Begomovirus-whitefly mutualism is achieved through repression of plant defences by a virus pathogenicity factor

Plant-mediated interactions between herbivorous arthropods and pathogens transmitted by herbivores are important determinants of the population dynamics of both types of organisms in the field. The role of plant defence in mediating these types of tripartite interactions have been recognized but rarely examined especially at the physiological and molecular levels. Our previous work shows that a worldwide invasive whitefly can establish mutualism with the begomovirus Tomato yellow leaf curl China virus (TYLCCNV) via crop plants. Here, we show that TYLCCNV and betasatellite co-infection suppresses jasmonic acid defences in the plant. Impairing or enhancing defences mediated by jasmonic acid in the plant enhances or depresses the performance of the whitefly. We further demonstrate that the pathogenicity factor βC1 encoded in the betasatellite is responsible for the initiation of suppression on plant defences and contributes to the realization of the virus-vector mutualism. By integrating ecological, mechanistic and molecular approaches, our study reveals a major mechanism of the plant-mediated mutualism between a virus and its vector. As the test plant is an important economic crop, the results also have substantial implications for developing novel strategies for management of crop viruses and the insect vectors associated with them.     

Probing the behaviors of gold nanorods in metastatic breast cancer cells based on UV-vis-NIR absorption spectroscopy

In this work, behaviors of positively-charged AuNRs in a highly metastatic tumor cell line MDA-MB-231 are examined based on UV-vis-NIR absorption spectroscopy in combination with inductively coupled plasma mass spectrometry (ICP-MS), transmission electron microscopy (TEM) and dark-field microscopic observation. It is found that characteristic surface plasmon resonance (SPR) peaks of AuNRs can be detected using spectroscopic method within living cells that have taken up AuNRs. The peak area of transverse SPR band is shown to be proportionally related to the amount of AuNRs in the cells determined with ICP-MS, which suggests a facile and real time quantification method for AuNRs in living cells. The shape of longitudinal SPR band in UV-vis-NIR spectrum reflects the aggregation state of AuNRs in the cells during the incubation period, which is proved by TEM and microscopic observations. Experimental results reveal that AuNRs are internalized by the cells rapidly; the accumulation, distribution and aggregation of AuNRs in the cells compartments are time and dose dependent. The established spectroscopic analysis method can not only monitor the behaviors of AuNRs in living cells but may also be helpful in choosing the optimum laser stimulation wavelength for anti-tumor thermotherapy.     

Identification and functional analysis of a novel von Willebrand factor mutation in a family with type 2A von Willebrand disease

von Willebrand factor (VWF) is essential for normal hemostasis. VWF gene mutations cause the hemorrhagic von Willebrand disease (VWD). In this study, a 9-year-old boy was diagnosed as type 2A VWD, based on a history of abnormal bleeding, low plasma VWF antigen and activity, low plasma factor VIII activity, and lack of plasma high-molecular-weight (HMW) VWF multimers. Sequencing analysis detected a 6-bp deletion in exon 28 of his VWF gene, which created a mutant lacking D1529V1530 residues in VWF A2 domain. This mutation also existed in his family members with abnormal bleedings but not in >60 normal controls. In transfected HEK293 cells, recombinant VWF ΔD1529V1530 protein had markedly reduced levels in the conditioned medium (42±4% of wild-type (WT) VWF, p<0.01). The mutant VWF in the medium had less HMW multimers. In contrast, the intracellular levels of the mutant VWF in the transfected cells were significantly higher than that of WT (174±29%, p<0.05), indicating intracellular retention of the mutant VWF. In co-transfection experiments, the mutant reduced WT VWF secretion from the cells. By immunofluorescence staining, the retention of the mutant VWF was identified within the endoplasmic reticulum (ER). Together, we identified a unique VWF mutation responsible for the bleeding phenotype in a patient family with type 2A VWD. The mutation impaired VWF trafficking through the ER, thereby preventing VWF secretion from the cells. Our results illustrate the diversity of VWF gene mutations, which contributes to the wide spectrum of VWD.     

Targeted delivery of chemotherapy agents using a liver cancer-specific aptamer

Background

Using antibody/aptamer-drug conjugates can be a promising method for decreasing toxicity, while increasing the efficiency of chemotherapy.

