Hepatic interleukin 32 attenuates liver injury through repression of necroptosis in cholestasis.

OBJECTIVE: We aimed to evaluate the association between interleukin (IL)-32 and necroptosis in cholestatic liver injury. METHODS: Levels of necroptosis-related markers in cholestatic and control patients, including the receptor-interacting serine-threonine kinase 3 (RIPK3), receptor-interacting serine-threonine kinase 1 (RIPK1), and mixed lineage kinase domain-like (MLKL) were measured. Animal experiments in C57BL/6J and transgenic mice with IL32ß/γ overexpression were also conducted to confirm the effect of IL-32 on necroptosis in cholestasis, which was induced by α-naphthylisothiocyanate (ANIT) and 1% lithocholic acid (LCA). PLC/PRF/5-ASBT and primary mouse hepatocytes were utilized for the investigation of the regulation and mechanism of IL-32 in cholestasis. RESULTS: In the liver tissues of cholestatic patients, the mRNA and protein expressions of RIPK1, RIPK3, and MLKL were increased and associated with IL-32 expression. In addition, expressions of these indicators in the liver of 1% LCA- and ANIT-induced mouse models were significantly increased, while they were markedly decreased in hIL32ßLTg and hIL32γLTg mice. After bile acid stimulation, IL-32 and phosphorylated Akt (p-Akt) expressions significantly elevated in a dose-dependent manner. After treated with tumor necrosis factor (TNF)-α, IL-32 inhibited MLKL expression in primary mouse hepatocytes. CONCLUSION: IL-32 is negatively associated with necroptosis in cholestatic patients. Moreover, IL-32 is induced by p-Akt and effectively attenuates necroptosis in ANIT- or 1% LCA-induced cholestasis.

Plasmon Coupling Enhanced Micro-Spectroscopy and Imaging for Sensitive Discrimination of Membrane Domains of a Single Cell.

Different cell membrane domains play different roles in many cell processes, and the discrimination of these domains is of considerable importance for the elucidation of cellular functions. However, the strategies available for distinguishing these cell membrane domains are limited. A novel technique called plasmon coupling enhanced micro-spectroscopy and imaging to discriminate basal and lateral membrane domains of a single cell combines the application of an additional plasmonic silver film for surface plasmon (SP) excitation to selectively excite and enhance the basal membranes in the near-field with directional enhanced microscopic imaging and spectroscopy. The SP and critical evanescent fields are induced upon excitation through a silver-coated semitransparent coverslip at the surface plasmon resonance and critical angles, respectively. The basal and lateral membrane domains located within the SP and critical evanescent fields can be selectively excited and distinguished by adjusting the incident angle of laser irradiation. Moreover, the brighter images and more intense spectra of membrane-targeting fluorescence-Raman probes under directional excitation than in conventional EPI mode allow clear identification of the membrane domains.

Study on the COVID-19 infection status, prevention and control strategies among people entering Shenzhen.

BACKGROUND: The novel coronavirus disease 2019 (COVID-19) confirmed cases overseas have continued to rise in the last months, and many people overseas have chosen to return to China. This increases the risk of a large number of imported cases which may cause a relapse of the COVID-19 outbreak. In order to prevent imported infection, the Shenzhen government has implemented a closed-loop management strategy using nucleic acid testing (NAT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and requiring 14 days of medical observation for individuals with an overseas tour history (Hong Kong, Macao, Taiwan province and other countries). Our study aims to describe the status of COVID-19 infection among people entering Shenzhen, and to evaluate the effect of the closed-loop management strategy. METHODS: We undertook a descriptive study and risk analysis by the entry time, time of reporting, and local confirmed cases in countries of origin. The NAT were completed in Shenzhen Center for Disease Control and Prevention (CDC), ten district-level CDCs, and fever clinics. RESULTS: A total of 86,844 people from overseas entered Shenzhen from January 1 to April 18, 2020; there were 39 imported COVID cases and 293 close contacts. The infection rate of people entering was 4.49‰ [95% Confidence interval (CI): 3.26‰-6.05‰]. Fourteen imported cases (35.9%) came from the UK, and nine (23.08%) came from the USA. People entering from the USA since March 9 or from the UK since March 13 are the high-risk population. As of July 17, there have been no new confirmed cases in Shenzhen for 153 days, and the numbers of confirmed case, close contacts, and asymptomatic cases are 0. CONCLUSIONS: The closed-loop management has been effective in preventing imported infection and controlling domestic relapse. The distribution of entry time and report time for imported cases overseas was similar. This shows that it is important to implement closed-loop management at the port of entry.

