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Int. J. Mol. Sci., Volume 17, Issue 9 (September 2016) – 206 articles

Cover Story (view full-size image): Cellulose Binding Domain-Driven Silk–Cellulose Nanomaterial Ordered Assembly Cellulose binding domain (CBD) plays a central role in the higher molecular order of silk–cellulose nanocomposites. Its ability to form dimers and mimic the non-repetitive spider silk terminal function enables formation of aligned nano-silk fibers. At a higher level, CBD specifically binds cellulose and mediates silk–cellulose aligned composite assembly. Cover image by Dr. Noam Atias. View this paper.
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978 KiB  
Review
New Phase of Growth for Xenogeneic-Based Bioartificial Organs
by Zorina Pitkin
Int. J. Mol. Sci. 2016, 17(9), 1593; https://doi.org/10.3390/ijms17091593 - 21 Sep 2016
Cited by 7 | Viewed by 6334
Abstract
In this article, we examine the advanced clinical development of bioartificial organs and describe the challenges to implementing such systems into patient care. The case for bioartificial organs is evident: they are meant to reduce patient morbidity and mortality caused by the persistent [...] Read more.
In this article, we examine the advanced clinical development of bioartificial organs and describe the challenges to implementing such systems into patient care. The case for bioartificial organs is evident: they are meant to reduce patient morbidity and mortality caused by the persistent shortage of organs available for allotransplantation. The widespread introduction and adoption of bioengineered organs, incorporating cells and tissues derived from either human or animal sources, would help address this shortage. Despite the decades of development, the variety of organs studied and bioengineered, and continuous progress in the field, only two bioengineered systems are currently commercially available: Apligraf® and Dermagraft® are both approved by the FDA to treat diabetic foot ulcers, and Apligraf® is approved to treat venous leg ulcers. Currently, no products based on xenotransplantation have been approved by the FDA. Risk factors include immunological barriers and the potential infectivity of porcine endogenous retrovirus (PERV), which is unique to xenotransplantation. Recent breakthroughs in gene editing may, however, mitigate risks related to PERV. Because of its primary role in interrupting progress in xenotransplantation, we present a risk assessment for PERV infection, and conclude that the formerly high risk has been reduced to a moderate level. Advances in gene editing, and more broadly in the field, may make it more likely than ever before that bioartificial organs will alleviate the suffering of patients with organ failure. Full article
(This article belongs to the Special Issue Advances in Cell Transplantation)
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Graphical abstract

Graphical abstract
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<p>Transplantation procedures for various organ types performed in January–June 2016 in comparison to the number of candidates on the transplantation list as of 29 July 2016. The disparity between organ supply and demand is striking, particularly for kidney, liver, and pancreas. Data adopted from UNOS [<a href="#B1-ijms-17-01593" class="html-bibr">1</a>].</p> Full article ">Figure 2
<p>Renal Assist Device (RAD), containing human renal tubule cells (RTC) is part of the two circuit system: a standard hemofilter and a bioreactor (RAD). The ultrafiltrate produced by the hemofilter enters the RAD lumen (A) upon which the RTC have been grown, and then discarded (B); The blood from the hemofilter enters the extracapillary space of the hollow fiber cartridge (C); in the RAD, the blood is separated from the RTC by the semipermeable hollow fiber membrane and returned to the patient (D).</p> Full article ">Figure 3
<p>The hepatAssist liver support system.</p> Full article ">
404 KiB  
Article
McDonald Criteria 2010 and 2005 Compared: Persistence of High Oligoclonal Band Prevalence Despite Almost Doubled Diagnostic Sensitivity
by Philipp Schwenkenbecher, Anastasia Sarikidi, Ulrich Wurster, Paul Bronzlik, Kurt-Wolfram Sühs, Peter Raab, Martin Stangel, Refik Pul and Thomas Skripuletz
Int. J. Mol. Sci. 2016, 17(9), 1592; https://doi.org/10.3390/ijms17091592 - 21 Sep 2016
Cited by 32 | Viewed by 6404
Abstract
The 2010 McDonald criteria were developed to allow a more rapid diagnosis of relapsing-remitting multiple sclerosis (MS) by only one MRI of the brain. Although cerebrospinal fluid (CSF) is not a mandatory part of the latest criteria, the evidence of an intrathecal humoral [...] Read more.
The 2010 McDonald criteria were developed to allow a more rapid diagnosis of relapsing-remitting multiple sclerosis (MS) by only one MRI of the brain. Although cerebrospinal fluid (CSF) is not a mandatory part of the latest criteria, the evidence of an intrathecal humoral immunoreaction in the form of oligoclonal bands (OCB) is crucial in the diagnostic workup. To date, the impact of the 2010 McDonald criteria on the prevalence of OCB has not been investigated. We retrospectively evaluated data of 325 patients with a clinical relapse suggestive of demyelination that were treated in a German university hospital between 2010 and 2015. One hundred thirty-six patients (42%) were diagnosed with MS and 189 patients with CIS when the criteria of 2010 were applied. The criteria of 2005 allowed only 70 patients (22%) to be designated as MS. In contrast, the prevalence of OCB was marginal affected in MS patients with 96% for the criteria of 2010 and 98.5% for the criteria of 2005. In conclusion, OCB are prevalent in most MS patients and reflect the chronic inflammatory nature of the disease. We recommend CSF examination to exclude alternative diagnoses and reevaluation of the diagnosis MS in patients with negative OCB. Full article
(This article belongs to the Special Issue Advances in Multiple Sclerosis 2016)
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Graphical abstract
Full article ">Figure 1
<p>Cerebrospinal fluid results in patients with multiple sclerosis and clinically isolated syndrome according to the McDonald criteria 2010. Graphs show the distribution of cell count (<b>A</b>), lactate (<b>B</b>), total protein (<b>C</b>), and albumin CSF/serum quotients (<b>D</b>). Bars represent median values in each group.</p> Full article ">
2095 KiB  
Review
Structure-Based Reverse Vaccinology Failed in the Case of HIV Because it Disregarded Accepted Immunological Theory
by Marc H. V. Van Regenmortel
Int. J. Mol. Sci. 2016, 17(9), 1591; https://doi.org/10.3390/ijms17091591 - 21 Sep 2016
Cited by 29 | Viewed by 6932
Abstract
Two types of reverse vaccinology (RV) should be distinguished: genome-based RV for bacterial vaccines and structure-based RV for viral vaccines. Structure-based RV consists in trying to generate a vaccine by first determining the crystallographic structure of a complex between a viral epitope and [...] Read more.
