Crystal Structure of the Hendra Virus Attachment G Glycoprotein Bound to a Potent Cross-Reactive Neutralizing Human Monoclonal Antibody

The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.     

KSHV MicroRNAs Mediate Cellular Transformation and Tumorigenesis by Redundantly Targeting Cell Growth and Survival Pathways

Kaposi''s sarcoma-associated herpesvirus (KSHV) is causally linked to several human cancers, including Kaposi''s sarcoma, primary effusion lymphoma and multicentric Castleman''s disease, malignancies commonly found in HIV-infected patients. While KSHV encodes diverse functional products, its mechanism of oncogenesis remains unknown. In this study, we determined the roles KSHV microRNAs (miRs) in cellular transformation and tumorigenesis using a recently developed KSHV-induced cellular transformation system of primary rat mesenchymal precursor cells. A mutant with a cluster of 10 precursor miRs (pre-miRs) deleted failed to transform primary cells, and instead, caused cell cycle arrest and apoptosis. Remarkably, the oncogenicity of the mutant virus was fully restored by genetic complementation with the miR cluster or several individual pre-miRs, which rescued cell cycle progression and inhibited apoptosis in part by redundantly targeting IκBα and the NF-κB pathway. Genomic analysis identified common targets of KSHV miRs in diverse pathways with several cancer-related pathways preferentially targeted. These works define for the first time an essential viral determinant for KSHV-induced oncogenesis and identify NF-κB as a critical pathway targeted by the viral miRs. Our results illustrate a common theme of shared functions with hierarchical order among the KSHV miRs.     

Identification of methylated flavonoid regioisomeric metabolites using enzymatic semisynthesis and liquid chromatography-tandem mass spectrometry

Discrimination of isomeric methylated metabolites is an important step toward identifying genes responsible for methylation, but presents substantial challenges because authentic standards are often unavailable and mass spectra of isomers have been considered indistinguishable. In this report, an approach is described for identifying methyl group positions in multiply methylated flavonoid metabolites using combinations of tandem mass spectrometry, liquid chromatography retention, and site-selective methylation by recombinant O-methyltransferases from Solanum habrochaites LA1777. The basis for observed fragment ions in tandem mass spectra of multiply methylated myricetin was further established using enzymatic incorporation of deuterium-labeled methyl groups using S-adenosylmethionine-d ₃ as precursor.     

Flow infusion electrospray ionisation mass spectrometry for high throughput,non-targeted metabolite fingerprinting: a review

Producing a comprehensive overview of the chemical content of biologically-derived material is a major challenge. Apart from ensuring adequate metabolome coverage and issues of instrument dynamic range, mass resolution and sensitivity, there are major technical difficulties associated with data pre-processing and signal identification when attempting large scale, high-throughput experimentation. To address these factors direct infusion or flow infusion electrospray mass spectrometry has been finding utility as a high throughput metabolite fingerprinting tool. With little sample pre-treatment, no chromatography and instrument cycle times of less than 5 min it is feasible to analyse more than 1,000 samples per week. Data pre-processing is limited to aligning extracted mass spectra and mass-intensity matrices are generally ready in a working day for a month’s worth of data mining and hypothesis generation. ESI-MS fingerprinting has remained rather qualitative by nature and as such ion suppression does not generally compromise data information content as originally suggested when the methodology was first introduced. This review will describe how the quality of data has improved through use of nano-flow infusion and mass-windowing approaches, particularly when using high resolution instruments. The increasingly wider availability of robust high accurate mass instruments actually promotes ESI-MS from a merely fingerprinting tool to the ranks of metabolite profiling and combined with MS/MS capabilities of hybrid instruments improved structural information is available concurrently. We summarise current applications in a wide range of fields where ESI-MS fingerprinting has proved to be an excellent tool for “first pass” metabolome analysis of complex biological samples. The final part of the review describes a typical workflow with reference to recently published data to emphasise key aspects of overall experimental design.     