Positron emission computed tomography/single photon emission computed tomography in Parkinson disease.

Parkinson disease (PD) is the second-most common neurodegenerative disorder. Its main pathological mechanism is the selective degeneration and deletion of dopaminergic neurons in the dense part of the substantia nigra and the damage of dopaminergic neurons caused by the abnormal deposition of a Lewy body, leading to a decreased dopamine level. Positron emission computed tomography (PET)/single photon emission computed tomography (SPECT) is a molecular imaging technology that can directly or indirectly reflect changes in molecular levels by using a specific tracer. With the research and development on the tracers of related enzymes for labeling dopamine transporter and dopamine receptor and for being involved in dopamine formation, this imaging technology has been applied to all aspects of PD research. It not only contributes to clinical work but also provides an important theoretical basis for exploring the pathological mechanism of PD at a molecular level. Therefore, this review discusses the application value of PET/SPECT in PD in terms of early diagnosis, disease severity evaluation, clinical manifestations, differential diagnosis, and pathological mechanism.

In situ and sensitive monitoring of configuration-switching involved dynamic adsorption by surface plasmon-coupled directional enhanced Raman scattering.

Surface adsorption studies play a crucial role in numerous fields from surface catalysis to molecular separation. However, investigation on adsorption mechanisms has been restricted to limited analytes and approaches, which calls for an in situ and sensitive surface analysis technique capable of revealing the mechanisms as well as discriminating different adsorbates and their geometry at different adsorption stages. In this study, we employed surface plasmon-coupled directional enhanced Raman scattering (SPCR), a novel technique developed by coupling surface plasmon-coupled emission with SERS, to study conformation-switching involved dynamic adsorption with background suppression and improved sensitivity (nearly 30-fold). We obtained the isotherms for a conformation-changing Raman model analyte, malachite green. An S-type Langmuir model was fitted from the time-resolved SPCR signals sensitively and without any interference from the bulk solution. The reorientation of the analyte from a predominantly parallel configuration to a perpendicular one was captured by the dramatic increase in the intensity ratios of the adsorption-related peaks to the adsorption-unrelated peak. We believe that this new sensitive and selective SPCR technique will be a promising tool for surface adsorption kinetics analysis.

Norovirus Infection and Histo-blood Group Antigens in Children Hospitalized with Diarrhea in Lulong and Chenzhou in China.

Norovirus (NoV) is a pathogen that commonly causes viral diarrhea in children. Studies indicate that NoV recognizes human histo-blood group antigens (HBGAs) as cell attachment factors. In order to explore the correlation between of NoV infection and HBGAs, a cross-sectional study was conducted in children less than five years old who were hospitalized with diarrhea in two areas of China between November 2014 and February 2015. Of the paired stool and saliva samples taken from 424 children, NoV was detected in 24 (6%) children, with viral genotypes GII.3 (n=5), GII.4 (n=14), GII.12 (n=1), and GII.17 (n=4). All of the individuals having NoV infection were either secretors (Lea-b+/Lex-y+) or partial secretors (Lea+b+/Lex+y+) except one GII.3 infection of a non-secretor (Lea+b-/Lex+y-). These results suggest that secretor positive is associated with NoV infection, although non-secretors are not absolutely protected from NoV infection.

Dynamic Diffusion Tensor Imaging Reveals Structural Changes in the Bilateral Pyramidal Tracts after Brain Stem Hemorrhage in Rats.

BACKGROUND AND PURPOSE: Few studies have concentrated on pyramidal tract (PY) changes after brain stem hemorrhage (BSH). In this study, we used a diffusion tensor imaging (DTI) technique and histologic identification to investigate longitudinal PY changes on both the contralateral and ipsilateral sides after experimental BSH. METHODS: BSH was induced in 61 Sprague-Dawley rats by infusing 30 µl of autogenous tail blood into each rat's right pons. DTI and motor function examinations were performed repeatedly on days 1, 3, 7, 14, and 28 after surgery. Fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity, and radial diffusivity were measured in the bilateral PYs. The axon and myelin injury in the PY were evaluated by histologic study. RESULTS: As compared with normal controls, the bilateral PYs in rats with induced BSH showed an early decrease and a late increase in FA and an early increase and a late decrease in MD. A progressive decrease in axial diffusivity with dramatic axon loss from day 1 to day 28 after BSH was found bilaterally. The bilateral PYs showed an early increase and a late decrease in radial diffusivity. Early myelin injury and late repair were also detected pathologically in the bilateral PYs of rats with BSH. Thus, the early motor function deficits of rats with BSH began to improve on day 14 and had almost completely disappeared by day 28. CONCLUSIONS: DTI revealed dynamic changes in the bilateral PYs after BSH, which was confirmed by histologic findings and which correlated with motor function alteration. These findings support the idea that quantitative DTI can track structural changes in the bilateral PYs and that DTI may serve as a noninvasive tool to predict the prognoses of patients with BSH.

