MV-mimicking micelles loaded with PEG-serine-ACP nanoparticles to achieve biomimetic intra/extra fibrillar mineralization of collagen in vitro

Since their discovery, matrix vesicles (MVs) containing minerals have received considerable attention for their role in the mineralization of bone, dentin and calcified cartilage. Additionally, MVs' association with collagen fibrils, which serve as the scaffold for calcification in the organic matrix, has been repeatedly highlighted. The primary purpose of the present study was to establish a MVs–mimicking model (PEG-S-ACP/micelle) in vitro for studying the exact mechanism of MVs-mediated extra/intra fibrillar mineralization of collagen in vivo. In this study, high-concentration serine was used to stabilize the amorphous calcium phosphate (S-ACP), which was subsequently mixed with polyethylene glycol (PEG) to form PEG-S-ACP nanoparticles. The nanoparticles were loaded in the polysorbate 80 micelle through a micelle self-assembly process in an aqueous environment. This MVs–mimicking model is referred to as the PEG-S-ACP/micelle model. By adjusting the pH and surface tension of the PEG-S-ACP/micelle, two forms of minerals (crystalline mineral nodules and ACP nanoparticles) were released to achieve the extrafibrillar and intrafibrillar mineralization, respectively. This in vitro mineralization process reproduced the mineral nodules mediating in vivo extrafibrillar mineralization and provided key insights into a possible mechanism of biomineralization by which in vivo intrafibrillar mineralization could be induced by ACP nanoparticles released from MVs. Also, the PEG-S-ACP/micelle model provides a promising methodology to prepare mineralized collagen scaffolds for repairing bone defects in bone tissue engineering.     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation

1. Download : Download high-res image (253KB)
2. Download : Download full-size image

     

A study on giant panda recognition based on images of a large proportion of captive pandas

1. As a highly endangered species, the giant panda (panda) has attracted significant attention in the past decades. Considerable efforts have been put on panda conservation and reproduction, offering the promising outcome of maintaining the population size of pandas. To evaluate the effectiveness of conservation and management strategies, recognizing individual pandas is critical. However, it remains a challenging task because the existing methods, such as traditional tracking method, discrimination method based on footprint identification, and molecular biology method, are invasive, inaccurate, expensive, or challenging to perform. The advances of imaging technologies have led to the wide applications of digital images and videos in panda conservation and management, which makes it possible for individual panda recognition in a noninvasive manner by using image‐based panda face recognition method.
2. In recent years, deep learning has achieved great success in the field of computer vision and pattern recognition. For panda face recognition, a fully automatic deep learning algorithm which consists of a sequence of deep neural networks (DNNs) used for panda face detection, segmentation, alignment, and identity prediction is developed in this study. To develop and evaluate the algorithm, the largest panda image dataset containing 6,441 images from 218 different pandas, which is 39.78% of captive pandas in the world, is established.
3. The algorithm achieved 96.27% accuracy in panda recognition and 100% accuracy in detection.
4. This study shows that panda faces can be used for panda recognition. It enables the use of the cameras installed in their habitat for monitoring their population and behavior. This noninvasive approach is much more cost‐effective than the approaches used in the previous panda surveys.

     

Rabbit small intestine does not contain an annexin II/caveolin 1 complex as a target for 2-azetidinone cholesterol absorption inhibitors

Intestinal cholesterol absorption is specifically inhibited by the 2-azetidinone cholesterol absorption inhibitor ezetimibe. Photoreactive ezetimibe analogues specifically label a 145-kDa protein in the brush border membrane of enterocytes from rabbit small intestine identified as aminopeptidase N (CD13). In zebrafish and mouse small intestinal cytosol, a heterocomplex of M_r 52 kDa between annexin II and caveolin 1 was suggested as a target of ezetimibe. In contrast, in the cytosol and brush border membrane vesicles (BBMV) from rabbit small intestine of control animals or rabbits treated with the nonabsorbable cholesterol absorption inhibitor AVE 5530, both annexin II and caveolin 1 were exclusively present as monomers without any heterocomplex formation. Upon immunoprecipitation with annexin II a 52-kDa band was observed after immunostaining with annexin II antibodies, whereas no staining of a 52-kDa band occurred with anti-caveolin 1 antibodies. Vice versa, a 52-kDa band obtained by immunoprecipitation with caveolin 1 antibodies did not stain with annexin II-antibodies. The intensity of the 52-kDa band was dependent on the amount of antibody and was also observed with anti-actin or anti-APN antibodies suggesting that the 52-kDa band is a biochemical artefact. After incubation of cytosol or BBMV with radioactively labelled ezetimibe analogues, no significant amounts of the ezetimibe analogues could be detected in the immunoprecipitate with caveolin-1 or annexin II antibodies. Photoaffinity labelling of rabbit small intestinal BBMV with ezetimibe analogues did not result in labelling of proteins being immunoreactive with annexin II, caveolin 1 or a 52-kDa heterocomplex. These findings indicate that the rabbit small intestine does not contain an annexin II/caveolin 1 heterocomplex as a target for ezetimibe.     