Methodology/Principal Findings

In this study, the antitumor agent Doxorubicin (Dox) was incorporated into the modified DNA aptamer TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. Cell viability tests demonstrated that the TLS11a-GC-Dox conjugates exhibited both potency and target specificity. Importantly, intercalating Dox into the modified aptamer inhibited nonspecific uptake of membrane-permeable Dox to the non-target cell line. Since the conjugates are selective for cells that express higher amounts of target proteins, both criteria noted above are met, making TLS11a-GC-Dox conjugates potential candidates for targeted delivery to liver cancer cells.

Conclusions/Significance

Considering the large number of available aptamers that have specific targets for a wide variety of cancer cells, this novel aptamer-drug intercalation method will have promising implications for chemotherapeutics in general.     

Toolbox for non-intrusive structural and functional analysis of recombinant VLP based vaccines: a case study with hepatitis B vaccine

Background

Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine.

Methodology

The epitope-specific immune reactivity of rHBsAg epitopes to a given monoclonal antibody was monitored by surface plasmon resonance (SPR) and quantitatively analyzed on rHBsAg VLPs in-solution or bound to adjuvant with a competitive enzyme-linked immunosorbent assay (ELISA). The structure of recombinant rHBsAg particles was examined by cryo transmission electron microscopy (cryoTEM) and in-solution atomic force microscopy (AFM).

Principal Findings

SPR and competitive ELISA determined relative antigenicity in solution, in real time, with rapid turn-around, and without the need of dissolving the particulate aluminum based adjuvant. These methods demonstrated the nature of the clinically relevant epitopes of HBsAg as being responsive to heat and/or redox treatment. In-solution AFM and cryoTEM determined vaccine particle size distribution, shape, and morphology. Redox-treated rHBsAg enabled 3D reconstruction from CryoTEM images – confirming the previously proposed octahedral structure and the established lipid-to-protein ratio of HBsAg particles. Results from these non-intrusive biophysical and immunochemical analyses coalesced into a comprehensive understanding of rHBsAg vaccine epitope structure and function that was important for assuring the desired epitope formation, determinants for vaccine potency, and particle stability during vaccine design, development, and manufacturing.

Significance

Together, the methods presented here comprise a novel suite of non-intrusive VLP structural and functional characterization tools for recombinant vaccines. Key VLP structural features were defined and epitope-specific antigenicity was quantified while preserving epitope integrity and particle morphology. These tools should facilitate the development of other VLP-based vaccines.     

Use of praziquantel as an adjuvant enhances protection and Tc-17 responses to killed H5N1 virus vaccine in mice

Background

H5N1 is a highly pathogenic influenza A virus, which can cause severe illness or even death in humans. Although the widely used killed vaccines are able to provide some protection against infection via neutralizing antibodies, cytotoxic T-lymphocyte responses that are thought to eradicate viral infections are lacking.

Methodology/Principal Findings

Aiming to promote cytotoxic responses against H5N1 infection, we extended our previous finding that praziquantel (PZQ) can act as an adjuvant to induce IL-17-producing CD8⁺ T cells (Tc17). We found that a single immunization of 57BL/6 mice with killed viral vaccine plus PZQ induced antigen-specific Tc17 cells, some of which also secreted IFN-γ. The induced Tc17 had cytolytic activities. Induction of these cells was impaired in CD8 knockout (KO) or IFN-γ KO mice, and was even lower in IL-17 KO mice. Importantly, the inoculation of killed vaccine with PZQ significantly reduced virus loads in the lung tissues and prolonged survival. Protection against H5N1 virus infection was obtained by adoptively transferring PZQ-primed wild type CD8⁺ T cells and this was more effective than transfer of activated IFN-γ KO or IL-17 KO CD8⁺ T cells.

Conclusions/Significance

Our results demonstrated that adding PZQ to killed H5N1 vaccine could promote broad Tc17-mediated cytotoxic T lymphocyte activity, resulting in improved control of highly pathogenic avian influenza virus infection.     

