Self-assembly of tissue transglutaminase into amyloid-like fibrils using physiological concentration of Ca 2+

Tissue transglutaminase (tTG or TG2) is a member of the transglutaminase family that catalyzes calcium dependent formation of isopeptide bonds. It has been shown that the expression of TG2 is elevated in neurodegenerative diseases such as Parkinson's, Huntington's, and Alzheimer's. We have investigated the self-assembly of TG2 in vitro. First, using software, hot spots, which are prone for aggregation, were identified in domain 2 of the enzyme. Next we expressed and purified recombinant TG2 and its truncated version that contains only the catalytic domain, and examined their amyloidogenic behavior in various conditions including different temperatures and pHs, in the presence of metal ions and Guanosine triphosphate (GTP).To analyze various stages leading to TG2 fibrillation, we employed various techniques including Thioflavin T (ThT) binding assay, Congo-Red, birefringence, Circular Dichroism (CD), 8-anilino-1-naphthalene sulfonic acid (ANS) binding, Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM). Our results indicated that using low concentrations of Ca 2+, TG2 self-assembled into amyloid-like fibrils; this self-assembly occurred at the physiological temperature (37 °C) and at a higher temperature (57 °C). The truncated version of TG2 (domain 2) also forms amyloid-like fibrils only in the presence of Ca 2+. Because amyloid formation has occurred with domain 2 alone where no enzymatic activity was shown, self-cross-linking by the enzyme was ruled out as a mechanism of amyloid induction. The self-assembly of TG2 was not significant with magnesium and zinc ions, indicating specificity of the self-assembly for calcium ions. The calcium role in self-assembly of TG2 into amyloid may be extended to other proteins with similar biophysical properties to produce novel biomaterials. © 2011 American Chemical Society

Effects of different heat processing methods of rapeseed on ruminal and post-ruminal nutrient disappearance

Due to improving the nutritive value of oilseeds and changing their digestion site in ruminants, processing, including heat treatment, seems necessary. The present research was conducted to evaluate the effect of applying heat processing such as roasting, microwaving and autoclaving on nutritive values; the amount and rate of degradability in rumen, and the disappearance of rapeseed nutrients in rumen, post-rumen and total tract. This evaluation was performed using mobile nylon bags techniques; three-step method of digestion and protein CNCPS fraction. A completely randomized design was used to investigate the effect of applying heat processing, and SAS software was used to analyze the data. The field Emission Scanning Electron Microscope was used to monitored the effect of heat treatment on surface of rapeseed. The application of heat processing in this research (roasting, microwaving and autoclaving) had no significant effect on the chemical composition of rapeseed. The results obtained from mobile nylon bags method and three-step digestion method showed that raw rapeseed has the highest disappearance of DM and CP in rumen and therefore has a significant difference with processed seeds (P<0.05). Also, the disappearance of DM and CP of processed rapeseed in intestines was significantly higher than raw seed (P<0.05), and this was higher than other processing for autoclaved rapeseed. According to the results obtained from CNCPS protein fractionation, applying heat processing altering protein fractionation (P<0.05). Applying microwave processing has created cracks in the surface of the rapeseed wall, and this condition was not observed in the wall surface of other heated seeds. In general, it can be said that in addition to increasing the digestibility of rapeseed in the entire gastrointestinal tract, applying heat processing reduces its degradability in the rumen and has increased the disappearance of nutrients in the intestine, that it can be stated the digestion site is altered from rumen to intestine, which can prevent the loss of protein sources in ruminant feed

The general behavior of NLONLO unintegrated parton distributions based on the single-scale evolution and the angular ordering constraint

