression values.</p><p><b>RESULTS</b>Compared with the full-length prototype E2, removals of both N- and C-terminal domains abolished E2 transcriptional repressive activities. The point mutations in the transactivation domain (nt 3037), the internal hinge region (nt 3387) and DNA binding domain (nt 3697) showed remarkable inhibition on its transcriptional depression function.</p><p><b>CONCLUSION</b>The transcriptional regulation activity of HPV2 E2 is related with its DNA binding and transactivation domains. The exchanges of the single amino acid within E2, derived from a HPV2 isolate, abolish significantly the repressive effect on viral promoter in the context of full-length E2.</p>