Gene Knockdown in Human Rhinovirus 1B Using 2'-OMe-modified siRNAs Results in the Reactivation of the Interferon Response.

The aim of this study was to investigate the knockdown efficiency of 2'-O-methylated (2'-OMe)-modified small interfering RNAs (siRNAs) on human rhinovirus 1B (HRV1B) replication and the interferon response. Thus, 24 2'-OMe-modified siRNAs were designed to target HRV1B. The RNA levels of HRV1B, Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and interferons were determined in HRV1B-infected HeLa and BEAS-2B epithelial cells transfected with 2'-OMe-modified siRNAs. The results revealed that all 2'-OMe-modified siRNAs interfered with the replication of HRV1B in a cell-specific and transfection efficiency-dependent manner. Viral activation of Toll-like receptor 3, melanoma differentiation-associated gene 5, retinoic acid inducible gene-I, and the interferon response was detected. In conclusion, the 2'-OMe-modified siRNAs used in this study could interfere with HRV1B replication, possibly leading to the reactivation of the interferon response.

[Establishment and primary application of a novel resequencing pathogen microarray-based assay for detecting pathogens in patients with unexplained diarrhea].

In this study, a novel resequencing pathogen microarray (RPM)-based multi-pathogen detection assay was developed to simultaneously detect 14 rotaviruses, 7 caliciviruses, 8 astroviruses, 28 enteroviruses, and 16 rare diarrhea viruses in patients with diarrhea syndrome. The specificity of the assay was examined using confirmed virus-positive specimens, and the sensitivity was evaluated by serial ten-fold dilutions of in vitro transcribed RNA. RPM assay could detect and differentiate virus types/subtypes at 20-2000 copies/microL. The detection threshold of RPM was determined by adjusting the reference concentration, and the detection steps were optimized to type Enterovirus. The nucleic acids of 10 stool samples from patients with unexplained diarrhea were screened, and 6 of them showed positive results. The RPM results were further verified by singleplex PCR followed by sequencing, and no difference was found between the two assays. In conclusion, we have established a high-throughput RPM assay with high specificity and sensitivity, which demonstrates a great potential for the identification of pathogens in patients with unexplained diarrhea and the management of emerging epidemic.

[Establish A duplex fluorescent quantitative one-step RT-PCR system for the detection of norovirus genogroup I and II].

The object of this study is to develop a duplex fluorescent quantitative one-step RT-PCR assay for detection and quantitation of GI and GII norovirus. The specific primers, Taqman probes, optimized reaction solution and condition were used to develop the duplex fluorescent quantitative one-step RT-PCR assay. The sensitivity, specificity and reproducibility of the assay were evaluated. The assay was evaluated by testing the 100 specimen samples and compared with the reference assay conventional RT-PCR. The assay possessed high specificity for norovirus detection without any evident cross-reaction with enteric adenovirus, rotavirus or astrovirus. The detection limit of the real-time RT-PCR assay, for GI and GII norovirus was up to 10(3) copy/microL respectively. Compared with the conventional RT-PCR assay, the assay in this study had higher sensitivity with higher detection rate of norovirus in stool specimens. The duplex fluorescent quantitative one-step RT-PCR assay provides rapid, sensitive and reliable detection of GI and GII norovirus, and could be used as a laboratory diagnosis of norovirus in acute gastroenteritis patients.

Simultaneous detection of seven enteric viruses associated with acute gastroenteritis by a multiplexed Luminex-based assay.