Two types of reverse vaccinology (RV) should be distinguished: genome-based RV for bacterial vaccines and structure-based RV for viral vaccines. Structure-based RV consists in trying to generate a vaccine by first determining the crystallographic structure of a complex between a viral epitope and a neutralizing monoclonal antibody (nMab) and then reconstructing the epitope by reverse molecular engineering outside the context of the native viral protein. It is based on the unwarranted assumption that the epitope designed to fit the nMab will have acquired the immunogenic capacity to elicit a polyclonal antibody response with the same protective capacity as the nMab. After more than a decade of intensive research using this type of RV, this approach has failed to deliver an effective, preventive HIV-1 vaccine. The structure and dynamics of different types of HIV-1 epitopes and of paratopes are described. The rational design of an anti-HIV-1 vaccine is shown to be a misnomer since investigators who claim that they design a vaccine are actually only improving the antigenic binding capacity of one epitope with respect to only one paratope and not the immunogenic capacity of an epitope to elicit neutralizing antibodies. Because of the degeneracy of the immune system and the polyspecificity of antibodies, each epitope studied by the structure-based RV procedure is only one of the many epitopes that the particular nMab is able to recognize and there is no reason to assume that this nMab must have been elicited by this one epitope of known structure. Recent evidence is presented that the trimeric Env spikes of the virus possess such an enormous plasticity and intrinsic structural flexibility that it is it extremely difficult to determine which Env regions are the best candidate vaccine immunogens most likely to elicit protective antibodies. Full article
(This article belongs to the Special Issue Reverse Vaccinology)
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Figure 1

Figure 1
<p>Discontinuous epitope of the outer surface protein A of the spirochete <span class="html-italic">Borrelia burdorferi</span> elucidated by X-ray crystallography from a complex with Mab 184.1. (<b>A</b>) Outline of the epitope in yellow; (<b>B</b>) position in space of the residues comprising the epitope. This set of residues cannot be isolated as such from the protein to demonstrate that it possesses binding activity in its own; (<b>C</b>) Parts of the discontinuous epitope and other peptide segments of the protein that may be able to bind Mab 184.1, in which case they would be called continuous epitopes (courtesy of Pernille Haste-Andersen, Danish Technical University).</p> Full article ">Figure 2
<p>The same residues of an antigen can contribute to different overlapping discontinuous epitopes. Two overlapping discontinuous epitopes of lysozyme are recognized by Mabs F9-13.7 (<b>a</b>); and HyHEL10 (<b>b</b>) elucidated by X-ray crystallography. Thirteen residues of lysozyme (in gray) are recognized by both antibodies, albeit with different bonding patterns. The rounded rectangle in gray represents the lysozyme α-helix. The two sets of CDRs are shown in color and have different orientations on the lysozyme surface. Three residues (N77, T89, and G102 highlighted with red circles) are not shared by the two epitopes. Intermolecular contacts are shown by arrows. Mab HyHEL10 forms a salt bridge between lysozyme K97 and residue D32 of the H1 antibody loop. Mab F9-13.7 forms salt bridges between lysozyme residues K97, K96, and H15 and respectively residues E50, D52, and D54 of the H2 antibody loop (adapted from Lescar et al., 1995 [<a href="#B47-ijms-17-01591" class="html-bibr">47</a>]).</p> Full article ">Figure 2 Cont.
<p>The same residues of an antigen can contribute to different overlapping discontinuous epitopes. Two overlapping discontinuous epitopes of lysozyme are recognized by Mabs F9-13.7 (<b>a</b>); and HyHEL10 (<b>b</b>) elucidated by X-ray crystallography. Thirteen residues of lysozyme (in gray) are recognized by both antibodies, albeit with different bonding patterns. The rounded rectangle in gray represents the lysozyme α-helix. The two sets of CDRs are shown in color and have different orientations on the lysozyme surface. Three residues (N77, T89, and G102 highlighted with red circles) are not shared by the two epitopes. Intermolecular contacts are shown by arrows. Mab HyHEL10 forms a salt bridge between lysozyme K97 and residue D32 of the H1 antibody loop. Mab F9-13.7 forms salt bridges between lysozyme residues K97, K96, and H15 and respectively residues E50, D52, and D54 of the H2 antibody loop (adapted from Lescar et al., 1995 [<a href="#B47-ijms-17-01591" class="html-bibr">47</a>]).</p> Full article ">Figure 3
<p>3D structure at 1.8 Å resolution of the anti-lysozyme Fv fragment of Mab D1.3. <b>Upper</b> frame: van der Waals surface of antibody residues (in <b>green</b>) interacting with residues of lysozyme (in <b>pink</b>); <b>Lower</b> frame: the same model showing cavities and channels filled with water molecules (in <b>blue</b>) after complex formation. There are more water molecules at the interface than in the free binding sites and they contribute H-bonds that stabilize the complex. The association is not driven by an hydrophobic entropy effect arising from the extrusion of water from the interface but by a large negative enthalpy arising from H-bonds and van der Waals interactions (Bhat et al., 1994 [<a href="#B65-ijms-17-01591" class="html-bibr">65</a>]).</p> Full article ">Figure 4
<p>Immune serum specificity as a population phenomenon. Individual B cell receptors are shown as having properties similar to immunoglobulin combining regions. For illustrative purposes, these are drawn as being each complementary to four different epitopes or antigens; we suppose that this number is in fact much larger. Stimulation by antigen A causes the cells with A specificity to divide and produce antibodies directed against A. The immune serum produced will therefore react in high titer with antigen A. Each produced immunoglobulin also has other specificities, but because these need not be the same in every molecule, the other specificities, B to Z, will be diluted out and will react only in low titer (from Richards et al., 1975 [<a href="#B182-ijms-17-01591" class="html-bibr">182</a>]).</p> Full article ">Figure 5
<p>Structure of gp120 in different ligation states. (<b>A</b>–<b>C</b>) Comparisons of gp120 structures in (<b>A</b>) the CD4-bound state; (<b>B</b>) the b12 antibody-bound state; and (<b>C</b>) the F105 antibody-bound state. Structures are rendered as ribbon diagrams colored spectrally along the sequence from blue at the N-termini to red at the C-termini. The view is into the face occupied by CD4 interactions. (Figure from Korkut &amp; Hendrickson [<a href="#B211-ijms-17-01591" class="html-bibr">211</a>].)</p> Full article ">
3002 KiB  
Article
Involvement of Ca2+ Signaling in the Synergistic Effects between Muscarinic Receptor Antagonists and β2-Adrenoceptor Agonists in Airway Smooth Muscle
by Kentaro Fukunaga, Hiroaki Kume, Tetsuya Oguma, Wataru Shigemori, Yuji Tohda, Emiko Ogawa and Yasutaka Nakano
Int. J. Mol. Sci. 2016, 17(9), 1590; https://doi.org/10.3390/ijms17091590 - 21 Sep 2016
Cited by 11 | Viewed by 10043
Abstract
Long-acting muscarinic antagonists (LAMAs) and short-acting β2-adrenoceptor agonists (SABAs) play important roles in remedy for COPD. To propel a translational research for development of bronchodilator therapy, synergistic effects between SABAs with LAMAs were examined focused on Ca2+ signaling using simultaneous [...] Read more.
