Mesenchymal stem cells contribute to endogenous FVIII:c production

Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative‐RT‐PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord‐derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Aerobic transformation of zinc into metal sulfide by photosynthetic microorganisms

Industrial activity over the last two centuries has increased heavy metal contamination worldwide, leading to greater human exposure. Zinc is particularly common in industrial effluents and although an essential nutrient, it is highly toxic at elevated concentrations. Photoautotrophic microbes hold promise for heavy metal bioremediation applications because of their ease of culture and their ability to produce sulfide through metabolic processes that in turn are known to complex with the metal ion, Hg(II). The green alga Chlamydomonas reinhardtii, the red alga Cyanidioschyzon merolae, and the cyanobacterium Synechococcus leopoliensis were all able to synthesize sulfide and form zinc sulfide when exposed to Zn(II). Supplementation of their respective media with sulfite and cysteine had deleterious effects on growth, although ZnS still formed in Cyanidioschyzon cells to the same extent as in unsupplemented cells. The simultaneous addition of sulfate and Zn(II) had similar effects to that of Zn(II) alone in all three species, whereas supplying sulfate prior to exposure to Zn(II) enhanced metal sulfide production. The coupled activities of serine acetyltransferase and O-acetylserine(thiol)lyase (SAT/OASTL) did not increase significantly in response to conditions in which enhanced ZnS formation occurred; sulfate added prior to and simultaneously with Zn(II). However, even low activity could provide sufficient sulfate assimilation over this relatively long-term study. Because the extractable activity of cysteine desulfhydrase was elevated in cells that produced higher amounts of zinc sulfide, cysteine is the probable source of the sulfide in this aerobic process.     

Vps1 in the late endosome-to-vacuole traffic

Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1Δ cells accumulated FM4-64 to a greater extent than wild-type (WT) cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1’s implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.     

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.     

Impacts of inundation and drought on eukaryote biodiversity in semi‐arid floodplain soils

Floodplain ecosystems are characterized by alternating wet and dry phases and periodic inundation defines their ecological character. Climate change, river regulation and the construction of levees have substantially altered natural flooding and drying regimes worldwide with uncertain effects on key biotic groups. In southern Australia, we hypothesized that soil eukaryotic communities in climate change affected areas of a semi‐arid floodplain would transition towards comprising mainly dry‐soil specialist species with increasing drought severity. Here, we used 18S rRNA amplicon pyrosequencing to measure the eukaryote community composition in soils that had been depleted of water to varying degrees to confirm that reproducible transitional changes occur in eukaryotic biodiversity on this floodplain. Interflood community structures (3 years post‐flood) were dominated by persistent rather than either aquatic or dry‐specialist organisms. Only 2% of taxa were unique to dry locations by 8 years post‐flood, and 10% were restricted to wet locations (inundated a year to 2 weeks post‐flood). Almost half (48%) of the total soil biota were detected in both these environments. The discovery of a large suite of organisms able to survive nearly a decade of drought, and up to a year submerged supports the concept of inherent resilience of Australian semi‐arid floodplain soil communities under increasing pressure from climatic induced changes in water availability.     