Rapid and broad diagnostic methods are needed for the identification of viral agents of gastroenteritis. In this study, we used Luminex xMAP technology to develop a multiplexed assay for the simultaneous identification of major enteric viral pathogens, including rotavirus A (RVA), noroviruses (NoVs) (including genogroups GI and GII), sapoviruses (SaV), human astrovirus (HAstV), enteric adenoviruses (EAds), and human bocavirus 2 (HBoV2). The analytical sensitivity allowed detection of 10(3) (EAds, HBoV2, and RVA) and 10(4) (NoV GI and GII, SaV, and HAstV) copies per reaction mixture. Compared to conventional PCR, the Luminex-based assay yielded greater than 75% sensitivity and 97% specificity for each virus, and the kappa correlation for detection of all viruses ranged from 0.75 to 1.00. In conclusion, this multiplexed Luminex-based assay provides a potentially rapid, high-throughput, and maneuverable diagnostic tool for major viral pathogens associated with gastroenteritis.

[Colorimetric detection of norovirus genotype GII by reverse transcription loop-mediated isothermal amplification].

A simple, rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established to detect norovirus genotype GII. The method employed a set of six specially designed primers that recognized eight distinct sequences of RNA-dependant RNA polymerase and capsid protein gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for 60 minutes. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP was validated by detecting several different diarrhea viruses including norovirus genotype GII. The sensitivity was determined by serial dilutions of RNA molecules from in vitro transcription of norovirus genotype GII in parallel with conventional RT-PCR detection. The assay was further evaluated with 93 clinical specimens of diarrhea patients. The results showed that the sensitivity of RT-LAMP was 1 000 copies/microL with a high specificity and the relative sensitivity was at the same level as that of conventional RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of conventional RT-PCR as well. This colorimetric RT-LAMP assay was potential for rapid detection of norovirus genotype GII on spot due to the observation of visual result with high specificity and sensitivity, time-saving and cost benefit.

High prevalence of human bocavirus 2 and its role in childhood acute gastroenteritis in China.

BACKGROUND: Acute gastroenteritis (AGE) is one of the leading causes of death in children worldwide. Human bocavirus 2 (HBoV2) was recently identified in stool samples and is involved in the pathogenesis of AGE, but the current data were too limited to clarify this issue. OBJECTIVE: We conducted a case-control study on 632 children with diarrhea and 162 healthy controls in Lanzhou, China, to assess the role of HBoV2 in gastroenteritis. STUDY DESIGN: Viruses known or suspected to be agents of AGE, including RV, HucV, AdV, AstV, and HBoVs, were detected. Viral loads of HBoV2 were quantified by Real-time PCR. RESULTS: HBoV2 was detected in 129 (20.4%) and 20 (12.3%) of the gastroenteritis and control samples, respectively. The association between HBoV2 and gastroenteritis was weaker (OR = 1.269, CI= 0.704-2.288) than that between gastroenteritis and RV, HucV, AdV, or AstV, as determined by multivariate logistic regression analysis. The data also suggested that infection with HBoV2 did not exacerbate the clinical symptoms of gastroenteritis. Mean HBoV2 viral load in the case and control groups was fewer than 55 copies/ml extract. CONCLUSIONS: HBoV2 exhibit different epidemiological features from HBoV1 and HBoV3. The data presented herein do not support a causative role for HBoV2 in AGE, despite its high prevalence in stool samples.

[A novel method for multiplex detection of gastroenteritis-associated viruses].

To develop and optimize a simultaneous detection method of RotavirusA, Norovirus GI, GII, Sapovirus, human astrovirus, enteric adenoviruses and HBoV2 with GenomeLab GeXP analysis system. The sensitivity was verified to be 10(4) copies/microL with plasmids containing the viral targets in triplicate on different days, and no cross-reaction with enterovirus71, human Parechovirus and PicobirnavirusII was observed. Finally, we successfully developed a high throughout, rapid and maneuverable multiplex RT-PCR assay for simultaneous detection of seven viruses related with viral gastroenteritis, which provide a novel method for the molecular diagnosis of diarrhea-associated virus.

[Detection and molecular characterization of human parechovirus (HPeV) in children with acute gastroenteritis].