Long-acting muscarinic antagonists (LAMAs) and short-acting β2-adrenoceptor agonists (SABAs) play important roles in remedy for COPD. To propel a translational research for development of bronchodilator therapy, synergistic effects between SABAs with LAMAs were examined focused on Ca2+ signaling using simultaneous records of isometric tension and F340/F380 in fura-2-loaded tracheal smooth muscle. Glycopyrronium (3 nM), a LAMA, modestly reduced methacholine (1 μM)-induced contraction. When procaterol, salbutamol and SABAs were applied in the presence of glycopyrronium, relaxant effects of these SABAs are markedly enhanced, and percent inhibition of tension was much greater than the sum of those for each agent and those expected from the BI theory. In contrast, percent inhibition of F340/F380 was not greater than those values. Bisindolylmaleimide, an inhibitor of protein kinase C (PKC), significantly increased the relaxant effect of LAMA without reducing F340/F380. Iberiotoxin, an inhibitor of large-conductance Ca2+-activated K+ (KCa) channels, significantly suppressed the effects of these combined agents with reducing F340/F380. In conclusion, combination of SABAs with LAMAs synergistically enhances inhibition of muscarinic contraction via decreasing both Ca2+ sensitization mediated by PKC and Ca2+ dynamics mediated by KCa channels. PKC and KCa channels may be molecular targets for cross talk between β2-adrenoceptors and muscarinic receptors. Full article
(This article belongs to the Collection G Protein-Coupled Receptor Signaling and Regulation)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>The intracellular mechanisms of the synergistic effects between β<sub>2</sub>-adrenoceptor agonists and muscarinic receptor antagonists. Not only Ca<sup>2+</sup> sensitization but also Ca<sup>2+</sup> dynamics contributes to the synergistic effect of β<sub>2</sub>-adrenoceptor agonists and muscarinic receptor antagonists (cross talk between β<sub>2</sub>-adrenoceptors and muscarinic receptors). In Ca<sup>2+</sup> dynamic, Ca<sup>2+</sup> influx by VDCE is involved in this phenomenon. VDCE is regulated by membrane potential via K<sub>Ca</sub> channel activity, which is augmented by G<sub>s</sub> coupled to β<sub>2</sub>-adrenoceptor, in contrast, attenuated by G<sub>i</sub> coupled to muscarinic M<sub>2</sub> receptors (dual regulation by G proteins). Increased intracellular Ca<sup>2+</sup> concentration causes contraction by activation of MLCK via Ca<sup>2+</sup>/CAM processes (Ca<sup>2+</sup>-dependent contraction). In Ca<sup>2+</sup> sensitization, PKC is involved in this phenomenon. PKC is activated by muscarinic M<sub>3</sub> receptors. PKC inhibits MP activity via CPI-17 processes. Inactivation of MP causes contraction by increased sensitivity to intracellular Ca<sup>2+</sup> (Ca<sup>2+</sup>-independent contraction). CPI-17 MP is activated by PKC, in contrast, inhibited by PKA. K<sub>Ca</sub> channels and CPI-17 are key molecules for this synergistic effect via the cross talk between these two receptors. This synergism may be caused by Ca<sup>2+</sup> dynamics (tone with changes in concentration of intracellular Ca<sup>2+</sup>) via K<sub>Ca</sub> channel activity reciprocally regulated by G proteins (G<sub>s</sub> and G<sub>i</sub>), and caused by Ca<sup>2+</sup> sensitization (tone without changes in concentration of intracellular Ca<sup>2+</sup>) via CPI-17 reciprocally regulated by protein kinases (PKA and PKC). K<sub>Ca</sub>: large-conductance Ca<sup>2+</sup>-activated K<sup>+</sup> channels. VDCE: voltage-dependent Ca<sup>2+</sup> entry. ACh: acetylcolin, LAMA: long-acting muscarinic receptor antagonist. AC: adenylate cyclase. PKA: protein kinase A. PKC: protein kinase C. CPI-17: C-kinase-potentiated protein phosphatase-1 inhibitor. CaM: calmodulin. MLCK: myosin light chain kinase. MP: myosin phosphatase.</p> Full article ">Figure 2
<p>Involvement of Ca<sup>2+</sup> dynamics and Ca<sup>2+</sup> sensitization in the relaxant effect of β<sub>2</sub>-adrenoceptor agonists. (<b>a</b>,<b>b</b>) Typical samples of continuous recording of tension and F340/F380 demonstrating the inhibitory effect of procaterol (0.1–30 nM) (<b>a</b>) and salbutamol (1–100 nM) (<b>b</b>) on MCh (1 µM)-induced smooth muscle contraction; (<b>c</b>,<b>d</b>) Concentration–response curve for procaterol (0.1–10 nM) (<b>c</b>) and salbutamol (1–100 nM) (<b>d</b>) in tension (□) and F340/F380 (■) in 1 µM MCh-precontracted smooth muscle. Resting state tension and F340/F380 were taken as 0%, and those in each MCh-stimulated state were taken as 100%. MCh, methacholine; PRO, procaterol; SB, salbutamol. **** <span class="html-italic">p</span> &lt; 0.0001; *** <span class="html-italic">p</span> &lt; 0.001; ** <span class="html-italic">p</span> &lt; 0.01; * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 3
