Phospholipid-induced secondary structural changes of lysozyme polymorphic amyloid fibrils studied using vacuum-ultraviolet circular dichroism.

The hallmark of amyloidosis, such as Alzheimer's disease and Parkinson's disease, is the deposition of amyloid fibrils in various internal organs. The onset of the disease is related to the strength of cytotoxicity caused by toxic amyloid species. Furthermore, amyloid fibrils show polymorphism, where some types of fibrils are cytotoxic while others are not. It is thus essential to understand the molecular mechanism of cytotoxicity, part of which is caused by the interaction between amyloid polymorphic fibrils and cell membranes. Here, using amyloid polymorphs of hen egg white lysozyme, which is associated with hereditary systemic amyloidosis, showing different levels of cytotoxicity and liposomes of DMPC and DMPG, changes in the secondary structure of the polymorphs and the structural state of phospholipid membranes caused by the interaction were investigated using vacuum-ultraviolet circular dichroism (VUVCD) and Laurdan fluorescence measurements, respectively. Analysis has shown that the more cytotoxic polymorph increases the antiparallel ß-sheet content and causes more disorder in the membrane structure while the other less cytotoxic polymorph shows the opposite structural changes and causes less structural disorder in the membrane. These results suggest a close correlation between the structural properties of amyloid fibrils and the degree of structural disorder of phospholipid membranes, both of which are involved in the fundamental process leading to amyloid cytotoxicity.

Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide.

INTRODUCTION: The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost. MATERIALS AND METHODS: We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene. RESULTS: We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells. CONCLUSION: 2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

Lipopolysaccharides affect compressed periodontal ligament cells via Eph-ephrin signaling.

OBJECTIVES: The aim of this study is to investigate the underlying mechanism of the recovery of periodontal ligament cells (PDLCs) sequentially exposed to inflammation and mechanical loading. MATERIALS AND METHODS: We divided PDLCs into four groups: control; compressive force (CF) alone (2.0 g/cm2 ); lipopolysaccharides (LPS) pretreatment (0.1 µg/ml) followed by simultaneous LPS and CF stimulation, simulating uncontrolled periodontitis; and LPS pretreatment followed by CF exposure, simulating controlled periodontitis. The expression of EphB4-ephrinB2 and EphA2-ephrinA2, and the level of osteoclastogenesis and osteogenesis were evaluated. RESULTS: Simultaneous stimulation by LPS and CF, compared with CF alone and sequential LPS and CF exposure, significantly suppressed EphB4 and enhanced ephrinA2 expression. Similarly, the most intense osteoclastic differentiation was observed under simultaneous LPS and CF stimulation, while sequential exposure to LPS and CF only slightly increased osteoclastic cell numbers. Both the activation of EphB4 signaling and ephrinA2 silencing lowered osteoclastic differentiation, which had previously been upregulated by simultaneous LPS and CF stimulation. These treatments also increased osteogenic differentiation. CONCLUSIONS: Simultaneous LPS and CF stimulation critically enhances osteoclastogenesis in PDLCs through the suppression of EphB4 and the induction of ephrinA2 signaling. Sequential LPS and CF exposure partially abolishes the osteolytic effects of simultaneous stimulation.

Conformation of myelin basic protein bound to phosphatidylinositol membrane characterized by vacuum-ultraviolet circular-dichroism spectroscopy and molecular-dynamics simulations.

The 18.5-kDa isoform of myelin basic protein (MBP) interacts with the membrane surface of the myelin sheath to construct its compact multilamellar structure. This study characterized the conformation of MBP in the membrane by measuring the vacuum-ultraviolet circular-dichroism (VUVCD) spectra of MBP in the bilayer liposome comprising the following essential lipid constituents of the myelin sheath: phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2). The spectra of MBP exhibited the characteristic peaks of the helix structure in the presence of PI liposome, and the intensity increased markedly in the presence of PIP and PIP2 liposomes to show an isodichroic point. This suggests that the amount of the membrane-bound conformation of MBP enhanced due to the increased number of negative net charges on the liposome surfaces. Secondary-structure analysis revealed that MBP in the membrane comprised approximately 40% helix contents and eight helix segments. Molecular-dynamics (MD) simulations of the eight segments were conducted for 250 ns in the presence of PI membrane, which predicted two amphiphilic and three nonamphiphilic helices as the membrane-interaction sites. Further analysis of the distances of the amino-acid residues in each segment from the phosphate group suggested that the nonamphiphilic helices interact with the membrane surface electrostatically, while the amphiphilic ones invade the inside of the membrane to produce electrostatic and hydrophobic interactions. These results show that MBP can interact with the PI membrane via amphiphilic and nonamphiphilic helices under the control of a delicate balance between electrostatic and hydrophobic interactions.

