High-throughput identification of refolding conditions for LXRbeta without a functional assay

The expression of human genes in bacteria is often one of the most efficient systems for generating proteins for drug discovery efforts. However, expression of mammalian cDNAs in Escherichia coli often results in the production of protein that is insoluble and misfolded and thus requires the development of a successful refolding procedure to generate active protein. To accelerate the process of developing protein refolding protocols, we have developed a semi-automated screening and assay system that utilizes an incomplete factorial approach to sample a large "space" of refolding conditions based on parameters known to influence protein stability and solubility. Testing of these conditions is performed readily in a 96-well plate format with minimal sample manipulation. The folded protein is resolved and detected using an HPLC equipped with a mini-column and a highly sensitive fluorescence detector. This simple method requires only a small amount of protein for the entire screen (<1 mg), and most importantly, a functional assay is not required to assess the refolding yields. Here, we validate the utility of this screening system using two model proteins, IL13 and MMP13, and demonstrate its successful application to the refolding of our target protein, the ligand-binding domain of rat liver X receptor beta.     

Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers – OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 – in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3–5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.     

Fluorescent dye ProteoStat to detect and discriminate intracellular amyloid‐like aggregates in Escherichia coli

The formation of amyloid aggregates is linked to the onset of an increasing number of human disorders. Thus, there is an increasing need for methodologies able to provide insights into protein deposition and its modulation. Many approaches exist to study amyloids in vitro, but the techniques available for the study of amyloid aggregation in cells are still limited and non‐specific. In this study we developed a methodology for the detection of amyloid‐like aggregates inside cells that discriminates these ordered assemblies from other intracellular aggregates. We chose bacteria as model system, since the inclusion bodies formed by amyloid proteins in the cytosol of bacteria resemble toxic amyloids both structurally and functionally. Using confocal microscopy, fluorescence spectroscopy, and flow cytometry, we show that the recently developed red fluorescent dye ProteoStat can detect the presence of intracellular amyloid‐like deposits in living bacterial cells with high specificity, even when the target proteins are expressed at low levels. This methodology allows quantitation of the intracellular amyloid content, shows the potential to replace in vitro screenings in the search for therapeutic anti‐amyloidogenic compounds, and might be useful for identifying conditions that prevent the aggregation of therapeutic recombinant proteins.     

Protein Bodies in the Pine Megagametophyte in vitro Culture: Ultrastructural, Histochemical and Electrophoretic Observations

The megagametophytes of the European black pine (Pinus nigra Arn.) were cultured on modified MS medium. After 10 d, protein bodies showed well-marked degradation on freeze-etched replicas and in preparations observed by scanning electron microscopy. After 20 d of cultivation, the megagametophyte cells were completely empty. Proteins secreted into the agar medium were determined by electrophoresis and 15 different proteins, in the range of 6.5 to 71 kDa, were identified.     

Oleosin fusion expression systems for the production of recombinant proteins

For the production of recombinant proteins, product purification is potentially difficult and expensive. Plant oleosins are capable of anchoring onto the surface of natural or artificial oil bodies. The oleosin fusion expression systems allow products to be extracted with oil bodies. In vivo, oleosin fusions are produced and directly localized to natural oil bodies in transgenic plant seeds. Via the oleosin fusion technology the thrombin inhibitor hirudin has been successfully produced and commercially used in Canada. In vitro, artificial oil bodies have been used as “carriers” for the recombinant proteins expressed in transformed microbes. In this article, plant oleosins, strategies and limitations of the oleosin fusion expression systems are summarized, alongside with progress and applications. The oleosin fusion expression systems reveal an available way to produce recombinant biopharmaceuticals at large scale.     

Viral suppression of innate immunity via spatial isolation of TBK1/IKKε from mitochondrial antivirai platform

For antiviral signaling mediated by retinoic acid-inducible gene I （RiG-I）-like receptors （RLRs）, the recruitment of cytosoUc RLRs and downstream molecules （such as TBK1 and IKKε） to mitochondriaL platform is a central event that facilitates the establishment of host antiviral state. Here, we present an example of viral targeting for immune evasion through spatial isolation of TBK1/IKKε from mitochond riai antiviral platform, which was employed by severe fever with thrombocytopenia syndrome virus （SFTSV）, a deadly bunyavirus emerging recently. We showed that SFTSV nonstructural protein NSs functions as the interferon （IFN） antagonist, mainly via suppressing TBK1/IKKε-IRF3 signaling. NSs mediates the formation of cytoplasmic inclusion bodies （IBs）, and the blockage of IB formation impairs IFN-inhibiting activity of NSs. We next demonstrate that I Bs are utilized to compartmentalize TBK1/I KKε. The compartmentalization results in spatial isolation of the kinases from mitochondria, and deprived TBK1/IKKε may participate in antiviral complex assembly, leadingto the blockage of lFN ind uction. This study proposes a new role of viral I Bs as virus-built＇jail＇ for imprisoning cellular factors and presents a novel and likely common mechanism of viral immune evasion through spatial isolation of critical signaling molecules from the mitochondrial antiviral platform.     

Use of the water-level fluctuation analysis tool (Regcel) in hydrological status assessment of Finnish lakes

Regulation of water flow constitutes the most important hydromorphological burden to Finnish lakes. The total area of regulated lakes is nearly 11,000 km², equalling one-third of the total area of Finnish inland waters. Extensive research projects have been carried out since the end of the 1980s to find out opportunities to mitigate harmful effects of the regulation of watercourses. A water-level fluctuation analysis tool, known as Regcel, has been developed to study water level data and to identify the most significant impacts. Results of the Regcel analysis give an overall picture of the impact of lake regulation in northern climate. The model is based on relationships between the water-level fluctuation and factors related to environmental, social and economical effects. Regcel has been used in 12 Lake Regulation Development Projects in Finland. In this article, we show how the Regcel model was applied in two cases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Guest editors: K. M. Wantzen, K.-O. Rothhaupt, M. M?rtl, M. Cantonati, L. G.-Tóth & P. Fischer Ecological Effects of Water-Level Fluctuations in Lakes     

Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.     

Maxent modeling for predicting the potential distribution of Sanghuang,an important group of medicinal fungi in China

The ability to identify the spatial distribution of economically important fungal species is crucial for understanding the environmental factors that affect them and for conservation management. A potentially valuable approach for this is maximum entropy (Maxent) spatial distribution modeling, which was applied here to map the potential distribution of three “Sanghuang” mushrooms in China, which include Phellinus baumii, Phellinus igniarius and Phellinus vaninii. Nineteen WorldClim bioclimatic variables, with corresponding altitude data, and 89 spatially well-dispersed species occurrence records were used in the modeling. The relative importance of the environmental variables was evaluated by Jackknife tests in the modeling analysis. The maximum entropy models obtained have high Area Under Receiver Operating Characteristic Curve (AUC) values: 0.956, 0.967 and 0.960, for P. baumii, P. igniarius and P. vaninii, respectively. The bioclimatic variable that most strongly affected distributions of P. baumii and P. vaninii was precipitation in the warmest quarter, while the mean temperature in the warmest quarter affected the distribution of P. igniarius most strongly. Overall, these models could provide valuable help in searching for the target species in areas where it is hitherto unknown, and be the reference of conservation measures for these medicinal fungal species.