<p>Involvement of intracellular Ca<sup>2+</sup> dynamics in the relaxant effect of glycopyrronium. (<b>a</b>) Typical sample of continuous recording of tension and F340/F380 demonstrating the inhibitory effect of glycopyrronium (0.3–10 nM) on MCh (1 µM)-induced contraction; (<b>b</b>) Concentration–response curve for glycopyrronium (0.1–10 nM) in tension (□) and F340/F380 (■) induced by MCh (1 µM). GB, glycopyrronium bromide; MCh, methacholine. ** <span class="html-italic">p</span> &lt; 0.01; * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 4
<p>Effects of combination of procaterol and glycopyrronium on tension and intracellular Ca<sup>2+</sup> concentration induced by muscarinic activation. (<b>a</b>) Typical traces of tension (<b>upper side</b> and F340/F380 (<b>lower side</b>) showing the inhibitory effect of procaterol (1 nM) in the presence of glycopyrronium (3 nM) on MCh (1 µM)-induced contraction; (<b>b</b>–<b>d</b>) Percent inhibition of tension (white columns) and F340/F380 (black columns) in 1 µM MCh-induced contraction under the experimental conditions of 1 nM procaterol (<b>b</b>); 0.1 nM procaterol (<b>c</b>); and 0.3 nM procaterol (<b>d</b>) in the presence of glycopyrronium (3 nM). <b>Left</b>, sum of percent inhibition of tension and F340/F380 by the two agents; <b>Center</b>, BI, expected percent inhibition of tension and F340/F380 calculated by the Bliss independence theory; <b>Right</b>, percent inhibition of tension and F340/F380 with the two agents in combination. PRO, procaterol; GB, glycopyrronium bromide; MCh, methacholine. **** <span class="html-italic">p</span> &lt; 0.0001; *** <span class="html-italic">p</span> &lt; 0.001; ** <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">Figure 5
<p>Effects of salbutamol and glycopyrronium in combination on tension and intracellular Ca<sup>2+</sup> concentration induced by muscarinic activation. (<b>a</b>) Typical sample record of tension (<b>upper side</b>) and F340/F380 (<b>lower side</b>) showing the inhibitory effect of salbutamol (10 nM) in the presence of glycopyrronium (3 nM) against 1 µM MCh-induced contraction; (<b>b</b>,<b>c</b>) Percent inhibition of tension (white columns) and F340/F380 (black columns) in 1 µM MCh-precontracted tissue incubated with 3 nM salbutamol (<b>b</b>) and 10 nM salbutamol (<b>c</b>) in the presence of glycopyrronium (3 nM). <b>Left</b>, sum of percent inhibition of tension and F340/F380 by the two agents; <b>Center</b>, expected percent inhibition of tension and F340/F380 calculated by the Bliss independence theory; <b>Right</b>, percent inhibition of tension and F340/F380 with the two agents in combination. SB, salbutamol; GB, glycopyrronium bromide; MCh, methacholine; BI, Bliss independence. **** <span class="html-italic">p</span> &lt; 0.0001; ** <span class="html-italic">p</span> &lt; 0.01; * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 6
<p>Effects of combination of procaterol and tiotropium on tension and <span class="html-italic">intracellular Ca<sup>2+</sup> concentration</span> induced by muscarinic activation. (<b>a</b>) A typical sample record of tension (<b>upper side</b>) and F340/F380 (<b>lower side</b>) showing the inhibitory effect of procaterol (1 nM) in the presence of tiotropium (1 nM) against 1 µM MCh-induced contraction; (<b>b</b>,<b>c</b>) Percent inhibition of tension (white columns) and F340/F380 (black columns) in 1 µM MCh-precontracted tissue incubated with 1 nM procaterol (<b>b</b>, <span class="html-italic">n</span> = 5) and 0.3 nM procaterol (<b>c</b>, <span class="html-italic">n</span> = 4) in the presence of tiotropium (1 nM). <b>Left</b>, sum of percent inhibition of tension and F340/F380 by the two agents; <b>Center</b>, expected percent inhibition of tension and F340/F380 calculated by the Bliss independence theory; <b>Right</b>, percent inhibition of tension and F340/F380 with the two agents in combination. PRO, procaterol; TIO, tiotropium; MCh, methacholine; BI, Bliss independence. ** <span class="html-italic">p</span> &lt; 0.01; * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 7
<p>Involvement of protein kinase C-induced Ca<sup>2+</sup> sensitization in synergy between β<sub>2</sub>-adrenoceptor agonists and muscarinic receptor antagonists. (<b>a</b>) Typical record of tension (<b>upper side</b>) and F340/F380 (<b>lower side</b>) of the inhibitory effect in combination with procaterol (0.3 nM) and glycopyrronium (3 nM) in the presence of bisindolylmaleimide (10 µM) against 1 µM MCh-induced contraction. In each panel, percent inhibition of tension is represented by white columns, and F340/F380 is represented by black columns; (<b>b</b>) <b>Left</b>, percent inhibition in tension and F340/F380 by bisindolylmaleimide (10 μM); <b>Center</b> , sum of percent inhibition of tension and F340/F380 with bisindolylmaleimide (10 µM) and glycopyrronium (3 nM); <b>Right</b>, percent inhibition of tension and F340/F380 with combination of bisindolylmaleimide (10 µM) and glycopyrronium (3 nM); (<b>c</b>) <b>Left</b>, sum of percent inhibition of tension and F340/F380 with procaterol (0.3 nM) and glycopyrronium (3 nM); <b>Center</b>, percent inhibition of tension and F340/F380 in combination with procaterol (0.3 nM) and glycopyrronium (3 nM); <b>Right</b>, percent inhibition of tension and F340/F380 with combination of procaterol (0.3 nM) and glycopyrronium (3 nM) in the presence of bisindolylmaleimide (10 µM). GB, glycopyrronium bromide; PRO, procaterol; MCh, methacholine; BIS, bisindolylmaleimide. **** <span class="html-italic">p</span> &lt; 0.0001; ** <span class="html-italic">p</span> &lt; 0.01; * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 8