[Updates on rickets and osteomalacia: mechanism and regulation of bone mineralization].

Bone is mineralized when hydroxyapatite crystals derived from calcium ions and inorganic phosphate (Pi) grow along collagen fibrils in the extracellular matrix. Mineralization is initiated by nucleation of those crystals. Mature osteoblasts secrete matrix vesicles into osteoid, which contain growing hydroxyapatite crystal seeds. After rupture of the lipid bilayer of those vesicles, crystals continue to grow as a mineralized nodule and adhere to collagen fibrils. It remains controversial whether nucleation occurs mainly in matrix vesicles or also extra-vesicularly around collagen fibrils. Mineralization is inhibited by pyrophosphate (PPi) and by SIBLING family proteins, which carry an acidic serine- and aspartate-rich motif (ASARM) and include osteopontin, dentin matrix protein 1 and MEPE. Intracellular and extracellular activity of these factors is regulated by the PPi-generating ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1) , the PPi-transporter progressive ankylosis (ANK) protein, the PPi-degrading/Pi-generating ectoenzyme alkaline phosphatase (ALPL, TNAP) , and PHEX endopeptidase. Gain- or loss-of-function mutations in genes encoding these proteins are associated with mineralization disorders such as ectopic calcification and other pathologies.

Lipid-membranes interaction, structural assessment, and sustainable production of polyhydroxyalkanoate by Priestia filamentosa AZU-A6 from sugarcane molasses.

This study presented for the first time the PHA-lipid interactions by circular dichroism (CD) spectroscopy, besides a sustainable PHA production strategy using a cost-effective microbial isolate. About 48 bacterial isolates were selected from multifarious Egyptian sites and screened for PHAs production. The Fe(AZU-A6) was the most potent isolate, and identified genetically as Priestia filamentosa AZU-A6, while the intracellular PHA granules were visualized by TEM. Sugarcane molasses (SCM) was used an inexpensive carbon source and the production conditions were optimized through a Factor-By-Factor strategy and a Plackett-Burman statistical model. The highest production (6.84 g L-1) was achieved at 8.0 % SCM, pH 8.0, 35 °C, 250 rpm, and 0.5 g L-1 ammonium chloride after 72 h. The complementary physicochemical techniques (e.g., FTIR, NMR, GC-MS, DSC, and TGA) have ascertained the structural identity as poly-3-hydroxybutyrate (P3HB) with a characteristic melting temperature of 174.5 °C. The circular dichroism analysis investigated the existence of interactions between the PHB and the different lipids, particularly 1,2-dimyristoyl-sn-glycero-3-phosphocholine. The ATR technique for the lipid-PHB films suggested that both the hydrophobic and electrostatic forces control the lipid-PHB interactions that might induce changes in the structuration of PHB.

IL-27 abrogates receptor activator of NF-kappa B ligand-mediated osteoclastogenesis of human granulocyte-macrophage colony-forming unit cells through STAT1-dependent inhibition of c-Fos.

