Enhanced dual contrast agent,Co-doped NaYF4:Yb,Tm nanorods,for near infrared-to-near infrared upconversion luminescence and magnetic resonance imaging

Dual-modality imaging with magnetic resonance (MR) and upconversion luminescence (UCL) is a promising technique for molecular imaging in biomedical research. Multifunctional lanthanide-based nanoparticles have been widely investigated as agents for contrast enhanced MR and fluorescence imaging. However, the use of rare earth fluoride nanoparticles for dual-modality imaging of T2-weighted MR and UCL is rarely reported. We find that NaYF₄:Yb³⁺,Tm³⁺,Co²⁺ (MUC) nanorods can be applied as a high-performance dual contrast agent for both T2-weighted MR and UCL dual-modality imaging. After modification with 6-O-carboxymethyl chitosan (OCC), MUC nanorods can be endocytosed by cells without showing signs of cytotoxicity. High-quality UCL images of living cells incubated with MUC-OCC nanorods were acquired on a near-infrared (NIR) confocal microscopy under the excitation at 980 nm. Moreover, MUC-OCC nanorods display high transverse (r2) relaxivities in vitro. The application of low-dose MUC-OCC nanorods for NIR-to-NIR UCL and MR dual-modality in vivo imaging was also carried out successfully. In addition, the toxicity of MUC-OCC nanorods was evaluated by MTT assay, serological tests and histological analysis of visceral organs.     

Three Isothermal Amplification Techniques for Rapid Identification of Cladophialophora carrionii,an Agent of Human Chromoblastomycosis

In this study, we developed rapid and sensitive assays for the detection of Cladophialophora carrionii, a common agent of human chromoblastomycosis. The isothermal techniques evaluated were rolling-circle amplification (RCA), multiplex ligation-dependent probe amplification (MLPA), and loop-mediated isothermal amplification (LAMP). The probes for RCA and MLPA were designed with target sequences in the rDNA internal transcribed spacer gene (ITS) region, and LAMP primers were designed using the elongation factor 1α gene (EF1); these probes and primers specifically amplified DNA of isolates of the species. The three techniques were sufficiently specific and sensitive for discriminating target DNA of C. carrionii from that of related Cladophialophora species and other agents of chromoblastomycosis. RCA, MLPA, and LAMP are advantageous in their reliability and ease of operation compared to standard PCR and conventional methods.     

Trigger-responsive,fast-degradable poly(β-amino ester)s for enhanced DNA unpackaging and reduced toxicity

Poly(β-amino ester)s (PBAEs) represent an important class of cationic gene delivery materials which, however, suffer from uncontrolled DNA release due in part to the slow degradation of their polyester backbone. Additionally, PBAEs with high molecular weight (MW) also show considerable toxicities. In this study, we designed and developed PBAEs with trigger-responsive domains built-in polymer backbones that can be rapidly cleaved upon external UV light triggering to promote intracellular DNA release as well as reduce material toxicity. Photo-responsive PBAEs were prepared via polyaddition of (2-nitro-1,3-phenylene)bis(methylene) diacrylate and a bifunctional amine. The nitrobenzene moiety was placed in each repeating unit of the PBAE to allow fast response to external UV irradiation, and thus the ester linkers were cleaved and the polymers were degraded within several minutes upon UV irradiation. Cationic PBAEs with high MWs were able to mediate effective intracellular gene delivery, while upon UV irradiation post-transfection, enhanced DNA unpackaging and reduced material toxicity were observed, which collectively contributed to greatly improved transfection efficiencies in various mammalian cell types tested. This strategy allows precise manipulation of material toxicity and gene release profiles of PBAEs, and thus provides an effective design approach to address critical issues in non-viral gene delivery.     

Specific lipase-responsive polymer-coated gadolinium nanoparticles for MR imaging of early acute pancreatitis

Currently, available methods for diagnosis of acute pancreatitis (AP) are mainly dependent on serum enzyme analysis and imaging techniques that are too low in sensitivity and specificity to accurately and promptly diagnose AP. The lack of early diagnostic tools highlights the need to search for a highly effective and specific diagnostic method. In this study, we synthesized a conditionally activated, gadolinium-containing, nanoparticle-based MRI nanoprobe as a diagnostic tool for the early identification of AP. Gadolinium diethylenetriaminepentaacetic fatty acid (Gd-DTPA-FA) nanoparticles were synthesized by conjugation of DTPA-FA ligand and gadolinium acetate. Gd-DTPA-FA exhibited low cytotoxicity and excellent biocompatibility when characterized in vitro and in vivo studies. L-arginine induced a gradual increase in the intensity of the T1-weighted MRI signal from 1 h to 36 h in AP rat models. The increase in signal intensity was most significant at 1 h, 6 h and 12 h. These results suggest that the Gd-DTPA-FA as an MRI contrast agent is highly efficient and specific to detect early AP.     

