Cells can control the assembly and disassembly of membraneless organelles by enzymatic processes, but similar control has not been achieved in vitro yet. Here we develop ATP-based coacervate droplets ...as artificial membraneless organelles that can be fully controlled by two cooperating enzymes. Droplets can be generated within a minute following the addition of phosphoenolpyruvate as a substrate, and they can be dissolved within tens of seconds by adding glucose as the second substrate. We show how the rates of droplet generation and dissolution can be tuned by varying the enzyme and substrate concentrations, and we support our findings with a kinetic model of the underlying enzymatic reaction network. As all steps of the coacervate droplet life cycle, including nucleation, coarsening, and dissolution, occur under the same reaction conditions, the cycle can be repeated multiple times. In addition, by carefully balancing the rates of both enzymatic reactions, our system can be programmed to either form or dissolve droplets at specified times, acting as a chemical timer.
Biochemical processes inside the cell take place in a complex environment that is highly crowded, heterogeneous, and replete with interfaces. The recently recognized importance of biomolecular ...condensates in cellular organization has added new elements of complexity to our understanding of chemistry in the cell. Many of these condensates are formed by liquid-liquid phase separation (LLPS) and behave like liquid droplets. Such droplet organelles can be reproduced and studied
by using coacervates and have some remarkable features, including regulated assembly, differential partitioning of macromolecules, permeability to small molecules, and a uniquely crowded environment. Here, we review the main principles of biochemical organization in model membraneless compartments. We focus on some promising
coacervate model systems that aptly mimic part of the compartmentalized cellular environment. We address the physicochemical characteristics of these liquid phase separated compartments, and their impact on biomolecular chemistry and assembly. These model systems enable a systematic investigation of the role of spatiotemporal organization of biomolecules in controlling biochemical processes in the cell, and they provide crucial insights for the development of functional artificial organelles and cells.
Active coacervate droplets are liquid condensates coupled to a chemical reaction that turns over their components, keeping the droplets out of equilibrium. This turnover can be used to drive active ...processes such as growth, and provide an insight into the chemical requirements underlying (proto)cellular behaviour. Moreover, controlled growth is a key requirement to achieve population fitness and survival. Here we present a minimal, nucleotide-based coacervate model for active droplets, and report three key findings that make these droplets into evolvable protocells. First, we show that coacervate droplets form and grow by the fuel-driven synthesis of new coacervate material. Second, we find that these droplets do not undergo Ostwald ripening, which we attribute to the attractive electrostatic interactions and translational entropy within complex coacervates, active or passive. Finally, we show that the droplet growth rate reflects experimental conditions such as substrate, enzyme and protein concentration, and that a different droplet composition (addition of RNA) leads to altered growth rates and droplet fitness. These findings together make active coacervate droplets a powerful platform to mimic cellular growth at a single-droplet level, and to study fitness at a population level.
ABA is a major phytohormone that regulates a broad range of plant traits and is especially important for adaptation to environmental conditions. Our understanding of the molecular basis of ABA ...responses in plants improved dramatically in 2009 and 2010, banner years for ABA research. There are three major components; PYR/PYL/ RCAR (an ABA receptor), type 2C protein phosphatase (PP2C; a negative regulator) and SNF1-related protein kinase 2 (SnRK2; a positive regulator), and they offer a double negative regulatory system, PYR/PYL/RCAR--| PP2C--| SnRK2. In the absence of ABA, PP2C inactivates SnRK2 by direct dephosphorylation. In response to environmental or developmental cues, ABA promotes the interaction of PYR/PYL/RCAR and PP2C, resulting in PP2C inhibition and SnRK2 activation. This signaling complex can work in