IL-27 was first discovered as a factor supporting initial Th1 immune responses. Subsequent studies revealed that this cytokine has pleiotropic effects, including inhibition of certain immune cells, a regulatory role in hemopoietic stem cell differentiation, and antitumor activities. However, the role of human IL (hIL)-27 in human osteoclast precursors and inflammatory bone disease is unclear. Here, we examined the direct effect of hIL-27 on human osteoclastogenesis. Human bone marrow cells cultured in MethoCult medium containing human (h) GM-CSF, human stem cell factor, and hIL-3 expressed Mac-1, c-kit, and c-Fms. These cells, called hCFU-GMs, also expressed the IL-27 receptor, an IL-27Ralpha (WSX-1)/gp130 heterodimer. Cultivation in hM-CSF and human receptor activator of NF-kappaB ligand induced the differentiation of tartrate-resistant acid phosphatase-positive multinucleated cells (osteoclasts) from hCFU-GMs, and hIL-27 inhibited this osteoclastogenesis in a dose-dependent manner. hIL-27 also repressed bone resorption by osteoclasts on a dentine slice. hIL-27 caused a remarkable increase in STAT1 phosphorylation and enhanced the STAT1 protein level. It also inhibited the expression of receptor activator of NF-kappaB ligand-induced c-Fos and cytoplasmic, calcineurin-dependent 1 NFAT (NFATc1), which are indispensable transcription factors for osteoclastogenesis. Fludarabine, a STAT1 inhibitor, and STAT1 small interfering RNA partially rescued the inhibition of osteoclastogenesis by IL-27. A WSX-1 deficiency caused severe inflammatory bone destruction primed by Escherichia coli cell wall lysate in vivo. Therefore, hIL-27 may act as an anti-inflammatory cytokine in human bone destruction, by inhibiting osteoclastogenesis from hCFU-GMs via STAT1-dependent down-regulation of the transcription factor c-Fos. Our results suggest that hIL-27 may prove useful as a therapeutic target for inflammatory bone destruction.

RT-PCR Screening Tests for SARS-CoV-2 with Saliva Samples in Asymptomatic People: Strategy to Maintain Social and Economic Activities while Reducing the Risk of Spreading the Virus.

The year 2020 will be remembered for the coronavirus disease 2019 (COVID-19) pandemic, which continues to affect the whole world. Early and accurate identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is fundamental to combat the disease. Among the current diagnostic tests, real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) is the most reliable and frequently used method. Herein, we discuss the interpretation of RT-qPCR results relative to viral infectivity. Although nasopharyngeal swab samples are often used for RT-qPCR testing, they require collection by trained medical staff. Saliva samples are emerging as an inexpensive and efficient alternative for large-scale screening. Pooled-sample testing of saliva has been applied for mass screening of SARS-CoV-2 infection. Current policies recommend isolating people with borderline cycle threshold (Ct) values (35

Odontoblast death drives cell-rich zone-derived dental tissue regeneration.

Severe dental tissue damage induces odontoblast death, after which dental pulp stem and progenitor cells (DPSCs) differentiate into odontoblast-like cells, contributing to reparative dentin. However, the damage-induced mechanism that triggers this regeneration process is still not clear. We aimed to understand the effect of odontoblast death without hard tissue damage on dental regeneration. Herein, using a Cre/LoxP-based strategy, we demonstrated that cell-rich zone (CZ)-localizing Nestin-GFP-positive and Nestin-GFP-negative cells proliferate and differentiate into odontoblast-like cells in response to odontoblast depletion. The regenerated odontoblast-like cells played a role in reparative dentin formation. RNA-sequencing analysis revealed that the expression of odontoblast differentiation- and activation-related genes was upregulated in the pulp in response to odontoblast depletion even without damage to dental tissue. In this regenerative process, the expression of type I parathyroid hormone receptor (PTH1R) increased in the odontoblast-depleted pulp, thereby boosting dentin formation. The levels of PTH1R and its downstream mediator, i.e., phosphorylated cyclic AMP response element-binding protein (Ser133) increased in the physically damaged pulp. Collectively, odontoblast death triggered the PTH1R cascade, which may represent a therapeutic target for inducing CZ-mediated dental regeneration.

Membrane-induced conformational change of alpha1-acid glycoprotein characterized by vacuum-ultraviolet circular dichroism spectroscopy.