<p>Y-27632-induced Ca<sup>2+</sup> sensitization is not involved in synergy between β<sub>2</sub>-adrenoceptor agonists and muscarinic receptor antagonists. In each panel, percent inhibition of tension is represented by white columns, and F340/F380 is represented by black columns. (<b>a</b>) <b>Left</b>, percent inhibition of tension and F340/F380 with Y-27632 (1 µM); <b>Center</b>, sum of percent inhibition of tension and F340/F380 with Y-27632 (1 µM) and glycopyrronium (3 nM); <b>Right</b>, percent inhibition of tension and F340/F380 with combination of Y-27632 (1 µM) and glycopyrronium (3 nM); (<b>b</b>) <b>Left</b>, sum of percent inhibition of tension and F340/F380 with procaterol (0.3 nM) and glycopyrronium (3 nM); <b>Center</b>, percent inhibition of tension and F340/F380 with combination of procaterol (0.3 nM) and glycopyrronium (3 nM); <b>Right</b>, percent inhibition of tension and F340/F380 with combination of procaterol (0.3 nM) and glycopyrronium (3 nM) in the presence of Y-27632 (1 µM). GB, glycopyrronium bromide; PRO, procaterol; MCh, methacholine. *** <span class="html-italic">p</span> &lt; 0.001; ** <span class="html-italic">p</span> &lt; 0.01; * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 9
<p>Involvement of large-conductance Ca<sup>2+</sup>-activated K<sup>+</sup> channel in synergy between β<sub>2</sub>-adrenoceptor agonists and muscarinic receptor antagonists. (<b>a</b>) Typical record of tension (<b>upper side</b>) and F340/F380 (<b>lower side</b>) showing the inhibitory effect of a combination of procaterol (1 nM) and glycopyrronium (3 nM) in the presence of iberiotoxin (30 nM) on MCh (1 µM)-induced contraction; (<b>b</b>) Percent inhibition of tension (white column) and F340/F380 (black column) with combination of procaterol (1 nM) and glycopyrronium (3 nM) in the absence (<b>left</b>) or presence (<b>right</b>) of iberiotoxin (30 nM); (<b>c</b>) Percent inhibition of tension (whitecolumn) and F340/F380 (black column) with combination of salbutamol (10 nM) and glycopyrronium (3 nM) in the absence (<b>left</b>) or presence (<b>right</b>) of iberiotoxin (30 nM). IbTX, iberiotoxin; GB, glycopyrronium bromide; PRO, procaterol; SB, salbutamol; MCh, methacholine. **** <span class="html-italic">p</span> &lt; 0.0001; *** <span class="html-italic">p</span> &lt; 0.001; ** <span class="html-italic">p</span> &lt; 0.01; * <span class="html-italic">p</span> &lt; 0.05.</p> Full article ">Figure 10
<p>Clinical effectiveness of Combination of β<sub>2</sub>-adrenoceptor agonists and muscarinic receptor antagonists is in the treatment for COPD via Ca<sup>2+</sup> signaling (Ca<sup>2+</sup> dynamics and Ca<sup>2+</sup> sensitization). Since addition of β<sub>2</sub>-adrenoceptor agonists to muscarinic receptor antagonists markedly enhance an inhibition of airway smooth muscle contraction, combination of these two agents are useful to suppression of excessive stimulation to muscarinic receptors induced by acetylcholine production in the airways, which is an fundamental characteristic for COPD. This phenomenon is due to cross talk between these two receptors via K<sub>Ca</sub> channel-induced Ca<sup>2+</sup> dynamics and PKC-induced Ca<sup>2+</sup> sensitization in airway smooth muscle. Therefore, this combination therapy leads to reducing symptoms such as dyspnea on exertion and frequency of exacerbations, and to improving health status and lung function in patients with COPD.</p> Full article ">
588 KiB  
Article
Multiplex Gene Expression Profiling of 16 Target Genes in Neoplastic and Non-Neoplastic Canine Mammary Tissues Using Branched-DNA Assay
by Florenza Lüder Ripoli, Susanne Conradine Hammer, Annika Mohr, Saskia Willenbrock, Marion Hewicker-Trautwein, Bertram Brenig, Hugo Murua Escobar and Ingo Nolte
Int. J. Mol. Sci. 2016, 17(9), 1589; https://doi.org/10.3390/ijms17091589 - 21 Sep 2016
Cited by 4 | Viewed by 5340
Abstract
Mammary gland tumors are one of the most common neoplasms in female dogs, and certain breeds are prone to develop the disease. The use of biomarkers in canines is still restricted to research purposes. Therefore, the necessity to analyze gene profiles in different [...] Read more.