A recombinant adenovirus expressing CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis elicits strong antigen-specific immune responses in mice

Tuberculosis (TB) is caused by an infection of Mycobacterium tuberculosis (Mtb) and remains an enormous and increasing health burden worldwide. To date, Mycobacterium bovis Bacillus Calmette Guerin (BCG) is the only licensed anti-TB vaccine worldwide, which provides an important but limited protection from the Mtb infection. The development of alternative anti-TB vaccines is therefore urgently needed. Here we report, the generation of Ad5-CEAB, a recombinant adenovirus expressing Mtb antigens of CFP10, ESAT6, Ag85A and Ag85B proteins in a form of mixture. In order to evaluate the immunogenicity of Ad5-CEAB, mice were immunized with Ad5-CEAB by intranasal instillation three times with 2-week intervals. The results demonstrated that Ad5-CEAB elicited a strong antigen-specific immune response, particularly of the Th1 immune responses that were characterized by an increased ratio of IgG2a/IgG1 and secretions of Th1 type cytokines, IFN-γ, TNF-α, IL-2 and IL-12. In addition, the Ad5-CEAB also showed an ability to enhance humoral responses with a dramatically augmented antigen-specific serum IgG. Furthermore, an elevated sIgA were also found in the bronchoalveolar lavage fluid of the immunized mice, suggesting the elicitation of mucosal immune responses. These data indicate that Ad5-CEAB can induce a broad range of antigen-specific immune responses in vivo, which provides a promising and novel route for developing anti-TB vaccines and warrants further investigation.     

Analysis of simple sequence repeats in the Gaeumannomyces graminis var. tritici genome and the development of microsatellite markers

Understanding the genetic structure of Gaeumannomyces graminis var. tritici is essential for the establishment of efficient disease control strategies. It is becoming clear that microsatellites, or simple sequence repeats (SSRs), play an important role in genome organization and phenotypic diversity, and are a large source of genetic markers for population genetics and meiotic maps. In this study, we examined the G. graminis var. tritici genome (1) to analyze its pattern of SSRs, (2) to compare it with other plant pathogenic filamentous fungi, such as Magnaporthe oryzae and M. poae, and (3) to identify new polymorphic SSR markers for genetic diversity. The G. graminis var. tritici genome was rich in SSRs; a total 13,650 SSRs have been identified with mononucleotides being the most common motifs. In coding regions, the densities of tri- and hexanucleotides were significantly higher than in noncoding regions. The di-, tri-, tetra, penta, and hexanucleotide repeats in the G. graminis var. tritici genome were more abundant than the same repeats in M. oryzae and M. poae. From 115 devised primers, 39 SSRs are polymorphic with G. graminis var. tritici isolates, and 8 primers were randomly selected to analyze 116 isolates from China. The number of alleles varied from 2 to 7 and the expected heterozygosity (He) from 0.499 to 0.837. In conclusion, SSRs developed in this study were highly polymorphic, and our analysis indicated that G. graminis var. tritici is a species with high genetic diversity. The results provide a pioneering report for several applications, such as the assessment of population structure and genetic diversity of G. graminis var. tritici.     

Genomic characterization of a bovine viral diarrhea virus 1 isolate from swine

The SD0803 strain of the bovine viral diarrhea virus (BVDV) was isolated from a piglet in China in 2008 and has been classified as a novel subgenotype of BVDV-1. To describe the molecular features of this novel subgenotype, we sequenced and characterized the complete genome of the SD0803 virus. The genome is 12,271 bp in length and contains 5′ and 3′ untranslated regions (UTRs) that flank an open reading frame (ORF) encoding a 3,898-amino-acid polypeptide. The full-length genome of the SD0803 strain shares 78.8 % to 83.3 % identity with those of other BVDV-1 strains, 70.0 % to 70.7 % identity with those of BVDV-2 strains, and less than 67.6 % identity with those of other pestiviruses. The highest level of shared identity was 83.3 % between the complete SD0803 genome and that of the ZM-95 strain of BVDV-1. Phylogenetic analysis of the 5′ UTR and the coding sequence for the N-terminal protease fragment of the SD0803 polyprotein indicated that the SD0803 virus is a member of the novel subgenotype BVDV-1q, isolates of which have been identified recently in dairy cattle and camels in China.     

The effect of temperature on the extrinsic incubation period and infection rate of dengue virus serotype 2 infection in Aedes albopictus

Dengue fever is an acute mosquito-borne viral disease caused by dengue virus (DENV). Temperature may affect the efficiency of the mosquito vectors in spreading DENV. Aedes albopictus mosquitoes were infected orally with a DENV2 suspension and incubated at different temperatures. Subsequently, DENV2 antigen was collected from salivary gland and thorax-abdomen samples on different days postinfection and tested using an immunofluorescence assay to determine the extrinsic incubation period and infection rate. As the temperature increased, the extrinsic DENV2 incubation period in Ae. albopictus gradually shortened, and infection rates showed a tendency to initially increase, followed by a subsequent decrease.