Liver in a dish

There exists a worldwide shortage of donor livers for transplant. This may not pose a problem in the future, as Takebe et al. have recently grown functional “liver buds” from stem cells in a dish.Since the discovery of human induced pluripotent stem cells (hiPSCs), the promise of generating organs from patients'' iPSCs has received considerable attention as an alternative to donor organ transplantation. Over the past few years, much progress has been made in the differentiation of various somatic cell types from human pluripotent stem cells (hPSCs). However, only a limited number of reports have described the generation of three-dimensional organoids from human stem cells in vitro, including the optic cup¹, the pituitary epithelium², and from adult stem cells — the gut epithelium³. These experimental systems share several common features: 1) they all begin with ES cells or adult stem cells, 2) the cells grow as floating aggregates, and 3) all three organoids (optic cup, pituitary epithelium, and gut crypt) are epithelial structures⁴. In addition, one particularly unexpected finding has emerged from each of these experiments, namely that a high level of self-organization seems to play a substantial role in establishing local tissue architecture and assembly of the resulting organoid.Despite these remarkable examples of organogenesis in vitro, the likelihood of growing a complex vascularized organ in dish, such as liver, has seemed less plausible. Takebe et al.⁵ have made the implausible possible by focusing on the first steps of organogenesis, namely the cellular interactions that occur during liver bud development. The earliest stage of liver organogenesis involves the outgrowth of a group of endodermal and mesenchymal cells from the posterior foregut that soon thereafter become vascularized to form a liver bud. During these morphogenetic changes, a key element to the formation of a liver bud is the orchestration of signals between three types of cells: liver, mesenchymal and endothelial progenitors. Takebe et al. posited that they might be able to recapitulate liver bud formation in vitro by mixing hepatic endoderm cells together with endothelial and mesenchymal cells. To test this idea, they prepared hepatic endoderm cells (hiPSC-HEs) from hiPSCs by directed differentiation, and then co-cultured them with human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (MSCs). Two days later, the cells had self-assembled into a 5-mm-long, three-dimensional tissue that was reminiscent of “liver bud” structures in vivo. To further mature these hiPSC-derived “liver buds” (hiPSC-LBs), they transplanted them into immune-compromised mice where the hiPSC-LBs connected with the host vasculature within 48 h and formed functional vascular networks similar in density and morphology to those of human adult livers. Transplanted hiPSC-LBs started functioning about 10 days later, producing human albumin and metabolizing drugs in a similar fashion to human liver. Perhaps most remarkably, Takebe et al. demonstrated that these hiPSC-LBs could rescue liver function when transplanted to mice with liver failure.The differences between Takebe and his colleagues'' study and other studies designed to reproduce organogenesis in vitro are that they started with several different cell types; the cells were grown initially in a two-dimensional petri dish; and the result was a solid liver organoid that can be vascularized and function after transplantation. For many, the most striking finding is the high level of self-organization in this experimental differentiation system. By analogy, it is equivalent to delivering all of the materials necessary to build a house to a construction site and returning several days later to find a fully assembled home. Clearly the principles of self-organization and self-assembly are playing much more profound roles during differentiation than we previously thought and it is likely what has been reported by Takebe et al. represents only the tip of the iceberg. One takeaway from the way that Takebe and his colleagues'' tackled the problem of in vitro organogenesis may be their focus on the earliest processes in organ development, as it is likely to identify the right combination of cell types for organogenesis to proceed. Nonetheless, this study has raised several new questions. How does self-organization and self-assembly occur in vitro? What is the molecular logic of this process? How can we manipulate a self-organizing system so that we might guide it in the direction we want it to go? And ultimately, could we use a similar strategy to produce other complex solid organs in vitro, e.g., lung, kidney, and pancreas?As summarized by Takebe et al., this study demonstrates a “proof-of-concept” that “organ-bud transplantation provides a promising new approach to study regenerative medicine”. However, a significant amount of work will be required before these findings can be translated into a therapy. First, these little liver buds do not form a complete adult liver. They are missing a number of critical cell types, chief among them biliary epithelial cells and thus bile ducts. How to produce a fully functional liver remains a challenge. Second, in order to translate these findings into human therapies, a key step will be to scale this process so that one can produce a liver bud large enough for transplantation into humans. Of course, there is always the question about safety when it comes to stem cell-based therapies. Undifferentiated stem cells left in transplants tend to form tumors and the use of oncogenes for iPS reprogramming needs to be resolved before iPS cells can be considered for human therapy. Despite the reality that clinical therapies based on this report remain a distant promise, it is inspirational to consider how quickly the field is moving and exciting to speculate about what might come next. If one considers that a drug has been identified to specifically eliminate pluripotent but not differentiated hPSCs⁶ and that a recent report showed that pluripotent stem cells could be induced from mouse somatic cells by using only small molecules⁷, we may have good reason to believe that one day in the not too distant future we could grow patient-customized organs for transplantation (Figure 1).Open in a separate windowFigure 1This figure outlines the strategy of generating organs from patients'' iPSCs as an alternative to transplantation. Patient-derived pluripotent stem cells (iPSCs) can be differentiated in vitro to desired cell types. As demonstrated by Takebe et al.⁵, different cell types can be co-cultured in dish to recapitulate the earliest process of organogenesis and form three-dimensional organ buds. These in vitro produced organ buds could be used for transplantation in the future.     

Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border (RB) end of the T-DNA is largely preserved whereas the left border (LB) end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61 % of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67 % of T-DNA integrations are integrations at a single chromosomal site and 31 % of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.     

Simulated impact of the renewable fuels standard on US Conservation Reserve Program enrollment and conversion

A socioeconomic model is used to estimate the land‐use implications on the U.S. Conservation Reserve Program from potential increases in second‐generation biofuel production. A baseline scenario with no second‐generation biofuel production is compared to a scenario where the Renewable Fuels Standard (RFS2) volumes are met by 2022. We allow for the possibility of converting expiring CRP lands to alternative uses such as conventional crops, dedicated second‐generation biofuel crops, or harvesting existing CRP grasses for biomass. Results indicate that RFS2 volumes (RFS2‐v) can be met primarily with crop residues (78% of feedstock demand) and woody residues (19% of feedstock demand) compared with dedicated biomass (3% of feedstock demand), with only minimal conversion of cropland (0.27 million hectares, <1% of total cropland), pastureland (0.28 million hectares of pastureland, <1% of total pastureland), and CRP lands (0.29 million hectares of CRP lands, 3% of existing CRP lands) to biomass production. Meeting RFS2 volumes would reduce CRP re‐enrollment by 0.19 million hectares, or 4%, below the baseline scenario where RFS2 is not met. Yet under RFS2‐v scenario, expiring CRP lands are more likely to be converted to or maintain perennial cover, with 1.78 million hectares of CRP lands converting to hay production, and 0.29 million hectares being harvested for existing grasses. A small amount of CRP is harvested for existing biomass, but no conversion of CRP to dedicated biomass crops, such as switchgrass, are projected to occur. Although less land is enrolled in CRP under RFS2‐v scenario, total land in perennial cover increases by 0.15 million hectares, or 2%, under RFS2‐v. Sensitivity to yield, payment and residue retention assumptions are evaluated.