The tertiary structure of alpha1-acid glycoprotein (AGP) remains unresolved despite its novel function because AGP is a hard target in X-ray and NMR analyses. To elucidate the membrane-induced conformational change of AGP, the vacuum-ultraviolet circular dichroism (VUVCD) spectra of AGP and its constituent sugars were measured down to 160 nm in the presence or absence of phosphoglyceride liposome using a synchrotron-radiation VUVCD spectrophotometer. The secondary-structure contents and numbers of segments of AGP were estimated from the VUVCD spectra of the protein moiety obtained by subtracting the contributions of the glycan moiety. Further, the positions of secondary structures on the amino acid sequence were predicted by combining the VUVCD data with a neural network algorithm. These comprehensive secondary-structure analyses revealed that AGP consists of 11.4% alpha-helices (3 segments) and 39.9% beta-strands (12 segments) in the absence of liposome (pH 4.5), which are close to the proportions in the secondary structure of native AGP (pH 7.4) predicted by homology modeling, and that it consists of 47.5% alpha-helices (7 segments) and 2.7% beta-strands (2 segments) in the presence of liposome (pH 4.5). Detailed characterization of these alpha-helices of AGP bound to liposome suggested that two alpha-helices (residues 15-27 and 161-175) in the N- and C-terminal regions strongly interact with liposome. Most of the progesterone-binding residues of AGP were involved in the sequences transferring from beta-strands to alpha-helices or unordered structures, which coincided with the large decrease in progesterone-binding capacity of liposome-bound AGP. These results provide the first sequence-level information on the membrane-binding mechanism and structure-function relationship of AGP.

Bone Marrow Cells Inhibit BMP-2-Induced Osteoblast Activity in the Marrow Environment.

Bone morphogenetic protein 2 (BMP-2) is widely known as a potent growth factor that promotes bone formation. However, an increasing number of studies have demonstrated side effects of BMP-2 therapy. A deeper understanding of the effect of BMP-2 on cells other than those involved directly in bone remodeling is of fundamental importance to promote a more effective delivery of BMP-2 to patients. In this study, we aimed to investigate the effect of BMP-2 in the marrow environment. First, BMP-2 adsorbed onto titanium implants was delivered at the tooth extraction socket (marrow-absent site) or in the mandible marrow of beagle dogs. BMP-2 could induce marked bone formation around the implant at the tooth extraction socket. Surprisingly, however, no bone formation was observed in the BMP-2-coated titanium implants inserted in the mandible marrow. In C57BL/6 mice, BMP-2 adsorbed in freeze-dried collagen pellets could induce bone formation in marrow-absent calvarial bone. However, similar to the canine model, BMP-2 could not induce bone formation in the femur marrow. Analysis of osteoblast differentiation using Col1a1(2.3)-GFP transgenic mice revealed a scarce number of osteoblasts in BMP-2-treated femurs, whereas in the control group, osteoblasts were abundant. Ablation of femur marrow recovered the BMP-2 ability to induce bone formation. In vitro experiments analyzing luciferase activity of C2C12 cells with the BMP-responsive element and alkaline phosphatase activity of MC3T3-E1 osteoblasts further revealed that bone marrow cells inhibit the BMP-2 effect on osteoblasts by direct cell-cell contact. Collectively, these results showed that the effect of BMP-2 in inducing bone formation is remarkably repressed by marrow cells via direct cell-cell contact with osteoblasts; this opens new perspectives on the clarification of the side-effects associated with BMP-2 application. © 2018 American Society for Bone and Mineral Research.

Involvement of Charcot-Marie-Tooth disease gene mitofusin 2 expression in paclitaxel-induced mechanical allodynia in rats.

Paclitaxel induces peripheral neuropathy, which is dose-limiting and results in loss of quality of life. Therefore, the prevention and treatment of paclitaxel-induced peripheral neuropathy are major concerns in clinical cancer therapy. However, the detailed mechanisms have not been fully elucidated. It has recently been reported that allelic variability in the Charcot-Marie-Tooth disease (CMT) genes, mitofusin 2 (MFN2), Rho guanine nucleotide exchange factor 10 (ARHGEF10), and periaxin (PRX), affected paclitaxel-induced peripheral neuropathy in clinical cases. Therefore, we hypothesized that paclitaxel may induce peripheral neuropathy due to changes in Mfn2, Arhgef10, and Prx mRNA expression. Paclitaxel (6mg/kg) was administered intraperitoneally, on two consecutive days per week for 4 weeks in rats. Paclitaxel-induced peripheral neuropathy was measured by the von Frey test and acetone test, mechanical allodynia, and cold hyperalgesia, respectively, on days 0, 3, 10, 17, and 24. Mfn2, Arhgef10, and Prx mRNA expression in the spinal cord were analyzed by qRT-PCR on days 3 and 24. Paclitaxel induced mechanical allodynia from days 17-24, but did not induce cold hyperalgesia. In addition, paclitaxel reduced Mfn2 mRNA expression, but not Arhgef10 or Prx mRNA expression, on days 3 and 24. In addition, Mfn2 mRNA level was decreased before the appearance of mechanical allodynia. The results of the present study suggest that a reduction in Mfn2 mRNA expression contributes to paclitaxel-induced mechanical allodynia.