To overcome the complexity of generalized two hard scale ($k_t$,$\mu$) evolution equation, well known as the $Ciafaloni$, $Catani$, $Fiorani$ and $Marchsini$ ($CCFM$) evolution equations, and calculate the unintegrated parton distribution functions ($UPDF$), $Kimber$, $Martin$ and $Ryskin$ ($KMR$) proposed a procedure based on ($i$) the inclusion of single-scale ($\mu$) only at the last step of evolution and ($ii$) the angular ordering constraint ($AOC$) on the $DGLAP$ terms (the $DGLAP$ collinear approximation), to bring the second scale, $k_t$ into the $UPDF$ evolution equations. In this work we intend to use the $MSTW 2008$ (Martin et al) parton distribution functions (PDF) and try to calculate $UPDF$ for various values of $x$ (the longitudinal fraction of parton momentum), $\mu$ (the probe scale) and $k_t$ (the parton transverse momentum) to see the general behavior of three dimensional $UPDF$ at the $NLO$ level up to the $LHC$ working energy scales ($\mu^2)$. It is shown that there exits some pronounced peaks for the three dimensional $UPDF$ $(f_a(x,k_t))$ with respect to the two variables $x$ and $k_t$ at various energies ($\mu$). These peaks get larger and move to larger values of $k_t$, as the energy ($\mu$) is increased. We hope these peaks could be detected in the $LHC$ experiments at $CERN$ and other laboratories in the less exclusive processes

Electrospun PLLA Nanofiber Scaffolds and Their Use in Combination with BMP-2 for Reconstruction of Bone Defects

Introduction Adequate migration and differentiation of mesenchymal stem cells is essential for regeneration of large bone defects. To achieve this, modern graft materials are becoming increasingly important. Among them, electrospun nanofiber scaffolds are a promising approach, because of their high physical porosity and potential to mimic the extracellular matrix (ECM). Materials and Methods The objective of the present study was to examine the impact of electrospun PLLA nanofiber scaffolds on bone formation in vivo, using a critical size rat calvarial defect model. In addition we analyzed whether direct incorporation of bone morphogenetic protein 2 (BMP-2) into nanofibers could enhance the osteoinductivity of the scaffolds. Two critical size calvarial defects (5 mm) were created in the parietal bones of adult male Sprague-Dawley rats. Defects were either (1) left unfilled, or treated with (2) bovine spongiosa, (3) PLLA scaffolds alone or (4) PLLA/BMP-2 scaffolds. Cranial CT-scans were taken at fixed intervals in vivo. Specimens obtained after euthanasia were processed for histology, histomorphometry and immunostaining (Osteocalcin, BMP-2 and Smad5). Results PLLA scaffolds were well colonized with cells after implantation, but only showed marginal ossification. PLLA/BMP-2 scaffolds showed much better bone regeneration and several ossification foci were observed throughout the defect. PLLA/BMP-2 scaffolds also stimulated significantly faster bone regeneration during the first eight weeks compared to bovine spongiosa. However, no significant differences between these two scaffolds could be observed after twelve weeks. Expression of osteogenic marker proteins in PLLA/BMP-2 scaffolds continuously increased throughout the observation period. After twelve weeks osteocalcin, BMP-2 and Smad5 were all significantly higher in the PLLA/BMP-2 group than in all other groups. Conclusion Electrospun PLLA nanofibers facilitate colonization of bone defects, while their use in combination with BMP-2 also increases bone regeneration in vivo and thus combines osteoconductivity of the scaffold with the ability to maintain an adequate osteogenic stimulus

The expression pattern of VISTA in the PBMCs of relapsing-remitting multiple sclerosis patients: A single-cell RNA sequencing-based study

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS). Dysregulated immune responses have been implicated in MS development. Growing evidence has indicated that inhibitory immune checkpoint molecules can substantially regulate immune responses and maintain immune tolerance. V-domain Ig suppressor of T cell activation (VISTA) is a novel inhibitory immune checkpoint molecule that can suppress immune responses; however, its expression pattern in the peripheral blood mononuclear cells (PBMCs) of relapsing-remitting multiple sclerosis (RRMS) has not thoroughly been studied. Herein, we evaluated Vsir expression in PBMCs of RRMS patients and characterized the expression pattern of the Vsir in the PBMCs of MS patients. Besides, we investigated the effect of fingolimod, IFNβ-1α, glatiramer acetate (GA), and dimethyl fumarate (DMF) on Vsir expression in PBMCs of RRMS patients. Our results have shown that Vsir expression is significantly downregulated in the PBMCs of patients with RRMS. Besides, the single-cell RNA sequencing results have demonstrated that Vsir expression is downregulated in classical monocyte, intermediate monocytes, non-classical monocytes, myeloid DCs (mDC), Plasmacytoid dendritic cells (pDCs), and naive B-cells of PBMCs of MS patients compared to the control. In addition, DMF, IFNβ-1α, and GA have significantly upregulated Vsir expression in the PBMCs of RRMS patients. Collectively, the current study has shed light on Vsir expression in the PBMCs of MS patients; however, further studies are needed to elucidate the significance of VISTA in the mentioned immune cells

