Phase separation, crystallization and interactions in biopolymer blends studied in situ by macro ATR-FTIR spectroscopic imaging

Ubiquitous applications of biopolymer blends require a broader range of accurate information about their behaviour. For this purpose, macro attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy and spectroscopic imaging are the main characterisation methods of choice to provide reliable information for both qualitative and quantitative analysis. The main objective of this project is to realise their full potential to investigate biopolymers and biopolymer blends with and without high-pressure CO2. In situ high-pressure ATR-FTIR spectroscopic imaging was applied to visualize the dynamic process of phase separation in biopolymer Polycaprolactone (PCL)/Poly (lactic acid) (PLA) blends under high-pressure CO2. It was demonstrated that the extent of phase separation in PCL/PLA blends under high-pressure CO2 is enhanced with increasing exposure time, CO2 pressure and temperature. The mechanisms of polymer-polymer interactions were investigated using 2D correlation analysis and 2D disrelation mapping. Evidence of the break of some of the existing inter- and intramolecular dipole-dipole interaction (C=O···C=O) between polymer molecules under high-pressure CO2 was provided. The simultaneous visualization of crystallization and phase separation in Poly (3-hydroxybutyrate) PHB/PLA blends has been realized for the first time through utilizing in situ ATR-FTIR spectroscopic imaging. Decreasing the annealing temperature is proved to be an effective method to constrain phase separation between these two blend components and tailor the final morphology in upper critical solution temperature (UCST) crystallizable polymer blends for practical applications. Spectroscopic images based on the band position were successfully used to investigate changes in intra- and intermolecular interactions in PHB/PLA blends. During the isothermal melt crystallization, the disappearance of the intermolecular interaction between PHB and PLA and the appearance of the inter- and intramolecular interactions within the PHB crystal were both visualized. Overall, this thesis has demonstrated further the use of ATR-FTIR spectroscopy and macro ATR-FTIR spectroscopic imaging as a valuable analytical approach to investigate biopolymers and biopolymer blends with and without high-pressure CO2, which can aid the design of biopolymer products and biopolymer process.Open Acces

EpCAM Is an Endoderm-Specific Wnt Derepressor that Licenses Hepatic Development

SummaryMechanisms underlying cell-type-specific response to morphogens or signaling molecules during embryonic development are poorly understood. To learn how response to the liver-inductive Wnt2bb signal is achieved, we identify an endoderm-enriched, single transmembrane protein, epithelial-cell-adhesion-molecule (EpCAM), as an endoderm-specific Wnt derepressor in zebrafish. hi2151/epcam mutants exhibit defective liver development similar to prt/wnt2bb mutants. EpCAM directly binds to Kremen1 and disrupts the Kremen1-Dickkopf2 (Dkk2) interaction, which prevents Kremen1-Dkk2-mediated removal of Lipoprotein-receptor-related protein 6 (Lrp6) from the cell surface. These data lead to a model in which EpCAM derepresses Lrp6 and cooperates with Wnt ligand to activate Wnt signaling through stabilizing membrane Lrp6 and allowing Lrp6 clustering into active signalosomes. Thus, EpCAM cell autonomously licenses and cooperatively activates Wnt2bb signaling in endodermal cells. Our results identify EpCAM as the key molecule and its functional mechanism to confer endodermal cells the competence to respond to the liver-inductive Wnt2bb signal

Cell-Cycle-Regulated Interaction between Mcm10 and Double Hexameric Mcm2-7 Is Required for Helicase Splitting and Activation during S Phase

Mcm2-7 helicase is loaded onto double-stranded origin DNA as an inactive double hexamer (DH) in G1 phase. The mechanisms of Mcm2-7 remodeling that trigger helicase activation in S phase remain unknown. Here, we develop an approach to detect and purify the endogenous DHs directly. Through cellular fractionation, we provide in vivo evidence that DHs are assembled on chromatin in G1 phase and separated during S phase. Interestingly, Mcm10, a robust MCM interactor, co-purifies exclusively with the DHs in the context of chromatin. Deletion of the main interaction domain, Mcm10 C terminus, causes growth and S phase defects, which can be suppressed through Mcm10-MCM fusions. By monitoring the dynamics of MCM DHs, we show a significant delay in DH dissolution during S phase in the Mcm10-MCM interaction-deficient mutants. Therefore, we propose an essential role for Mcm10 in Mcm2-7 remodeling through formation of a cell-cycle-regulated supercomplex with DHs

Flexibly-oriented double Cdc45-MCM-GINS intermediates during eukaryotic replicative helicase maturation

The core of the eukaryotic helicase MCM is loaded as an inactive double hexamer (DH). How it is assembled into two active Cdc45-MCM-GINS (CMG) helicases remains elusive. Here, we report that at the onset of S phase, both Cdc45 and GINS are loaded as dimers onto MCM DH, resulting in formation of double CMG (d-CMG). As S phase proceeds, d-CMGs gradually mature into two single CMG-centered replisome progression complexes (RPCs). Mass spectra reveal that RPA and DNA Pol α/primase co-purify exclusively with RPCs, but not with d-CMGs. Consistently, d-CMGs are not able to catalyze either the unwinding or de novo DNA synthesis, while RPCs can do both. Using single-particle electron microscopy, we have obtained 2D class averages of d-CMGs. Compared to MCM DHs, they display heterogeneous, flexibly orientated and partially loosened conformations with changed interfaces. The dumbbell-shaped d-CMGs are mediated by Ctf4, while other types of d-CMGs are independent of Ctf4. These data suggest CMG dimers as bona fide intermediates during MCM maturation, providing an additional quality control for symmetric origin activation and bidirectional replication

