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.     

Contributions of the two accessory subunits,RNASEH2B and RNASEH2C,to the activity and properties of the human RNase H2 complex

Eukaryotic RNase H2 is a heterotrimeric enzyme. Here, we show that the biochemical composition and stoichiometry of the human RNase H2 complex is consistent with the properties previously deduced from genetic studies. The catalytic subunit of eukaryotic RNase H2, RNASEH2A, is well conserved and similar to the monomeric prokaryotic RNase HII. In contrast, the RNASEH2B and RNASEH2C subunits from human and Saccharomyces cerevisiae share very little homology, although they both form soluble B/C complexes that may serve as a nucleation site for the addition of RNASEH2A to form an active RNase H2, or for interactions with other proteins to support different functions. The RNASEH2B subunit has a PIP-box and confers PCNA binding to human RNase H2. Unlike Escherichia coli RNase HII, eukaryotic RNase H2 acts processively and hydrolyzes a variety of RNA/DNA hybrids with similar efficiencies, suggesting multiple cellular substrates. Moreover, of five analyzed mutations in human RNASEH2B and RNASEH2C linked to Aicardi-Goutières Syndrome (AGS), only one, R69W in the RNASEH2C protein, exhibits a significant reduction in specific activity, revealing a role for the C subunit in enzymatic activity. Near-normal activity of four AGS-related mutant enzymes was unexpected in light of their predicted impairment causing the AGS phenotype.     

Synthesis of Superhelical Simian Virus 40 Deoxyribonucleic Acid in Cell Lysates*.

In vivo-labeled SV40 replicating DNA molecules can be converted into covalently closed superhelical SV40 DNA (SV40(I) using a lysate of sv40-infected monkey cells containing intact nuclei. Replication in vitro occurred at one-third the in vivo rate for 30 min at 30 degrees. After 1 hour of incubation, about 54% of the replicating molecules had been converted to SV40(I), 5% to nicked, circular molecules (SV40(II), 5% to covalently closed dimers; the remainder failed to complete replication although 75% of the prelabeled daughter strands had been elongated to one-genome length. Density labeling in vitro showed that all replicating molecules had participated during DNA synthesis in vitro. Velocity and equilibrium sedimentation analysis of pulse-chased and labeled DNA using radioactive and density labels suggested that SV40 DNA synthesis in vitro was a continuation of normal ongoing DNA synthesis. Initiation of new rounds of SV40 DNA replication was not detectable.     

The dual roles of geminin during trophoblast proliferation and differentiation

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.     

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.     

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.     

ORIGIN AND BIOGEOGRAPHY OF AESCULUS L. (HIPPOCASTANACEAE): A MOLECULAR PHYLOGENETIC PERSPECTIVE

Sequences of chloroplast gene matK and internal transcribed spacers of nuclear ribosomal RNA genes were used for phylogenetic analyses of Aesculus, a genus currently distributed in eastern Asia, eastern and western North America, and southeastern Europe. Phylogenetic relationships inferred from these molecular data are highly correlated with the geographic distributions of species. The identified lineages closely correspond to the five sections previously recognized on the basis of morphology. Ancestral character-state reconstruction, a molecular clock, and fossil evidence were used to infer the origin and biogeographic history of the genus within a phylogenetic framework. Based on the molecular phylogenetic reconstruction of the genus, sequence divergence, and paleontological evidence, we infer that the genus originated during the transition from the Cretaceous to the Tertiary (~65 M.Y.B.P.) at a high latitude in eastern Asia and spread into North America and Europe as an element of the “boreotropical flora”; the current disjunct distribution of the genus resulted from geological and climatic changes during the Tertiary.