The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.     

Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

Projecting terrestrial biodiversity intactness with GLOBIO 4

Scenario‐based biodiversity modelling is a powerful approach to evaluate how possible future socio‐economic developments may affect biodiversity. Here, we evaluated the changes in terrestrial biodiversity intactness, expressed by the mean species abundance (MSA) metric, resulting from three of the shared socio‐economic pathways (SSPs) combined with different levels of climate change (according to representative concentration pathways [RCPs]): a future oriented towards sustainability (SSP1xRCP2.6), a future determined by a politically divided world (SSP3xRCP6.0) and a future with continued global dependency on fossil fuels (SSP5xRCP8.5). To this end, we first updated the GLOBIO model, which now runs at a spatial resolution of 10 arc‐seconds (~300 m), contains new modules for downscaling land use and for quantifying impacts of hunting in the tropics, and updated modules to quantify impacts of climate change, land use, habitat fragmentation and nitrogen pollution. We then used the updated model to project terrestrial biodiversity intactness from 2015 to 2050 as a function of land use and climate changes corresponding with the selected scenarios. We estimated a global area‐weighted mean MSA of 0.56 for 2015. Biodiversity intactness declined in all three scenarios, yet the decline was smaller in the sustainability scenario (?0.02) than the regional rivalry and fossil‐fuelled development scenarios (?0.06 and ?0.05 respectively). We further found considerable variation in projected biodiversity change among different world regions, with large future losses particularly for sub‐Saharan Africa. In some scenario‐region combinations, we projected future biodiversity recovery due to reduced demands for agricultural land, yet this recovery was counteracted by increased impacts of other pressures (notably climate change and road disturbance). Effective measures to halt or reverse the decline of terrestrial biodiversity should not only reduce land demand (e.g. by increasing agricultural productivity and dietary changes) but also focus on reducing or mitigating the impacts of other pressures.     

Bioprospecting the African Renaissance: The new value of <Emphasis Type="Italic">muthi</Emphasis> in South Africa

This article gives an overview of anthropological research on bioprospecting in general and of available literature related to bioprospecting particularly in South Africa. It points out how new insights on value regimes concerning plant-based medicines may be gained through further research and is meant to contribute to a critical discussion about the ethics of Access and Benefit Sharing (ABS). In South Africa, traditional healers, plant gatherers, petty traders, researchers and private investors are assembled around the issues of standardization and commercialization of knowledge about plants. This coincides with a nation-building project which promotes the revitalization of local knowledge within the so called African Renaissance. A social science analysis of the transformation of so called Traditional Medicine (TM) may shed light onto this renaissance by tracing social arenas in which different regimes of value are brought into conflict. When medicinal plants turn into assets in a national and global economy, they seem to be manipulated and transformed in relation to their capacity to promote health, their market value, and their potential to construct new ethics of development. In this context, the translation of socially and culturally situated local knowledge about muthi into global pharmaceuticals creates new forms of agency as well as new power differentials between the different actors involved.     

Codon-anticodon interaction in tRNAPhe: II. A nuclear magnetic resonance study of the binding of the codon UUC

The effect of binding of the codon UUC to yeast tRNA^Phe was investigated by means of n.m.r.² spectroscopy and analytical ultracentrifugation. Binding of UUC to the transfer RNA anticodon tends to promote the aggregation of tRNA molecules; this is manifest from a line broadening in the n.m.r. experiments as well as from an increase in s_20,w the ultracentrifuge experiments. Such an aggregation of tRNA molecules was not observed upon addition of different oligonucleotides, as described in the accompanying paper. In addition to the general broadening observed in the n.m.r. spectra, specific resonances in the methyl proton spectrum as well as in the hydrogen-bonded proton spectrum are broadened or shifted upon binding of UUC.These results are explained on the basis of the premise that two different tRNA-UUC complexes can exist in solution. It is suggested that the binding of UUC tends to promote a disruption of the m⁷G₄₆ · m²₂G₂₂ base-pair and its neighbouring base-pairs.In studying the binding of U-U-U-U to yeast tRNA^Phe no resonances of protons hydrogen-bonded between the oligonucleotide and the tRNA could be detected at low temperatures. This indicates, that at these temperatures the lifetime of the tRNA-U-U-U-U complex is substantially shorter than the lifetime of the other tRNA-oligonucleotide complexes studied in this and the accompanying paper under these conditions.     

Solution structure of the single-stranded DNA binding protein of the filamentous Pseudomonas phage Pf3: similarity to other proteins binding to single-stranded nucleic acids.

The three-dimensional structure of the homodimeric single-stranded DNA binding protein encoded by the filamentous Pseudomonas bacteriophage Pf3 has been determined using heteronuclear multidimensional NMR techniques and restrained molecular dynamics. NMR experiments and structure calculations have been performed on a mutant protein (Phe36 --> His) that was successfully designed to reduce the tendency of the protein to aggregate. The protein monomer is composed of a five-stranded antiparallel beta-sheet from which two beta-hairpins and a large loop protrude. The structure is compared with the single-stranded DNA binding protein encoded by the filamentous Escherichia coli phage Ff, a protein with a similar biological function and DNA binding properties, yet quite different amino acid sequence, and with the major cold shock protein of Escherichia coli, a single-stranded DNA binding protein with an entirely different sequence, biological function and binding characteristics. The amino acid sequence of the latter is highly homologous to the nucleic acid binding domain (i.e. the cold shock domain) of proteins belonging to the Y-box family. Despite their differences in amino acid sequence and function, the folds of the three proteins are remarkably similar, suggesting that this is a preferred folding pattern shared by many single-stranded DNA binding proteins.     

31P saturation transfer spectroscopy predicts differential intracellular macromolecular association of ATP and ADP in skeletal muscle

The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo ³¹P magnetic resonance spectroscopy using magnetization transfer. However, magnetization transfer effects seen on the signals of ATP also could arise from intramolecular cross-relaxation. This relaxation process carries information on the association state of ATP in the cell. To disentangle contributions of chemical exchange and cross-relaxation to magnetization transfer effects seen in ³¹P magnetic resonance spectroscopy of skeletal muscle, we performed saturation transfer experiments on wild type and double-mutant mice lacking the cytosolic muscle creatine kinase and adenylate kinase isoforms. We find that cross-relaxation, observed as nuclear Overhauser effects (NOEs), is responsible for magnetization transfer between ATP phosphates both in wild type and in mutant mice. Analysis of ³¹P relaxation properties identifies these effects as transferred NOEs, i.e. underlying this process is an exchange between free cellular ATP and ATP bound to slowly rotating macromolecules. This explains the β-ATP signal decrease upon saturation of the γ-ATP resonance. Although this usually is attributed to β-ADP ↔ β-ATP phosphoryl exchange, we did not detect an effect of this exchange on the β-ATP signal as expected for free [ADP], derived from the creatine kinase equilibrium reaction. This indicates that in resting muscle, conditions prevail that prevent saturation of β-ADP spins and puts into question the derivation of free [ADP] from the creatine kinase equilibrium. We present a model, matching the experimental result, for ADP ↔ ATP exchange, in which ADP is only transiently present in the cytosol.