Single-cell analyses identify distinct and intermediate states of zebrafish pancreatic islet development.

Pancreatic endocrine islets are vital for glucose homeostasis. However, the islet developmental trajectory and its regulatory network are not well understood. To define the features of these specification and differentiation processes, we isolated individual islet cells from TgBAC(neurod1:EGFP) transgenic zebrafish and analyzed islet developmental dynamics across four different embryonic stages using a single-cell RNA-seq strategy. We identified proliferative endocrine progenitors, which could be further categorized by different cell cycle phases with the G1/S subpopulation displaying a distinct differentiation potential. We identified endocrine precursors, a heterogeneous intermediate-state population consisting of lineage-primed alpha, beta and delta cells that were characterized by the expression of lineage-specific transcription factors and relatively low expression of terminally differentiation markers. The terminally differentiated alpha, beta, and delta cells displayed stage-dependent differentiation states, which were related to their functional maturation. Our data unveiled distinct states, events and molecular features during the islet developmental transition, and provided resources to comprehensively understand the lineage hierarchy of islet development at the single-cell level.

DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding.

Viral infections have caused numerous diseases and deaths worldwide. Due to the emergence of new viruses and frequent virus variation, conventional antiviral strategies that directly target viral or cellular proteins are limited because of the specificity, drug resistance and rapid clearance from the human body. Therefore, developing safe and potent antiviral agents with activity against viral infection at multiple points in the viral life cycle remains a major challenge. In this report, we propose a new modality to inhibit viral infection by fabricating DNA conjugated gold nanoparticle (DNA-AuNP) networks on cell membranes as a protective barrier. The DNA-AuNPs networks were found, via a plaque formation assay and viral titers, to have potent antiviral ability and protect host cells from human respiratory syncytial virus (RSV). Confocal immunofluorescence image analysis showed 80 ± 3.8% of viral attachment, 91.1 ± 0.9% of viral entry and 87.9 ± 2.8% of viral budding were inhibited by the DNA-AuNP networks, which were further confirmed by real-time fluorescence imaging of the RSV infection process. The antiviral activity of the networks may be attributed to steric effects, the disruption of membrane glycoproteins and limited fusion of cell membrane bilayers, all of which play important roles in viral infection. Therefore, our results suggest that the DNA-AuNP networks have not only prophylactic effects to inhibit virus attachment and entry, but also therapeutic effects to inhibit viral budding and cell-to-cell spread. More importantly, this proof-of-principle study provides a pathway for the development of a universal, broad-spectrum antiviral therapy.

Acute gastroenteritis outbreak caused by a GII.6 norovirus.

AIM: To report an acute gastroenteritis outbreak caused by a genogroup 2 genotype 6 (GII.6) strain norovirus in Shanghai, China. METHODS: Noroviruses are responsible for approximately half of all reported gastroenteritis outbreaks in many countries. Genogroup 2 genotype 4 strains are the most prevalent. Rare outbreaks caused by GII.6 strains have been reported. An acute gastroenteritis outbreak occurred in an elementary school in Shanghai in December of 2013. Field and molecular epidemiologic investigations were conducted. RESULTS: The outbreak was limited to one class in an elementary school located in southwest Shanghai. The age of the students ranged from 9 to 10 years. The first case emerged on December 10, 2013, and the last case emerged on December 14, 2013. The cases peaked on December 11, 2013, with 21 new cases. Of 45 students in the class, 32 were affected. The main symptom was gastroenteritis, and 15.6% (5/32) of the cases exhibited a fever. A field epidemiologic investigation showed the pathogen may have been transmitted to the elementary school from employees in a delicatessen via the first case student, who had eaten food from the delicatessen one day before the gastroenteritis episodes began. A molecular epidemiologic investigation identified the cause of the gastroenteritis as norovirus strain GII.6; the viral sequence of the student cases showed 100% homology with that of the shop employees. Genetic relatedness analyses showed that the new viral strain is closely related to previously reported GII.6 sequences, especially to a strain reported in Japan. CONCLUSION: This is the first report to show that norovirus strain GII.6 can cause a gastroenteritis outbreak. Thus, the prevalence of GII.6 noroviruses requires attention.

Metal-enhanced fluorescence of nano-core-shell structure used for sensitive detection of prion protein with a dual-aptamer strategy.

Metal-enhanced fluorescence (MEF) as a newly recognized technology is widespread throughout biological research. The use of fluorophore-metal interactions is recognized to be able to alleviate some of fluorophore photophysical constraints, favorably increase both the fluorophore emission intensity and photostability. In this contribution, we developed a novel metal-enhanced fluorescence (MEF) and dual-aptamer-based strategy to achieve the prion detection in solution and intracellular protein imaging simultaneously, which shows high promise for nanostructure-based biosensing. In the presence of prion protein, core-shell Ag@SiO2, which are functionalized covalently by single stranded aptamer (Apt1) of prions and Cyanine 3 (Cy3) decorated the other aptamer (Apt2) were coupled together by the specific interaction between prions and the anti-prion aptamers in solution. By adjusting shell thickness of the pariticles, a dual-aptamer strategy combined MEF can be realized by the excitation and/or emission rates of Cy3. It was found that the enhanced fluorescence intensities followed a linear relationship in the range of 0.05-0.30 nM, which is successfully applied to the detection of PrP in mice brain homogenates.

[Zebrafish as the model system to study organogenesis and regeneration].