Use of a generalized additive model to investigate key abiotic factors affecting microcystin cellular quotas in heavy bloom areas of Lake Taihu

Lake Taihu is the third largest freshwater lake in China and is suffering from serious cyanobacterial blooms with the associated drinking water contamination by microcystin (MC) for millions of citizens. So far, most studies on MCs have been limited to two small bays, while systematic research on the whole lake is lacking. To explain the variations in MC concentrations during cyanobacterial bloom, a large-scale survey at 30 sites across the lake was conducted monthly in 2008. The health risks of MC exposure were high, especially in the northern area. Both Microcystis abundance and MC cellular quotas presented positive correlations with MC concentration in the bloom seasons, suggesting that the toxic risks during Microcystis proliferations were affected by variations in both Microcystis density and MC production per Microcystis cell. Use of a powerful predictive modeling tool named generalized additive model (GAM) helped visualize significant effects of abiotic factors related to carbon fixation and proliferation of Microcystis (conductivity, dissolved inorganic carbon (DIC), water temperature and pH) on MC cellular quotas from recruitment period of Microcystis to the bloom seasons, suggesting the possible use of these factors, in addition to Microcystis abundance, as warning signs to predict toxic events in the future. The interesting relationship between macrophytes and MC cellular quotas of Microcystis (i.e., high MC cellular quotas in the presence of macrophytes) needs further investigation.     

The distribution profile and oxidation states of biometals in APP transgenic mouse brain: dyshomeostasis with age and as a function of the development of Alzheimer's disease

The enrichment of transition metals in the brain and the dyshomeostasis of metals are thought to be important etiological factors for elderly people in a number of neurodegenerative diseases, including Alzheimer's disease (AD). However, the understanding of how biometals dynamically dysregulate in the stages of AD development, such as the exact time-dependent and site-dependent accumulation in the brain with AD progression, is still limited. Herein, by using the APP/V717I transgenic mouse model and age-matched mice as control, we offer distinctive in situ and quantitative images of metals (Cu, Fe, Zn and Ca) in brain sections by synchrotron radiation micro beam X-ray fluorescence (SR-μXRF). The images show that Fe and Ca increased with brain aging in both AD and control (CNT) mice, and Cu, Fe, Zn and Ca appeared significantly elevated in AD mice and showed an obvious age-dependent rise. Fe, Cu and Zn were obviously specifically enriched in the cortex and hippocampus, which were also the plaque-formation sensitive brain regions. Our results demonstrate that the enrichment of transition metals with age and metals' dyshomeostasis in specific regions may contribute together to the etiology and development of AD in elderly people. The XANES measurements of Cu and Fe show evidence that Cu may have redox properties in the AD brain.     

Current opportunities and challenges: genome-wide association studies on pigmentation and skin cancer

Genome-wide association studies (GWAS) have become a widely used approach for genetic association studies of various human traits. A few GWAS have been conducted with the goal of identifying novel loci for pigmentation traits, melanoma, and non-melanoma skin cancer. Nevertheless, the phenotype variation explained by the genetic markers identified so far is limited. In this review, we discuss the GWAS study design and its application in pigmentation and skin cancer research. Furthermore, we summarize recent developments in post-GWAS activities such as meta-analysis, pathway analysis, and risk prediction.     

Enhanced thermostability of a Rhizopus chinensis lipase by in vivo recombination in Pichia pastoris

ABSTRACT: BACKGROUND: Lipase from Rhizopus chinensis is a versatile biocatalyst for various bioconversions and has been expressed at high-level in Pichia pastoris. However, the use of R. chinensis lipase in industrial applications is restricted by its low thermostability. Directed evolution has been proven to be a powerful and efficient protein engineering tool for improvement of biocatalysts. The present work describes improvement of the thermostability of R. chinensis lipase by directed evolution using P. pastoris as the host. RESULTS: An efficient, fast and highly simplified method was developed to create a mutant gene library in P. pastoris based on in vivo recombination, whose recombination efficiency could reach 2.3 x 105 /mug DNA. The thermostability of r27RCL was improved significantly by two rounds of error-prone PCR and two rounds of DNA shuffling in P. pastoris. The S4-3 variant was found to be the most thermostable lipase, under the conditions tested. Compared with the parent, the optimum temperature of S4-3 was two degrees higher, Tm was 22 degrees higher and half-lives at 60degreesC and 65degreesC were 46- and 23- times longer. Moreover, the catalytic efficiency kcat/Km of S4-3 was comparable to the parent. Stabilizing mutations probably increased thermostability by increasing the hydrophilicity and polarity of the protein surface and creating hydrophobic contacts inside the protein. CONCLUSIONS: P. pastoris was shown to be a valuable cell factory to improve thermostability of enzymes by directed evolution and it also could be used for improving other properties of enzymes. In this study, by using P. pastoris as a host to build mutant pool, we succeeded in obtaining a thermostable variant S4-3 without compromising enzyme activity and making it a highly promising candidate for future applications at high temperatures.