A genome walking strategy for the identification of nucleotide sequences adjacent to known regions

To identify the transposon insertion sites in a soil actinomycete, Saccharopolyspora spinosa, a genome walking approach, termed SPTA-PCR, was developed. In SPTA-PCR, a simple procedure consisting of TA cloning and a high stringency PCR, following the single primer-mediated, randomly-primed PCR, can eliminate non-target DNA fragments and obtain target fragments specifically. Using SPTA-PCR, the DNA sequence adjacent to the highly conserved region of lectin coding gene in onion plant, Allium chinense, was also cloned.     

Submillisecond Elastic Recoil Reveals Molecular Origins of Fibrin Fiber Mechanics

Fibrin fibers form the structural scaffold of blood clots. Thus, their mechanical properties are of central importance to understanding hemostasis and thrombotic disease. Recent studies have revealed that fibrin fibers are elastomeric despite their high degree of molecular ordering. These results have inspired a variety of molecular models for fibrin’s elasticity, ranging from reversible protein unfolding to rubber-like elasticity. An important property that has not been explored is the timescale of elastic recoil, a parameter that is critical for fibrin’s mechanical function and places a temporal constraint on molecular models of fiber elasticity. Using high-frame-rate imaging and atomic force microscopy-based nanomanipulation, we measured the recoil dynamics of individual fibrin fibers and found that the recoil was orders of magnitude faster than anticipated from models involving protein refolding. We also performed steered discrete molecular-dynamics simulations to investigate the molecular origins of the observed recoil. Our results point to the unstructured αC regions of the otherwise structured fibrin molecule as being responsible for the elastic recoil of the fibers.     

Potato purple top phytoplasma‐induced disruption of gibberellin homeostasis in tomato plants

Phytoplasmas are phloem‐inhabiting, cell wall‐less bacteria that cause numerous plant diseases worldwide. Plants infected by phytoplasmas often exhibit various symptoms indicative of hormonal imbalance. In this study, we investigated the effects of potato purple top (PPT) phytoplasma infection on gibberellin homeostasis in tomato plants. We found that PPT phytoplasma infection caused a significant reduction in endogenous levels of gibberellic acid (GA₃). The decrease in GA₃ content in diseased plants was correlated with down regulation of genes responsible for biosynthesis of bioactive GAs ( GA20ox1 and GA3ox1) and genes involved in formation of GA precursors [geranyl diphosphate synthase (GPS) and copalyldiphosphate synthase (CPS)]. Exogenous application of GA₃ at 200 µmol L^?1 was able to restore the GA content in infected plants to levels comparable to those in healthy controls, and to attenuate the characteristic ‘big bud’ symptoms induced by the phytoplasma. The interesting observation that PPT phytoplasma‐infected plants had prolonged low expression of key GA biosynthesis genes GA20ox1 and GA3ox1 under GA deficiency conditions led us to hypothesise that there was a diminished sensitivity of the GA metabolism feedback regulation, especially GA biosynthesis negative feedback regulation, in those affected plants, and such diminished sensitization in early stages of infection may represent a central element of the phytoplasma‐induced disruption of GA homeostasis and pathogenesis.     

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.     

Improved synchronous light scattering method for measuring baker’s yeast biomass using thickened suspensions

Measuring yeast biomass is important in the processes of microbial fermentations. It has been demonstrated that synchronous light scattering (SLS) signals could be applied for the quantification of model bioparticles such as Saccharomyces cerevisiae. In this study, an improved synchronous light scattering method was developed for yeast biomass estimation. The settlement of yeast cells during SLS signals measuring process was studied, and hydrolysis anionic polyacrylamide was added into yeast suspensions to increase the stability of the cells in liquid environment. By simultaneously scanning both the excitation and emission monochromators of a common spectrofluorometer with same starting excitation and emission wavelength (namely, ?λ = 0), the SLS intensity was found to be proportional to the yeast concentration in the range from 0 to 4.9 × 10⁶ cell/mL (R ² = 0.9907), the detection limit is 8.1 × 10³ cell/mL. The developed method exhibited good stability and sensitivity in the recovery test and growth curve drawing process, demonstrating the potential of the method in practical application of biomass estimation.     

Grape seed proanthocyanidin extracts prevent high glucose-induced endothelia dysfunction via PKC and NF-κB inhibition

In our study, it has been detected in vivo and in vitro that GSPE reversed high glucose-induced the increase of ICAM-1 and VCAM-1. It is shown that by western blotting detection, GSPE significantly inhibited the activation of NF-κB induced by high glucose while there was significant decrease of the expression of PKC with GSPE intervention. By adding the NF-κB blocker PDTC and the PKC inhibitor peptide 19–31(10^?6 M), no significant difference was found in the levels of VCAM-1 and ICAM-1 among GSPE group, the PKC inhibitor peptide 19–31-added GSPE group and the PDTC-added GSPE group. So the conclusion could be drawn that PKC inhibition must be involved in GSPE decreasing the level of ICAM-1 and VCAM-1.We proved for the first time that GSPE prevented high glucose-induced the increase of ICAM-1 and VCAM-1 by PKC and NF-κB inhibition. These findings show a novel mechanism of the action GSPE preventing endothelial dysfunction, which may have clinical application values.     

Potential antitumor mechanisms of phenothiazine drugs

In this study, three kinds of phenothiazine drugs were analyzed to explore their potential antitumor mechanisms. First, target proteins that could interact with chlorpromazine, fluphenazine and trifluoperazine were predicted. Then, the target proteins of the three drugs were intersected. Cell signaling pathway enrichment and related disease enrichment were conducted for the intersected proteins to extract the enrichment categories associated with tumors. By regulation network analysis of the protein interactions, the mechanisms of action of these target proteins in tumor tissue were clarified, thus confirming the potential antitumor mechanisms of the phenothiazine drugs. The final results of cell signaling pathway enrichment and related disease enrichment showed that the categories with the highest score were all found in tumors. Target proteins belonging to the tumor category included signaling pathway members such as Wnt, MAPK and retinoic acid receptor. Moreover, another target protein, MAPK8, could indirectly act on target proteins CDK2, IGF1R, GSK3B, RARA, FGFR2 and MAPK10, thereby affecting tumor cell division and proliferation. Therefore, phenothiazine drugs may have potential antitumor effects, and tumor-associated target proteins play important roles in the process of cell signaling transduction cascades.