Mammary gland tumors are one of the most common neoplasms in female dogs, and certain breeds are prone to develop the disease. The use of biomarkers in canines is still restricted to research purposes. Therefore, the necessity to analyze gene profiles in different mammary entities in large sample sets is evident in order to evaluate the strength of potential markers serving as future prognostic factors. The aim of the present study was to analyze the gene expression of 16 target genes (BRCA1, BRCA2, FOXO3, GATA4, HER2, HMGA1, HMGA2, HMGB1, MAPK1, MAPK3, MCL1, MYC, PFDN5, PIK3CA, PTEN, and TP53) known to be involved in human and canine mammary neoplasm development. Expression was analyzed in 111 fresh frozen (FF) and in 170 formalin-fixed, paraffin-embedded (FFPE) specimens of neoplastic and non-neoplastic canine mammary tissues using a multiplexed branched-DNA (b-DNA) assay. TP53, FOXO3, PTEN, and PFDN5 expression revealed consistent results with significant low expression in malignant tumors. The possibility of utilizing them as predictive factors as well as for assisting in the choice of an adequate gene therapy may help in the development of new and improved approaches in canine mammary tumors. Full article
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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Figure 1
<p>(<b>A</b>) Exemplary box plot indicating the normalized gene expression of <span class="html-italic">PFDN5</span> in fresh frozen samples in the different histologic groups. Statistically significant differences are indicated by an asterisk (*). Results are classified as significant (* <span class="html-italic">p</span> &lt; 0.05) and very significant (** <span class="html-italic">p</span> &lt; 0.01). The box includes cases from the 25th to the 75th percentile. The horizontal line within the box represents the median and the upper and lower bars are the largest and lowest observed values. Significant differences are revealed between the groups healthy tissue vs. malignant tumors and benign tumors vs. malignant tumors; (<b>B</b>) Normalized expression fold changes (mean) indicating the percentage change of <span class="html-italic">PFDN5</span> in fresh frozen samples in the different histologic groups.</p> Full article ">Figure 2
<p>(<b>A</b>) Exemplary box plot indicating the normalized gene expression of <span class="html-italic">PFDN5</span> in formalin-fixed, paraffin-embedded samples in the different histologic groups. Statistically significant differences are indicated by an asterisk (*). Results are classified as very significant (** <span class="html-italic">p</span> &lt; 0.01) and extremely significant (*** <span class="html-italic">p</span> &lt; 0.001). The box includes cases from the 25th to the 75th percentile. The horizontal line within the box represents the median and the upper and lower bars are the largest and lowest observed values. Significant differences are revealed between the groups lobular hyperplasias vs. malignant tumors and benign tumors vs. malignant tumors. A lower level of expression can be clearly identified when comparing the expression of <span class="html-italic">PFDN5</span> in fresh frozen samples (<a href="#ijms-17-01589-f001" class="html-fig">Figure 1</a>); (<b>B</b>). Normalized expression fold changes (mean) indicating the percentage change of <span class="html-italic">PFDN5</span> in formalin-fixed, paraffin-embedded samples in the different histologic groups.</p> Full article ">
3166 KiB  
Article
Anthocyanin Attenuates Doxorubicin-Induced Cardiomyotoxicity via Estrogen Receptor-α/β and Stabilizes HSF1 to Inhibit the IGF-IIR Apoptotic Pathway
by Pei-Chen Huang, Wei-Wen Kuo, Chia-Yao Shen, Yu-Feng Chen, Yueh-Min Lin, Tsung-Jung Ho, V. Vijaya Padma, Jeng-Fan Lo, Chih-Yang Huang and Chih-Yang Huang
Int. J. Mol. Sci. 2016, 17(9), 1588; https://doi.org/10.3390/ijms17091588 - 21 Sep 2016
Cited by 31 | Viewed by 7499
Abstract
Doxorubicin (Dox) is extensively used for chemotherapy in different types of cancer, but its use is limited to because of its cardiotoxicity. Our previous studies found that doxorubicin-induced insulin-like growth factor II receptor (IGF-IIR) accumulation causes cardiomyocytes apoptosis via down-regulation of HSF1 pathway. [...] Read more.
Doxorubicin (Dox) is extensively used for chemotherapy in different types of cancer, but its use is limited to because of its cardiotoxicity. Our previous studies found that doxorubicin-induced insulin-like growth factor II receptor (IGF-IIR) accumulation causes cardiomyocytes apoptosis via down-regulation of HSF1 pathway. In these studies, we demonstrated a new mechanism through which anthocyanin protects cardiomyoblast cells against doxorubicin-induced injury. We found that anthocyanin decreased IGF-IIR expression via estrogen receptors and stabilized heat shock factor 1 (HSF1) to inhibit caspase 3 activation and apoptosis of cardiomyocytes. Therefore, the phytoestrogen from plants has been considered as another potential treatment for heart failure. It has been reported that the natural compound anthocyanin (ACN) has the ability to reduce the risk of cardiovascular disease (CVD). Here, we demonstrated that anthocyanin acts as a cardioprotective drug against doxorubicin-induced heart failure by attenuating cardiac apoptosis via estrogen receptors to stabilize HSF1 expression and down-regulated IGF-IIR-induced cardiomyocyte apoptosis. Full article
(This article belongs to the Special Issue Anthocyanins)
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<p>Dox stimulated the insulin-like growth factor II receptor (IGF-IIR) apoptotic pathway and repressed the expression of the estrogen receptors (ERs). (<b>A</b>) H9c2 cells are treated with different concentrations of doxorubicin for 24 h; the protein level of CHIP, HSF1, IGF-IIR, active caspase 3, and p-Akt is measured by immunoblotting. Quantification of these results is shown right (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001; (<b>B</b>) H9c2 cells are treated with different concentrations of doxorubicin for 24 h. The cell viability was measured by MTT assay. Quantification of these results is shown (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01; (<b>C</b>) H9c2 cells are treated with different concentrations of doxorubicin for 24 h. The caspase-3 activities were measured by PhiPhiLux<sup>®</sup>-G1D2 assay. Quantification of these results is shown (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001; (<b>D</b>) H9c2 cells are treated with different concentrations of doxorubicin for 24 h. The apoptotic cells were measured by TUNEL assay. Quantification of these results is shown (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001; (<b>E</b>) H9c2 cells are treated with siRNA against CHIP for 24 h, and treated with 1 µM doxorubicin for further 24 hrs. The protein level of CHIP, HSF1, IGF-IIR and p-NFκB is measured by immunoblotting; and (<b>F</b>) H9c2 cells are treated with different concentrations of doxorubicin for 24 h, the protein level of the ERs is measured by immunoblotting. Quantification of these results is shown (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001. These data were obtained from at least three independent experiments and values represent the means ± S.D.</p> Full article ">Figure 2