OBJECTIVE: To study HPeV from stool samples of children with acute gastroenteritis under 5 years old. METHODS: We conducted a real-time PCR to detect HPeV from stool samples and to amply VP1 sequence by nested RT-PCR to identify HPeV type. RESULTS: The results showed that 27 of 306 (8.82%) children with acute gastroenteritis were infected HPeV. 11 strains were typed. 9 strains HPeV1, both HPeV2 and HPeV4 was 1 strain. HPeV was mostly identified in autumn season with a peak in July. HPeV seemed relevant in children >2 years old. The range of nucleotide identity between all isolated strains with reference strains was 79%-92%. CONCLUSION: Epidemiology characteristic of HPeV in Jilin was concordance with that of reports. HPeV3 wasnt detected. It's significant to conduct the large scale and long-term surveillance of HPeV.

[Study on concentration of nuorovirus genegroup II from environmental water].

A new viral sampling concentration device was designed which was equipped with a new cationic filter membrane-Nanoceram suitable for field sampling. Norovirus Genegroup II was detected from environmental water with the aid of this device. The effects on virus recovery of prefiltration, various second-concentration methods, and different eluants were investigated through pre-experiment. The concentration optimized process, and the optimal concentration process were then determined. The results showed that the prefiltration had a profound effect on virus recovery, and two second-concentration method: PEG-NaC1 precipitation and celite adsorption, had almost the same concentration effects. The Na2 HPO4 solution of 0.15 mol/L was selected as the final eluant to elute the adsorbed Nuorovirus from the celite. The virus recovery of Nanoceram was determined to be 3.02%. Finally, successful detection of Norovirus GII in sewage from Yangqiao River, Fengtai District, Beijing was acheived. All these data had shown that the Naneceram filter concentration method could concentrate Norovirus from environmental water with a steady effects.

Development of a real-time PCR assay for detecting and quantifying human bocavirus 2.

Human bocavirus 2 (HBoV2) is a parvovirus that has been recently identified in stool samples from children. Any association between the virus and clinical disease is unclear. A rapid, reliable diagnostic method is necessary to address this issue. In this study, we developed a sensitive and specific HBoV2 quantitative real-time PCR assay that targets the HBoV2 NP-1 gene, based on the TaqMan method. The assay could reproducibly detect 10 copies of a recombinant DNA plasmid containing a partial region of the HBoV2 genome, with a dynamic range of 8 log units (10(1) to 10(8) copies). A clinical evaluation detected HBoV2 in 85 (24.6%) of 345 children with gastroenteritis, with viral loads ranging from 1.67 × 10(2) to 4.27 × 10(9) copies per ml of stool specimen.

Analysis and characterization of the complete genome of a member of a new species of kobuvirus associated with swine.

A virus belonging to a new species in the genus Kobuvirus, family Picornaviridae, was first isolated in 2008 from apparently healthy pigs in Hungary and China. We report the complete genome sequence and the genetic organization of the novel porcine kobuvirus strain Y-1-CHI, which was identified in China. The RNA genome of strain Y-1-CHI contains 8210 nucleotides (nt) and has an organization similar to that of other picornaviruses. The full-length nucleotide sequence of Y-1-CHI was 88.62%, 58.66%, and 48.86% identical to those of S-1-HUN, U-1, and Aichi virus, respectively. No positive results were found in 454 stool samples from children with acute gastroenteritis. Dendrograms indicated that Y-1-CHI and S-1-HUN are most closely related to each other and belong to the same species. Our results suggest that members of this novel species have the typical genome characteristics of members of the genus Kobuvirus and may be distributed globally in swine.

[Analysis on epidemiologic characteristics of viral diarrhea among infants in Taiyuan, Shanxi province, 2007-2008].

OBJECTIVE: To study the epidemiologic characteristics of virus-induced acute diarrhea in children under 5 years old in Taiyuan, Shanxi province. METHODS: Stool specimens and clinical data were collected from 346 inpatients with acute diarrhea from children less than 5 years old. Rotavirus-positive specimens were identified by ELASA kit. Calicivirus and astrovirus were detected by reverse transcription-polymerase chain reaction (RT-PCR). Adenovirus was done by polymerase chain reaction (PCR). RESULTS: Of the 346 specimens, the percentage of samples with Rotavirus, Calicivirus, Astrovirus, and Adenovirus was 40.8%, 7.5%, 6.4% and 3.2%. Among 141 rotavirus positive samples, serotype G1 (42.6%) was the predominant strain. More than 95% of viral diarrhea patients under hospitalization occurred among children younger than 2 years. CONCLUSION: Rotavirus is the major pathogen contributing to the acute diarrhea. The disease generally peaks at autumn/winter. The predominant rotavirus strain circulated was G1P[8].