Zebrafish (Danio rerio) is becoming more and more popular as a model organism to study vertebrate organogenesis and regeneration, taking the advantage of its extra-uterine development, transparency, strong regeneration ability and suitability for the large scale genetic screen. In the past decades, a series of useful methods have been established in zebrafish, which allows studies of important questions in developmental biology including cell fate determination, differentiation, morphogenesis, and regeneration after tissue/organ injury. Recently, using large scale mutagenesis screen and improved live imaging techniques, many questions that had not been well understood in mice have been successfully investigated in zebrafish. Therefore, we are confident that the zebrafish model system will further help decipher the cellular and molecular mechanisms of organogenesis and regeneration in the future, providing valuable references for the treatment of related clinical diseases. This review just summarized the progress in the studies of the important organs development using zebrafish, such as central nervous system(CNS), liver and pancreas, blood and vessel, recently. Meanwhile, the updated informations about the regeneration of tail fin, heart, liver, retina and so on in zebrafish were also included in this paper.

[Establishment and application of multiplex PCR for non-O157 H7 STEC virulence genes detection].

OBJECTIVE: Traditional detection approaches for non-O157 STEC are both time and labour consuming in diseases surveillance. Virulence genes detection based on multiplex PCR could not only improve the detection efficiency but also increase the accuracy. METHODS: Six virulence genes of non-O157:H7 (stx1, stx2, eae, hly, etpD, katP6) were detected by two groups of trebling PCRs. The multiplex PCRs were optimized by melting curve analysis in SYBR Green I real-time PCR. Testing result of multiplex PCR was consistent with serological testing. RESULTS: The sensitivity limits of the multiplex PCR for stx1, stx2, eaeP, etpD, katP, and hly were 10 ng/ml, 120 ng/ml, 110 ng/ml,165 ng/ml, 85 ng/ml, and 15 ng/ml, respectively, which is similar with that of single PCR. When the multiplex PCR was applied in 120 adults and 90 children diarrhea samples detection, 13 cases were detected for non-O157 positive. CONCLUSION: The method we established can be used for non-O157 STEC virulence genes detection and screening with high efficiency and accuracy.

Aptamer-mediated nanoparticle-based protein labeling platform for intracellular imaging and tracking endocytosis dynamics.

Although nanoparticles have been widely used as optical contrasts for cell imaging, the complicated prefunctionalized steps and low labeling efficiency of nanoprobes greatly inhibit their applications in cellular protein imaging. In this study, we developed a novel and general strategy that employs an aptamer not only as a recognizer for protein recognition but also as a linker for nanoreporter targeting to specifically label membrane proteins of interest and track their endocytic pathway. With this strategy, three kinds of nanoparticles, including gold nanoparticles, silver nanoparticles, and quantum dots (QDs), have been successfully targeted to the membrane proteins of interest, such as nucleolin or prion protein (PrP(C)). The following investigations on the subcellular distribution with fluorescent immunocolocalization assay indicated that PrP(C)-aptamer-QD complexes most likely internalized into cytoplasm through a classical clathrin-dependent/receptor-mediated pathway. Further single-particle tracking and trajectory analysis demonstrated that PrP(C)-aptamer-QD complexes exhibited a complex dynamic process, which involved three types of movements, including membrane diffusion, vesicle transportation, and confined diffusion, and all types of these movements were associated with distinct phases of PrP(C) endocytosis. Compared with traditional multilayer methods, our proposed aptamer-mediated strategy is simple in procedure, avoiding any complicated probe premodification and purification. In particular, the new double-color labeling strategy is unique and significant due to its superior advantages of targeting two signal reporters simultaneously in a single protein using only one aptamer. What is more important, we have constructed a general and versatile aptamer-mediated protein labeling nanoplatform that has shown great promise for future biomedical labeling and intracellular protein dynamic analysis.

[Sealing of tracheoesophageal fistula using Sigma covered mental stents].

OBJECTIVE: To explore the possibilities and efficiency of Sigma covered stents for the treatment of tracheoesophageal fistula (TEF). METHODS: Between 2006 and 2009, 10 Sigma covered mental stents had been placed by guidance of bronchoscopy and/or fluoroscopy in 8 patients with 9 tracheoesophageal fistulas. Among them, 7 fistulas were caused by cancer and 2 by mechanical damage. Seven fistulas were located in lower part of trachea and 1 each in the middle and upper parts. RESULTS: Six Y-shaped, 2 L-shaped and 2 I-shaped stents were placed respectively in 8 patients with 9 TEFs. All the TEFs were effectively sealed by the stents except for 1 massive lower and 1 upper fistulas. One mechanical fistula was cured within 1 year of stent placement. The mean survival period of all patients was 8 months. CONCLUSION: By sealing the fistula with a Sigma covered stent, severe respiratory infection may be successfully controlled in patients with TEFs with a major improvement of quality of life.

[Research on Zhejiang blood information network and management system].

This research was aimed to develop the first level blood information centralized database and real time communication network at a province area in China. Multiple technology like local area network database separate operation, real time data concentration and distribution mechanism, allopatric backup, and optical fiber virtual private network (VPN) were used. As a result, the blood information centralized database and management system were successfully constructed, which covers all the Zhejiang province, and the real time exchange of blood data was realised. In conclusion, its implementation promote volunteer blood donation and ensure the blood safety in Zhejiang, especially strengthen the quick response to public health emergency. This project lays the first stone of centralized test and allotment among blood banks in Zhejiang, and can serve as a reference of contemporary blood bank information systems in China.