both the nucleus and cytosol, as it has been shown that SnRK2 phosphorylates basic-domain leucine zipper (bZIP) transcription factors or membrane proteins. Several structural analyses of PYR/PYL/RCAR have provided the mechanistic basis for this 'core signaling' model, by elucidating the mechanism of ABA binding of receptors, or the 'gate-latch-lock' mechanism of interaction with PP2C in inhibiting activity. On the other hand, intercellular ABA transport had remained a major issue, as had intracellular ABA signaling. Recently, two plasma membrane-type ABC transporters were identified and shed light on the influx/efflux system of ABA, resolving how ABA is transported from cell to cell in plants. Our knowledge of ABA responses in plants has been greatly expanded from intracellular signaling to intercellular transport of ABA.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Cyclic wet-dry is one of the influential weathering agents which can rapidly alter the mechanical properties of soils, limiting their durability and consistent performance. This study investigates ...the effect of wet-dry cycles on the mechanical behaviour of bio-cemented soil. Microbial-induced carbonate precipitation-based bio-cementation is an innovative soil improvement method, which is gaining increasing attention as a potential alternative for stabilizing slope surface. As the treated surfaces are exposed to repeated rainfalls and draughts, durability analysis is essential; cyclic wet-dry tests were therefore performed as a credible indicator of durability. The soil obtained from the Hokkaido expressway slope was treated at laboratory to varying cementation levels (% CaCO
3
) and subjected to 50 subsequent wet-dry cycles. Physical and mechanical changes were monitored using mass loss, shear wave velocities and needle penetration tests during wet-dry cycles. The results showed that the wet-dry cycles deteriorated the physical and mechanical at two stages. The mass and S-wave velocity of specimens significantly dropped after first few cycles and then tended to reach equilibrium. The second stage of notable deterioration was observed between 30 and 50 wet-dry cycles. It is suggested that the erosion of weak and powdery deposition of CaCO
3
causes the degradation at the early stage, whereas the degradation in the late stage was attributed to the microstructural deformations of intact carbonate bonds. It was also found that the increase in cementation level decreases the deterioration of bio-cemented soil under wet-dry cycles.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
FOLFIRI and FOLFOX have shown equivalent efficacy for metastatic colorectal cancer (mCRC), but their comparative effectiveness is unknown when combined with bevacizumab.
WJOG4407G was a randomized, ...open-label, phase III trial conducted in Japan. Patients with previously untreated mCRC were randomized 1:1 to receive either FOLFIRI plus bevacizumab (FOLFIRI + Bev) or mFOLFOX6 plus bevacizumab (mFOLFOX6 + Bev), stratified by institution, adjuvant chemotherapy, and liver-limited disease. The primary end point was non-inferiority of FOLFIRI + Bev to mFOLFOX6 + Bev in progression-free survival (PFS), with an expected hazard ratio (HR) of 0.9 and non-inferiority margin of 1.25 (power 0.85, one-sided α-error 0.025). The secondary end points were response rate (RR), overall survival (OS), safety, and quality of life (QoL) during 18 months. This trial is registered to the University Hospital Medical Information Network, number UMIN000001396.
Among 402 patients enrolled from September 2008 to January 2012, 395 patients were eligible for efficacy analysis. The median PFS for FOLFIRI + Bev (n = 197) and mFOLFOX6 + Bev (n = 198) were 12.1 and 10.7 months, respectively HR, 0.905; 95% confidence interval (CI) 0.723–1.133; P = 0.003 for non-inferiority. The median OS for FOLFIRI + Bev and mFOLFOX6 + Bev were 31.4 and 30.1 months, respectively (HR, 0.990; 95% CI 0.785–1.249). The best overall RRs were 64% for FOLFIRI + Bev and 62% for mFOLFOX6 + Bev. The common grade 3 or higher adverse events were leukopenia (11% in FOLFIRI + Bev/5% in mFOLFOX6 + Bev), neutropenia (46%/35%), diarrhea (9%/5%), febrile neutropenia (5%/2%), peripheral neuropathy (0%/22%), and venous thromboembolism (6%/2%). The QoL assessed by FACT-C (TOI-PFC) and FACT/GOG-Ntx was favorable for FOLFIRI + Bev during 18 months.
FOLFIRI plus bevacizumab was non-inferior for PFS, compared with mFOLFOX6 plus bevacizumab, as the first-line systemic treatment for mCRC.
