10 research outputs found
1âAminoanthracene Transduction into Liposomes Driven by Odorant-Binding Protein Proximity
In
this work, the anchorage of pig odorant binding protein (OBP-I)
into liposomal membrane was promoted by the fusion of OBP-I with the
anchor SP-DS3 peptide and with the (GQ)<sub>20</sub> spacer. The presence
of the (GQ)<sub>20</sub> spacer in the construct confers flexibility
to the protein and increases the distance between the OBP binding
site and the liposomal surface. The engineered proteins, OBP::SP-DS3
and OBP::(GQ)<sub>20</sub>::SP-DS3, were produced in <i>Escherichia
coli</i> BL21Â(DE3) and characterized by circular dichroism spectroscopy
and MALDI-TOF. The functionalization of liposomes with the OBP proteins
was performed through ethanol injection, and similar liposomal anchorage
(âŧ92â97%) was found for both OBP constructs. The effect
of OBPsâ proximity to the liposomes membrane on 1-aminoanthracene
(1-AMA, model ligand) transduction was evaluated by measuring the
amount of 1-AMA transduced into liposomes by fluorescence spectroscopy.
While protein flexibility, given by the presence of the (GQ)<sub>20</sub> spacer, seems to influence the binding efficiency, âŧ45% for
OBP::(GQ)<sub>20</sub>::SP-DS3 and âŧ29% for OBP::SP-DS3, the
distance between the proteinsâ binding site and the liposomal
membrane determines their ability to transduce the 1-AMA into the
liposomes (âŧ23% for OBP::SP-DS3 and âŧ19% for OBP::(GQ)<sub>20</sub>::SP-DS3). The anchorage capacity and proximity effect were
confirmed by an experimental control where the wild-type (wt) OBP
was added to the liposomes, resulting in low 1-AMA transduction (âŧ3.5%)
and low binding to OBPwt (âŧ9%). These findings evidence the
effect of anchorage, carrier proteinâs flexibility, and proximity
as key features for the entrapment of molecules into the liposomal
membrane. The developed OBP-based devices are thus promising anchorage
systems for the capture and storage of odors with potential applications
in textile and cosmetic industries
Phosphorylated Silk Fibroin Matrix for Methotrexate Release
Silk-based matrix was produced for delivery of a model
anticancer
drug, methotrexate (MTX). The calculation of net charge of silk fibroin
and MTX was performed to better understand the electrostatic interactions
during matrix formation upon casting. Silk fibroin films were cast
at pH 7.2 and pH 3.5. Protein kinase A was used to prepare phosphorylated
silk fibroin. The phosphorylation content of matrix was controlled
by mixing at specific ratios the phosphorylated and unphosphorylated
solutions. <i>In vitro</i> release profiling data suggest
that the observed interactions are mainly structural and not electrostatical.
The release of MTX is facilitated by use of proteolytic enzymes and
higher pHs. The elevated β-sheet content and crystallinity of
the acidified-cast fibroin solution seem not to favor drug retention.
All the acquired data underline the prevalence of structural interactions
over electrostatical interactions between methotrexate and silk fibroin
Lithium calix[4]arenes: structural studies and use in the ring opening polymerization of cyclic esters
We have structurally characterized a number of lithiated calix[4]arenes, where the bridge in the calix[4]arene is thia (âSâ, LS H4), sulfinyl (âSOâ, LSOH4), sulfonyl (âSO2â, LSO2H4), dimethyleneoxa (âCH2OCH2â, LCOCH4) or methylene (âCH2â, LH4). In the case of L4SH4, interaction with LiOtBu led to the isolation of the complex [Li8(L4S)2(THF)4]5THF (15THF), whilst similar interaction of L4SOH4 led to the isolation of [Li6(L4SOH)2(THF)2]5(THF) (22THF) and [Li6(LCOC)2(HOtBu)2]0.78THF1.22hexane (60.78THF1.22hexane), respectively. In the case of LH4, reaction with LiOtBu in THF afforded a monoclinic polymorph [LH2Li2(thf)(OH2)2]3THF (73THF) of a known triclinic form of the complex, whilst reaction of the de-butylated analogue of LH4, namely de-BuLH4, afforded a polymeric chain structure {[Li5(de-BuL)(OH)(NCMe)3]2MeCN}n (82MeCN). For comparative catalytic studies, the complex [Li6(LPr)2(H2O)2]hexane (9 hexane), where LPr2H2 Âŧ 1,3-di-n-propyloxycalix[4]areneH2, was also prepared. The molecular crystal structures of 1â9 are reported, and their ability to act as catalysts for the ring opening (co-)/polymerization (ROP) of the cyclic esters 3-caprolactone, d-valerolactone, and rac-lactide has been investigated. In most of the cases, complex 6 outperformed the other systems, allowing for higher conversions and/or greated polymer Mn
Insights on the Mechanism of Formation of Protein Microspheres in a Biphasic System
Microspheres of bovine serum albumin (BSA) and silk fibroin
are produced by applying ultrasound in a biphasic system consisting
of an aqueous protein solution and an organic solvent. The protein
microspheres are dispersed in an aqueous media where the protein remains
at the interface covering the organic solvent. This only occurs when
high shear forces are applied that induce changes to force the protein
to the interface. Fourier transform infrared results indicate a large
increase in the content of the β-sheet during the formation
of silk fibroin microspheres. Molecular dynamics simulations show
a clear adaption on the 3D structure of BSA when stabilized at the
interface, without major changes in secondary structure. Further studies
demonstrate that high water content, oil solvents, and larger peptides
with separated and clear hydrophobic and hydrophilic areas lead to
more stable and smaller spheres. This is the first time that these
results are presented. We also present herein the rationale to produce
tailored protein microspheres with a controlled size, controlled charge,
and increased stability
Radical conversion of (1) terephthalic acid to (2) 2-hydroxyterephthalic acid [16].