Cancer cachexia’s metabolic signature in a murine model confirms a distinct entity

Despite recent consensus definitions, lack of specific biomarkers remains a hurdle towards a more accurate and efficient diagnosis of cancer cachexia, distinguishing cachexia as a separate entity from other wasting syndromes. In a previous pilot study, we have shown that cancer-cachectic mice have a unique metabolic fingerprint with distinct glucose and lipid alterations compared to healthy controls. Further metabolomics studies were carried out to investigate differences in metabolic profiles of cancer-cachectic mice to tumor-bearing non-cachectic mice, calorie-restricted mice, and surgically treated cancer-cachectic mice. CD2F1 mice were divided into: (1) Cachexia Group received cachexia-inducing C26 undifferentiated colon carcinoma cells; (2) Tumor-Burden Group received, non-cachectic, P388 lymphoma cells; (3) Caloric-Restriction Group, remaining cancer-free, but subjected to caloric-restriction; (4) Surgery Group, similar to Cachexia Group, but tumors resected mid-experiment; and (5) Control Group aged intact. Baseline, mid-experiment and final serum samples were collected for ¹H NMR spectroscopic analysis. After data reduction, unsupervised principal component analysis and orthogonal projections to latent structures analyses demonstrate that the unique metabolic fingerprint is independent of tumor-burden and distinct from profiles of caloric-restriction and aging. Hyperlipidemia, hyperglycemia, and reduced branched-chain amino acids distinguish cachexia from other groups. Furthermore, the profile of surgically treated mice differs from that of cachectic mice, reverting to a profile more congruent with healthy controls indicating cachexia is amenable to correction where surgical cure is possible. That metabolomic analysis of murine serum is able to differentiate cachexia from tumor-burden and caloric-restriction warrants similar translational investigations in patients to explore cancer cachexia’s unique biomarkers.     

Population structure and distribution patterns of the rare and endangered Ardisia violacea (Myrsinaceae)

Ardisia violacea (Myrsinaceae), one of China’s rare and endangered species, occurs only in Zhejiang and Taiwan provinces. We examined the population dynamics of this species to reveal details related to the development of populations. Assembling intensity indices were used, such as V/m, K, m^*, PAI, CA and I. Age class structure and the time-specific life tables were analyzed by the contiguous grid quadrate method. The population structures and distribution patterns of A. violacea in different habitats, age classes and quadrat scales are discussed and the life parameters such as the survival curve, mortality curve and life expectancy were estimated. The results show the age class distribution of the entire population generally fits a reverse “J” type curve, suggesting a growing population. The population spatial distribution pattern was clumped in all three habitats, especially in the age classes I–III, and had a uniform distribution in the age classes VII and IX. The plants mainly occurred at microsites with generally adequate light, water, nutrients and air. Micro-environmental factors and human factors can also have a great impact. In the age class structure pattern, the individual percentage of the first three age classes accounted for 71.19% in H1 (habitat 1), 68.18% in H2 (habitat 2) and 88.24% in H3 (habitat 3), which indicated the number of individuals in the first three age classes were larger than any other age classes. The population size peaked in the age classes III and X and only 13.1% individuals survive age class III to enter class IV. As a result, fewer middle and advanced aged individuals were found. Age class IV had the highest life expectancy for A. violacea. The survival curve approached to Deevy-II. The spatial distribution pattern of A. violacea populations was decided mainly by the interaction of biological and ecological characteristics of its environment, habitat heterogeneities, microenvironments and human disturbance.     