Flexibly-oriented double Cdc45-MCM-GINS intermediates during eukaryotic replicative helicase maturation

The core of the eukaryotic helicase MCM is loaded as an inactive double hexamer (DH). How it is assembled into two active Cdc45-MCM-GINS (CMG) helicases remains elusive. Here, we report that at the onset of S phase, both Cdc45 and GINS are loaded as dimers onto MCM DH, resulting in formation of double CMG (d-CMG). As S phase proceeds, d-CMGs gradually mature into two single CMG-centered replisome progression complexes (RPCs). Mass spectra reveal that RPA and DNA Pol α/primase co-purify exclusively with RPCs, but not with d-CMGs. Consistently, d-CMGs are not able to catalyze either the unwinding or de novo DNA synthesis, while RPCs can do both. Using single-particle electron microscopy, we have obtained 2D class averages of d-CMGs. Compared to MCM DHs, they display heterogeneous, flexibly orientated and partially loosened conformations with changed interfaces. The dumbbell-shaped d-CMGs are mediated by Ctf4, while other types of d-CMGs are independent of Ctf4. These data suggest CMG dimers as bona fide intermediates during MCM maturation, providing an additional quality control for symmetric origin activation and bidirectional replication

Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells

Naive embryonic stem cells hold great promise for research and therapeutics as they have broad and robust developmental potential. While such cells are readily derived from mouse blastocysts it has not been possible to isolate human equivalents easily, although human naive-like cells have been artificially generated (rather than extracted) by coercion of human primed embryonic stem cells by modifying culture conditions or through transgenic modification. Here we show that a sub-population within cultures of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) manifests key properties of naive state cells. These naive-like cells can be genetically tagged, and are associated with elevated transcription of HERVH, a primate-specific endogenous retrovirus. HERVH elements provide functional binding sites for a combination of naive pluripotency transcription factors, including LBP9, recently recognized as relevant to naivety in mice. LBP9-HERVH drives hESC-specific alternative and chimaeric transcripts, including pluripotency-modulating long non-coding RNAs. Disruption of LBP9, HERVH and HERVH-derived transcripts compromises self-renewal. These observations define HERVH expression as a hallmark of naive-like hESCs, and establish novel primate-specific transcriptional circuitry regulating pluripotency

CMIP5-Based Projection of Decadal and Seasonal Sea Surface Temperature Variations in East China Shelf Seas

The East China Shelf Seas, comprising the Bohai Sea, the Yellow Sea, and the shelf region of East China Sea, play significant roles among the shelf seas of the Western North Pacific Ocean. The projection of sea surface temperature (SST) changes in these regions is a hot research topic in marine science. However, this is a very difficult task due to the lack of available long-term projection data. Recently, with the high development of simulation technology based on numerical models, the model intercomparison projects, e.g., Phase 5 of the Climate Model Intercomparison Project (CMIP5), have become important ways of understanding climate changes. CMIP5 provides multiple models that can be used to estimate SST changes by 2100 under different representative concentration pathways (RCPs). This paper developed a CMIP5-based SST investigation framework for the projection of decadal and seasonal variation of SST in East China Shelf Seas by 2100. Since the simulation results of CMIP5 models may have degrees of errors, this paper uses hydrological observation data from World Ocean Atlas 2018 (WOA18) for model validation and correction. This paper selects seven representative ones including ACCESS1.3, CCSM4, FIO-ESM, CESM1-CAM5, CMCC-CMS, NorESM1-ME, and Max Planck Institute Earth System Model of medium resolution (MPI-ESM-MR). The decadal and seasonal SST changes in the next 100 years (2030, 2060, 2090) are investigated by comparing with the present analysis in 2010. The experimental results demonstrate that SST will increase significantly by 2100: the decadal SST will increase by about 1.55 °C, while the seasonal SST will increase by 1.03–1.95 °C

Maximum safe pumping rate in sloping coastal aquifers

Analytical solutions for pumping-induced sawater intrusion in a sloping unconfined coastal aquifer are developed.</p

(&minus;)-Lariciresinol Isolated from the Roots of Isatis indigotica Fortune ex Lindl. Inhibits Hepatitis B Virus by Regulating Viral Transcription

Chronic hepatitis induced by hepatitis B virus (HBV) infection is a serious public health problem, leading to hepatic cirrhosis and liver cancer. Although the currently approved medications can reliably decrease the virus load and prevent the development of hepatic diseases, they fail to induce durable off-drug control of HBV replication in the majority of patients. The roots of Isatis indigotica Fortune ex Lindl., a traditional Chinese medicine, were frequently used for the prevention of viral disease in China. In the present study, (&minus;)-lariciresinol ((&minus;)-LRSL), isolated from the roots of Isatis indigotica Fortune ex Lindl., was found to inhibit HBV DNA replication of both wild-type and nucleos(t)ide analogues (NUCs)-resistant strains in vitro. Mechanism studies revealed that (&minus;)-LRSL could block RNA production after treatment, followed by viral proteins, and then viral particles and DNA. Promoter reporter assays and RNA decaying dynamic experiments indicated that (&minus;)-LRSL mediated HBV RNA reduction was mainly due to transcriptional inhibition rather than degradation. Moreover, (&minus;)-LRSL in a dose-dependent manner also inhibited other animal hepadnaviruses, including woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Combining the analysis of RNA-seq, we further found that the decrease in HBV transcriptional activity by (&minus;)-LRSL may be related to hepatocyte nuclear factor 1&alpha; (HNF1&alpha;). Taken together, (&minus;)-LRSL represents a novel chemical entity that inhibits HBV replication by regulating HNF1&alpha; mediated HBV transcription, which may provide a new perspective for HBV therapeutics