<p>Anthocyanin enhanced ER expression and attenuated the IGF-IIR apoptotic pathway. (<b>A</b>) After H9c2 cells are treated with 1 µM doxorubicin for 6 and 12 h, they are washed with PBS, and then, fresh medium is added, followed by post-treatment with anthocyanin 20 and 40 µg/mL and incubation of cells for 24 h after doxorubicin treatment. The ERα and ERβ protein levels were measured. Quantification of these results is shown right (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001; (<b>B</b>) after H9c2 cells are treated with 1 µM doxorubicin for 6 and 12 h, they are washed with PBS, and then, fresh medium is added, followed by post-treatment with anthocyanin 20 and 40 µg/mL and incubation of cells for 24 h after doxorubicin treatment. Proteins involved in IGF-IIR apoptotic pathway were measured by immunoblotting. Quantification of these results is shown right (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001; and (<b>C</b>) after H9c2 cells are treated with 1 µM doxorubicin for 6 h, they are washed with PBS and fresh medium is added, followed by post-treatment with anthocyanin at 20 and 40 µg/mL, and incubation of cells for 18 h. The detection of apoptotic cells was determined by a TUNEL assay. Bars = 10 µm. Quantification of these results is shown right (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05 and *** <span class="html-italic">p</span> &lt; 0.001. These data were obtained from at least three independent experiments and values represent the means ± S.D.</p> Full article ">Figure 3
<p>The effects of anthocyanin treatment were attenuated by the estrogen receptor antagonist ICI 182,780. (<b>A</b>) H9c2 cells were pre-treated with 1 µM ICI 182,780 for 1 h, followed by treatment with doxorubicin at 1 µM and incubation for 6 h. Then, the medium was changed to fresh medium, anthocyanin at 40 µg/mL was added, and the cells were incubated for 18 h before measurement by immunoblotting. Quantification of these results is shown right (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001; (<b>B</b>) the level of SIRT1 protein expression after different concentrations of doxorubicin for 24 h was investigated by immunoblotting. Quantification of these results is shown (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05; (<b>C</b>) H9c2 cells were pre-treated with 1 µM ICI 182,780 for 1 h, followed by treatment with doxorubicin at 1 µM and incubation for 6 h. Then, the medium was changed to fresh medium, anthocyanin at 40 µg/mL was added, and the cells were incubated for 18 h before measurement by immunoblotting. Quantification of SIRT1 epxression is shown right (<span class="html-italic">n</span> = 3). * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 4
<p>Echocardiographic assessments and histopathological analysis of rat left ventricular cells after doxorubicin and anthocyanin treatment. (<b>A</b>) The schematic procedure of DOX and HSF1A administration; (<b>B</b>) histopathologic analysis of heart tissue sections stained with H and E. Magnification: 200×; bars = 50 µm. An enlarged interstitium was observed in the doxorubicin-treated rat hearts, and the arrows indicate the myocardial interstitium. The expression of TUNEL + cardiomyocytes and SIRT1 expression were evaluated by immunohistochemistry (IHC) and TUNEL assay. Quantification of TUNEL + cardiomyocytes from each group is shown right (<span class="html-italic">n</span> = 3 per group). These data were obtained from at least three independent experiments and values represent the means ± S.D. ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p> Full article ">Figure 5
<p>Protein expression of the left ventricles of rat hearts after six weeks of doxorubicin and anthocyanin treatment. (<b>A</b>,<b>B</b>) The left ventricles of hearts were excised and homogenized. The cell lysates were quantified and analyzed via immunoblotting. The expression of the IGF-IIR signaling pathway protein and the expression of the apoptosis marker caspase-3 and ERs were estimated via immunoblotting. Quantification of the results is shown right (<span class="html-italic">n</span> = 3 per group). * <span class="html-italic">p</span> &lt; 0.05 and ** <span class="html-italic">p</span> &lt; 0.01; and (<b>C</b>) immunohistochemical detection of CHIP, active caspase-3 expression. Arrows indicated the expression of CHIP and active caspase-3, respectively. Magnification: 400×; bars = 10 µm.</p> Full article ">Figure 6
<p>Schematic diagram of how ACN attenuates doxorubicin-induced cardiomyotoxicity through ERα/β to up-regulate CHIP-mediated HSF1 nuclear translocation and SIRT1-mediated HSF1 activation to inhibit the IGF-IIR apoptotic pathway.</p> Full article ">
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Review
Cancer Cell Fusion: Mechanisms Slowly Unravel
by Felicite K. Noubissi and Brenda M. Ogle
Int. J. Mol. Sci. 2016, 17(9), 1587; https://doi.org/10.3390/ijms17091587 - 21 Sep 2016
Cited by 40 | Viewed by 6028
Abstract
Although molecular mechanisms and signaling pathways driving invasion and metastasis have been studied for many years, the origin of the population of metastatic cells within the primary tumor is still not well understood. About a century ago, Aichel proposed that cancer cell fusion [...] Read more.
Although molecular mechanisms and signaling pathways driving invasion and metastasis have been studied for many years, the origin of the population of metastatic cells within the primary tumor is still not well understood. About a century ago, Aichel proposed that cancer cell fusion was a mechanism of cancer metastasis. This hypothesis gained some support over the years, and recently became the focus of many studies that revealed increasing evidence pointing to the possibility that cancer cell fusion probably gives rise to the metastatic phenotype by generating widespread genetic and epigenetic diversity, leading to the emergence of critical populations needed to evolve resistance to the treatment and development of metastasis. In this review, we will discuss the clinical relevance of cancer cell fusion, describe emerging mechanisms of cancer cell fusion, address why inhibiting cancer cell fusion could represent a critical line of attack to limit drug resistance and to prevent metastasis, and suggest one new modality for doing so. Full article
(This article belongs to the Special Issue Cell Fusion in Cancer)
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<p>Unraveling and targeting mechanisms of cancer cell fusion. (<b>a</b>) Tumor cells and mesenchymal stem cells (MSCs) are capable of spontaneous fusion, which is augmented with hypoxia; (<b>b</b>) Fusion in hypoxic conditions can be facilitated by the engagement of the exposed phosphatidyl serine (PtdSer) of apoptotic cells with PtdSer receptors (PtdSerR) on tumor cells or MSCs. Engagement of this type facilitates podosome formation that ultimately leads to robust activation of the F actin of the attacking fusion partner and MyoII of the receiving cell; (<b>c</b>) Green arrows indicate resisting forces from the actomyosin network, and black arrows indicate pushing forces from invasive protrusions of the attacking cell; (<b>d</b>) Hybrids formed in this way represent an accelerated evolution of sorts, sometimes giving rise to cells with enhanced metastatic potential or the ability to resist drug treatment. Inhibiting the engagement of apoptotic cells via PtdSer represents one potential therapeutic approach to the prevention of tumor cell fusion.</p> Full article ">
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Article
Differential Impact of Hyperglycemia in Critically Ill Patients: Significance in Acute Myocardial Infarction but Not in Sepsis?