Effects of Comb Tooth Cap Medicinal Mushroom, Hericium ramosum (Higher Basidiomycetes) Mycelia on DPPH Radical Scavenging Activity and Nerve Growth Factor Synthesis.

The goal of this study was to evaluate the antioxidant effects of and nerve growth factor (NGF) synthesis caused by Hericium ramosum mycelia. Wild mushroom fruiting bodies were collected from nature to isolate their mycelia. Pieces of H. ramosum fruiting bodies were plated onto 90-mm Petri dishes with potato dextrose agar medium to isolate their mycelia. Antioxidant activity was measured using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging activity in vitro; the ethanol extract from H. ramosum mycelia (63.11 µmol Trolox/g) was more potent than that of other mushroom mycelia extracts. There was a proportional relationship (R2 = 0.7929) between DPPH radical scavenging activity and total phenolic content in extracts of different mushroom mycelia. We investigated the ability of H. ramosum mycelia to inducing NGF synthesis in vivo. Oral administration of H. ramosum mycelia significantly increased concentrations of NGF in the hippocampus of intact mice. These results are the first concerning antioxidant activity and NGF synthesis of H. ramosum mycelia. These mushroom mycelia could be useful as food and/or nutritional supplements because of certain biological functions.

Circular-dichroism and synchrotron-radiation circular-dichroism spectroscopy as tools to monitor protein structure in a lipid environment.

Circular-dichroism (CD) spectroscopy is a powerful tool for the secondary-structure analysis of proteins. The structural information obtained by CD does not have atomic-level resolution (unlike X-ray crystallography and NMR spectroscopy), but it has the great advantage of being applicable to both nonnative and native proteins in a wide range of solution conditions containing lipids and detergents. The development of synchrotron-radiation CD (SRCD) instruments has greatly expanded the utility of this method by extending the spectra to the vacuum-ultraviolet region below 190 nm and producing information that is unobtainable by conventional CD instruments. Combining SRCD data with bioinformatics provides new insight into the conformational changes of proteins in a membrane environment.

EphB/ephrin-B interactions mediate human MSC attachment, migration and osteochondral differentiation.

Bone marrow derived mesenchymal stem/stromal cells (MSC) contribute to skeletal tissue formation and the regulation of haematopoiesis. The Eph/ephrin family of receptor tyrosine kinases is potentially important in the maintenance of the stem cell niche within neural, intestinal and dental tissues and has recently been shown to play a role in regulating bone homeostasis. However, the contribution of EphB/ephrin-B molecules in human MSC function remains to be determined. In the present study, EphB and ephrin-B molecules were expressed by ex vivo expanded human MSC populations and within human bone marrow trephine samples. To elucidate the contribution of EphB/ephrin-B molecules in MSC recruitment, we performed functional spreading and migration assays and showed that reverse ephrin-B signalling inhibited MSC attachment and spreading by activating Src-, PI3Kinase- and JNK-dependent signalling pathways. In contrast, forward EphB2 signalling promoted MSC migration by activating the Src kinase- and Abl-dependent signalling pathways. Furthermore, activation of ephrin-B1 and/or ephrin-B2 molecules expressed by MSC was found to increase osteogenic differentiation, while ephrin-B1 activation promoted chondrogenic differentiation. These observations suggest that EphB/ephrin-B interactions may mediate the recruitment, migration and differentiation of MSC during bone repair.