<p>Radical conversion of (1) terephthalic acid to (2) 2-hydroxyterephthalic acid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0197537#pone.0197537.ref016" target="_blank">16</a>].</p
Normalized extraction efficiencies (%) for ultrasound-assisted extractions: (A) extraction without pretreatment, (B) extraction with one boiling water pretreatment and (C) extraction with two following boiling water pretreatments.
<p>Normalized extraction efficiencies (%) for ultrasound-assisted extractions: (A) extraction without pretreatment, (B) extraction with one boiling water pretreatment and (C) extraction with two following boiling water pretreatments.</p
(A) Relative increase [%] of (+)-pinoresinol measured by LC-ESI-TOF after extraction B (extraction with one boiling water pretreatment) and C (extraction with two following boiling water pretreatments) using an input power of 200 or 400 W with a 15 mm probe depth (B) Dimerization reaction of coniferyl alcohol radicals generated by ultrasound induced radical formation to (+)-pinoresinol; the control consisted on the extraction without ultrasound.
<p>(A) Relative increase [%] of (+)-pinoresinol measured by LC-ESI-TOF after extraction B (extraction with one boiling water pretreatment) and C (extraction with two following boiling water pretreatments) using an input power of 200 or 400 W with a 15 mm probe depth (B) Dimerization reaction of coniferyl alcohol radicals generated by ultrasound induced radical formation to (+)-pinoresinol; the control consisted on the extraction without ultrasound.</p
presentation_1_Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.PDF
<p>If misregulated, macrophage (MĪ)âT cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-MĪ (GM-CSF)- and MĪ colony-stimulating factor (M-CSF)-dependent MĪs have dichotomous effects on T cell activity. While GM-CSF-dependent MĪs show a highly stimulatory activity typical for M1 MĪs, M-CSF-dependent MĪs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory MĪs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβ<sup>+</sup>CD39<sup>+</sup>CD73<sup>+</sup> MĪs, which boosts adenosine production and curtails the dominance of proinflammatory MĪs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the MĪ extracellular purine metabolism as a novel checkpoint in MĪ cell fate decision-making and an attractive target to control pathological MĪs in immune-mediated diseases.</p
video_1_Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.mov
<p>If misregulated, macrophage (MĪ)âT cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-MĪ (GM-CSF)- and MĪ colony-stimulating factor (M-CSF)-dependent MĪs have dichotomous effects on T cell activity. While GM-CSF-dependent MĪs show a highly stimulatory activity typical for M1 MĪs, M-CSF-dependent MĪs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory MĪs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβ<sup>+</sup>CD39<sup>+</sup>CD73<sup>+</sup> MĪs, which boosts adenosine production and curtails the dominance of proinflammatory MĪs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the MĪ extracellular purine metabolism as a novel checkpoint in MĪ cell fate decision-making and an attractive target to control pathological MĪs in immune-mediated diseases.</p
video_2_Extracellular Purine Metabolism Is the Switchboard of Immunosuppressive Macrophages and a Novel Target to Treat Diseases With Macrophage Imbalances.mov
<p>If misregulated, macrophage (MĪ)âT cell interactions can drive chronic inflammation thereby causing diseases, such as rheumatoid arthritis (RA). We report that in a proinflammatory environment, granulocyte-MĪ (GM-CSF)- and MĪ colony-stimulating factor (M-CSF)-dependent MĪs have dichotomous effects on T cell activity. While GM-CSF-dependent MĪs show a highly stimulatory activity typical for M1 MĪs, M-CSF-dependent MĪs, marked by folate receptor β (FRβ), adopt an immunosuppressive M2 phenotype. We find the latter to be caused by the purinergic pathway that directs release of extracellular ATP and its conversion to immunosuppressive adenosine by co-expressed CD39 and CD73. Since we observed a misbalance between immunosuppressive and immunostimulatory MĪs in human and murine arthritic joints, we devised a new strategy for RA treatment based on targeted delivery of a novel methotrexate (MTX) formulation to the immunosuppressive FRβ<sup>+</sup>CD39<sup>+</sup>CD73<sup>+</sup> MĪs, which boosts adenosine production and curtails the dominance of proinflammatory MĪs. In contrast to untargeted MTX, this approach leads to potent alleviation of inflammation in the murine arthritis model. In conclusion, we define the MĪ extracellular purine metabolism as a novel checkpoint in MĪ cell fate decision-making and an attractive target to control pathological MĪs in immune-mediated diseases.</p