UMIN000001396.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In classical analyses of γ-ray data from imaging atmospheric Cherenkov telescopes (IACTs), such as the High Energy Stereoscopic System (H.E.S.S.), aperture photometry, or photon counting, is applied ...in a (typically circular) region of interest (RoI) encompassing the source. A key element in the analysis is to estimate the amount of background in the RoI due to residual cosmic ray-induced air showers in the data. Various standard background estimation techniques have been developed in the last decades, most of them rely on a measurement of the background from source-free regions within the observed field of view. However, in particular in the Galactic plane, source analysis and background estimation are hampered by the large number of, sometimes overlapping, γ-ray sources and large-scale diffuse γ-ray emission. For complicated fields of view, a three-dimensional (3D) likelihood analysis shows the potential to be superior to classical analysis. In this analysis technique, a spectromorphological model, consisting of one or multiple source components and a background component, is fitted to the data, resulting in a complete spectral and spatial description of the field of view. For the application to IACT data, the major challenge of such an approach is the construction of a robust background model. In this work, we apply the 3D likelihood analysis to various test data recently made public by the H.E.S.S. collaboration, using the open analysis frameworks ctools and Gammapy. First, we show that, when using these tools in a classical analysis approach and comparing to the proprietary H.E.S.S. analysis framework, virtually identical high-level analysis results, such as field-of-view maps and spectra, are obtained. We then describe the construction of a generic background model from data of H.E.S.S. observations, and demonstrate that a 3D likelihood analysis using this background model yields high-level analysis results that are highly compatible with those obtained from the classical analyses. This validation of the 3D likelihood analysis approach on experimental data is an important step towards using this method for IACT data analysis, and in particular for the analysis of data from the upcoming Cherenkov Telescope Array (CTA).
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FMFMET, NUK, UL, UM, UPUK
The effects of different transient liquid phase (TLP) bonding times on the microstructure and mechanical properties of Hastelloy X joints made by Ni–Cr–B–Si–Fe filler alloy were investigated. The ...specimens were TLP bonded at 1070 °C for holding times of 5, 20, 80, 320, and 640 min. The electron probe microanalysis (EPMA) results revealed that the main eutectic phases observed at the joints following incomplete isothermal solidification were Ni-rich borides, Ni-rich silicides, Ni–Si eutectic, and some Cr-rich borides. A high density of plate-like, blocky, and acicular (Mo and Cr)-rich borides were observed in the diffusion-affected zone (DAZ) of the samples; however, increasing the holding time decreased the contents of these phases. The solid-state diffusion was found to be a more effective transportation phenomenon than base metal dissolution at longer holding times. The increased DAZ thickness and the complete isothermal solidification as a result of the improved solid-state diffusion helped increase the uniformity of the hardness profile of the TLP bond at higher holding times (320 and 640 min). The results showed reverse relationship between the athermally solidified zone (ASZ) width and the bonding strength. The highest tensile strength (∼617 MPa) was achieved for the sample bonded at a holding time of 320 min; this strength was more than 80% of the base metal strength. A fractographic analysis of the tensile failure revealed a cellular fracture surface, exhibiting the characteristics of both brittle and ductile fractures. The sites prone to stress concentration and crack initiation were reduced with the completion of isothermal solidification.
•Bonding of Hastelloy X joints by TLP method using Ni-Cr-B-Si-Fe filler alloy.•Effect of time on joint microstructure and mechanical properties.•Reduction in the room-temperature tensile strength of the joint owing to a layer of continuous borides.•Higher strength of samples prepared at longer holding times due to complete isothermal solidification.•Change in the joint fracture from brittle to cellular at longer holding times.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Objective and Background
Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are ...commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3‐dimensional cultures such as spheroids, which are spherical clusters of cells formed by self‐assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo.
Material and Methods
Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real‐time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis‐related genes in monolayer and spheroid‐cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled‐related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid‐cultured hPDLMSCs with OIM were measured by real‐time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3‐deficient hPDLMSC spheroids.
Results
The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate “stemness” were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer‐cultured hPDLMSCs. ALP activity and expression of osteogenesis‐related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3‐mediated ALP activation.
Conclusion
Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine.
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CMK, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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