Phylogenetic diversity of endolithic bacteria in Bole granite rock in Xinjiang

The endolithic environment is a ubiquitous microbial habitat for microorganisms, such as lichens, Cyanobacteria and fungi, and it provides mineral nutrients and growth surfaces. In extremely environments, such as hot and cold desert, endolithic communities are often the main form of life. More recently, endolithic microbial communities have been observed inhabiting a variety of rock types ranging from hard granite to porous rocks such as basalt, dolomite, limestone, sandstone and granites. Regardless of geographic location and rock type, each of these habitats is characterized by a subsurface microclimate that prevents endolithic microorganisms growth. Photosynthesis-based endolithic microbial communities commonly inhabit the outer millimeters to centimeters of rocks exposed to the surface. The ability to fix carbon dioxide and in some cases atmospheric dinitrogen, gives the Cyanobacteria a clear competitive advantage over heterotrophic bacteria, so it is been called the main primary producer. Light quality and intensity appear to be the main determinant of the maximum depth to which growth occurs in endolithic phototrophic communities. Valleys of Fantastic Rocks in Bole is close to Alashankou Port of Xinjiang which belongs to extreme continental climate. In order to investigate the structure, composition and diversity of endolithic bacterial community in exposed granitic porphyry in the Valleys of Fantastic Rocks, environmental DNA was directly extracted from granite rock, the 16S rRNA genes were amplified from the total DNA by PCR with bacterial-specific primers, and an endolithic bacterial clone library was constructed. Positive clones were randomly selected from the library and identified by Restriction Fragment Length Polymorphism (RFLP). The unique rRNA types clones were sequenced, analysised and then constructed phylogenetic tree. In total, 129 positive clones were screened and grouped into 46 operational taxonomic unites (OTUs). The clone coverage C value was 89.15%, indicating that most of the estimated endolithic bacterial diversity was sampled. BLAST analysis indicated that 46 OTUs were divided into seven phyla (Acidobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Planctomycetes, Proteobacteria) and five unknown groups. Cyanobacteria (43%), especially the Gp I, form the functional basis for an endolithic bacteria community which contain a wide spectrum species of chemotrophic bacteria (33%) with mainly Actinobacteria, α-Proteobacteria, Acidobacteria. Additionally, most clones that derived from the endolithic bacteria clone library showed high similarity to the sequence deposited in GenBank database with 97%–99%. Besides, 35% of the clones showed less than 97% of sequence similarity, of which 12% sequences were affiliated to genus Rubrobacter. The results suggested that endolithic bacteria in Valleys of Fantastic Rocks in Xinjiang were highly diverse in species richness, and maybe have a diversity of potential novel species and lineages.     

TGF-β1 Regulation of Estrogen Production in Mature Rat Leydig Cells

Background

Besides androgens, estrogens produced in Leydig cells are also crucial for mammalian germ cell differentiation. Transforming growth factor-β1 (TGF-β1) is now known to have multiple effects on regulation of Leydig cell function. The objective of the present study is to determine whether TGF-β1 regulates estradiol (E₂) synthesis in adult rat Leydig cells and then to assess the impact of TGF-β1 on Cx43-based gap junctional intercellular communication (GJIC) between Leydig cells.

Methodology/Principal Findings

Primary cultured Leydig cells were incubated in the presence of recombinant TGF-β1 and the production of E₂ as well as testosterone (T) were measured by RIA. The activity of P450arom was addressed by the tritiated water release assay and the expression of Cyp19 gene was evaluated by Western blotting and real time RT-PCR. The expression of Cx43 and GJIC were investigated with immunofluorescence and fluorescence recovery after photo-bleaching (FRAP), respectively. Results from this study show that TGF-β1 down-regulates the level of E₂ secretion and the activity of P450arom in a dose-dependent manner in adult Leydig cells. In addition, the expression of Cx43 and GJIC was closely related to the regulation of E₂ and TGF-β1, and E₂ treatment in turn restored the inhibition of TGF-β1 on GJIC.

Conclusions

Our results indicate, for the first time in adult rat Leydig cells, that TGF-β1 suppresses P450arom activity, as well as the expression of the Cyp19 gene, and that depression of E₂ secretion leads to down-regulation of Cx43-based GJIC between Leydig cells.