Biological Designer Self-Assembling Peptide Nanofiber Scaffolds Significantly Enhance Osteoblast Proliferation, Differentiation and 3-D Migration

A class of self-assembling peptide nanofiber scaffolds has been shown to be an excellent biological material for 3-dimension cell culture and stimulating cell migration into the scaffold, as well as for repairing tissue defects in animals. We report here the development of several peptide nanofiber scaffolds designed specifically for osteoblasts. We designed one of the pure self-assembling peptide scaffolds RADA16-I through direct coupling to short biologically active motifs. The motifs included osteogenic growth peptide ALK (ALKRQGRTLYGF) bone-cell secreted-signal peptide, osteopontin cell adhesion motif DGR (DGRGDSVAYG) and 2-unit RGD binding sequence PGR (PRGDSGYRGDS). We made the new peptide scaffolds by mixing the pure RAD16 and designer-peptide solutions, and we examined the molecular integration of the mixed nanofiber scaffolds using AFM. Compared to pure RAD16 scaffold, we found that these designer peptide scaffolds significantly promoted mouse pre-osteoblast MC3T3-E1 cell proliferation. Moreover, alkaline phosphatase (ALP) activity and osteocalcin secretion, which are early and late markers for osteoblastic differentiation, were also significantly increased. We demonstrated that the designer, self-assembling peptide scaffolds promoted the proliferation and osteogenic differentiation of MC3T3-E1. Under the identical culture medium condition, confocal images unequivocally demonstrated that the designer PRG peptide scaffold stimulated cell migration into the 3-D scaffold. Our results suggest that these designer peptide scaffolds may be very useful for promoting bone tissue regeneration

Tuning chelation by the surfactant-like peptide A6H using predetermined pH values

We examine the self-assembly of a peptide A6H comprising a hexa-alanine sequence A6 with a histidine (H) “head group”, which chelates Zn2+ cations. We study the self assembly of A6H and binding of Zn2+ ions in ZnCl2 solutions, under acidic and neutral conditions. A6H self-assembles into nanotapes held together by a β-sheet structure in acidic aqueous solutions. By dissolving A6H in acidic ZnCl2 solutions, the carbonyl oxygen atoms in A6H chelate the Zn2+ ions and allow for β-sheet formation at lower concentrations, consequently reducing the onset concentration for nanotape formation. A6H mixed with water or ZnCl2 solutions under neutral conditions produces short sheets or pseudocrystalline tapes, respectively. The imidazole ring of A6H chelates Zn2+ ions in neutral solutions. The internal structure of nanosheets and pseudocrystalline sheets in neutral solutions is similar to the internal structure of A6H nanotapes in acidic solutions. Our results show that it is possible to induce dramatic changes in the self-assembly and chelation sites of A6H by changing the pH of the solution. However, it is likely that the amphiphilic nature of A6H determines the internal structure of the self-assembled aggregates independent from changes in chelation

Nonmonotonic fracture behavior of polymer nanocomposites

Polymer composite materials are widely used for their exceptional mechanical properties, notably their ability to resist large deformations. Here, we examine the failure stress and strain of rubbers reinforced by varying amounts of nano-sized silica particles. We find that small amounts of silica increase the fracture stress and strain, but too much filler makes the material become brittle and consequently fracture happens at small deformations. We thus find that as a function of the amount of filler there is an optimum in the breaking resistance at intermediate filler concentrations. We use a modified Griffith theory to establish a direct relation between the material properties and the fracture behavior that agrees with the experiment