Impact of the Arundo scale Rhizaspidiotus donacis (Hemiptera: Diaspididae) on the weight of Arundo donax (Poaceae: Arundinoideae) rhizomes in Languedoc southern France and Mediterranean Spain

Arundo donax L. (Poaceae) is native to Mediterranean Europe and invasive in the Rio Grande Basin of North America. Rhizomes from nine sites in France and Spain infested with a candidate control agent, the armoured scale Rhizaspidiotus donacis (Hemiptera: Diaspididae) weighed 50% less than those from nine sites without scale.     

Extracellular vesicles in bone and tooth: A state-of-art paradigm in skeletal regeneration

Bone and tooth, fundamental parts of the craniofacial skeleton, are anatomically and developmentally interconnected structures. Notably, pathological processes in these tissues underwent together and progressed in multilevels. Extracellular vesicles (EVs) are cell-released small organelles and transfer proteins and genetic information into cells and tissues. Although EVs have been identified in bone and tooth, particularly EVs have been identified in the bone formation and resorption, the concrete roles of EVs in bone and tooth development and diseases remain elusive. As such, we review the recent progress of EVs in bone and tooth to highlight the novel findings of EVs in cellular communication, tissue homeostasis, and interventions. This will enhance our comprehension on the skeletal biology and shed new light on the modulation of skeletal disorders and the potential of genetic treatment.     

Procedure using voltage-sensitive spin-labels to monitor dipole potential changes in phospholipid vesicles: The estimation of phloretin-induced conductance changes in vesicles

Summary Voltage-sensitive membrane potential probes were used to monitor currents resulting from positive or negative charge movement across small and large unilamellar phosphatidylcholine (PC) vesicles. Positive currents were measured for the paramagnetic phosphonium ion or for K⁺-valinomycin. Negative currents were indirectly measured for the anionic proton carriers CCCP and DNP by monitoring transmembrane proton currents. Phloretin, a compound that is believed to decrease dipole fields in planar bilayers, increases positive currents and decreases negative currents when added to egg PC vesicles. In these vesicles, positive currents are increased by phloretin addition to a much larger degree than CCCP currents are reduced. This asymmetry, with respect to the sign of the charge carrier, is apparently not the result of changes in the membrane dielectric constant. It is most easily explained by deeper binding minima at the membrane-solution interface for the CCCP anion, when compared to the phosphonium. The measured asymmetry and the magnitudes of the current changes are consistent with the predictions of a point dipole model. The use of potential-sensitive probes to estimate positive and negative currents, provides a methodology to monitor changes in the membrane dipole potential in vesicle systems.     

The association of human coagulation factors VIII,IXa and X with phospholipid vesicles involves both electrostatic and hydrophobic interactions

Blood coagulation factor X (FX) is converted to its active form (FXa) by a membrane bound multi-protein enzyme complex, comprised of factor VIII (FVIII), factor IXa (FIXa) and FX. Characterization of the molecular forces involved in the association of these proteins with phospholipids is crucial to understanding how these proteins bind to the lipid milieux of physiological membranes. In this report, the molecular forces involved in the association of FVIII, FIXa or FX with phospholipid vesicles (PLV) were characterized by ligand affinity chromatographic analyses. Treating FVIII-affinity columns with agents that disrupt electrostatic interactions caused elution of 15.2% of the total bound PLV, while agents that disrupt hydrophobic interactions caused elution of 84.8% of the total bound PLV. These results demonstrate that the association of PLV with FVIII is primarily hydrophobic. In contrast, the association of PLV with FIXa or FX is largely the result of electrostatic forces. This was established by observing that 71.3% and 78.9% of the total bound PLV was eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt electrostatic interactions. Of the total bound PLV, 28.7% and 21.2% were eluted from FIXa- and FX-affinity columns, respectively, by agents that disrupt hydrophobic interactions. These data demonstrate that hydrophobic forces play a heretofore unrecognized role in the association of PLV with FIXa or FX.