by Bernhard Wernly, Michael Lichtenauer, Marcus Franz, Bjoern Kabisch, Johanna Muessig, Maryna Masyuk, Malte Kelm, Uta C. Hoppe and Christian Jung
Int. J. Mol. Sci. 2016, 17(9), 1586; https://doi.org/10.3390/ijms17091586 - 21 Sep 2016
Cited by 13 | Viewed by 5791
Abstract
Hyperglycemia is a common condition in critically ill patients admitted to an intensive care unit (ICU). These patients represent an inhomogeneous collective and hyperglycemia might need different evaluation depending on the underlying disorder. To elucidate this, we investigated and compared associations of severe [...] Read more.
Hyperglycemia is a common condition in critically ill patients admitted to an intensive care unit (ICU). These patients represent an inhomogeneous collective and hyperglycemia might need different evaluation depending on the underlying disorder. To elucidate this, we investigated and compared associations of severe hyperglycemia (>200 mg/dL) and mortality in patients admitted to an ICU for acute myocardial infarction (AMI) or sepsis as the two most frequent admission diagnoses. From 2006 to 2009, 2551 patients 69 (58–77) years; 1544 male; 337 patients suffering from type 2 diabetes (T2DM)) who were admitted because of either AMI or sepsis to an ICU in a tertiary care hospital were investigated retrospectively. Follow-up of patients was performed between May 2013 and November 2013. In a Cox regression analysis, maximum glucose concentration at the day of admission was associated with mortality in the overall cohort (HR = 1.006, 95% CI: 1.004–1.009; p < 0.001) and in patients suffering from myocardial infarction (HR = 1.101, 95% CI: 1.075–1.127; p < 0.001) but only in trend in patients admitted to an ICU for sepsis (HR = 1.030, 95% CI: 0.998–1.062; p = 0.07). Severe hyperglycemia was associated with adverse intra-ICU mortality in the overall cohort (23% vs. 13%; p < 0.001) and patients admitted for AMI (15% vs. 5%; p < 0.001) but not for septic patients (39% vs. 40%; p = 0.48). A medical history of type 2 diabetes (n = 337; 13%) was not associated with increased intra-ICU mortality (15% vs. 15%; p = 0.93) but in patients with severe hyperglycemia and/or a known medical history of type 2 diabetes considered in combination, an increased mortality in AMI patients (intra-ICU 5% vs. 13%; p < 0.001) but not in septic patients (intra-ICU 38% vs. 41%; p = 0.53) could be evidenced. The presence of hyperglycemia in critically ill patients has differential impact within the different etiological groups. Hyperglycemia in AMI patients might identify a sicker patient collective suffering from pre-diabetes or undiagnosed diabetes with its’ known adverse consequences, especially in the long-term. Hyperglycemia in sepsis might be considered as adaptive survival mechanism to hypo-perfusion and consecutive lack of glucose in peripheral cells. AMI patients with hyperglycemic derailment during an ICU-stay should be closely followed-up and extensively screened for diabetes to improve patients’ outcome. Full article
(This article belongs to the Special Issue Diabetic Complications: Pathophysiology, Mechanisms, and Therapies)
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<p>In the overall population severe hyperglycemia (&gt;200 mg/dL) was associated with increased mortality in the long-term (HR 1.74, 95% CI: 1.44–2.09; <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">Figure 2
<p>Patients admitted for sepsis and suffering from severe hyperglycemia did not evidence increased mortality in the long-term (HR 1.13, 95% CI: 0.89–1.44; <span class="html-italic">p</span> = 0.32).</p> Full article ">Figure 3
<p>Myocardial infarction patients with severe hyperglycemia at admission day evidenced a significantly increased mortality in the long-term (HR 2.19, 95% CI: 1.66–2.89; <span class="html-italic">p</span> &lt; 0.001).</p> Full article ">
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Review
Protein Kinases and Parkinson’s Disease
by Syed Jafar Mehdi, Hector Rosas-Hernandez, Elvis Cuevas, Susan M. Lantz, Steven W. Barger, Sumit Sarkar, Merle G. Paule, Syed F. Ali and Syed Z. Imam
Int. J. Mol. Sci. 2016, 17(9), 1585; https://doi.org/10.3390/ijms17091585 - 20 Sep 2016
Cited by 25 | Viewed by 7585
Abstract
Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson’s disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved [...] Read more.
Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson’s disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson’s disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson’s disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules. Full article
(This article belongs to the Special Issue Neuroprotective Strategies 2016)
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<p>In response to various stressors, c-Jun N-terminal kinase (JNK) is activated and phosphorylates c-Jun, which increases the activity of activating protein-1 (AP-1). AP-1 modulates the transcription genes such as <span class="html-italic">Fas ligand</span> (<span class="html-italic">FasL</span>) to induce apoptosis via an extrinsic pathway. JNK also appears to activate non-nuclear substrates (such as Bcl-2 family members) to promote cell death via an intrinsic pathway. 6-OHDA: 6-hydroxydopamine.</p> Full article ">Figure 2
<p>Phosphorylated c-Abl tyrosine phosphorylates parkin at Y143, which leads to the loss of ubiquitin-ligase activity, the accumulation of toxic parkin substrates, and neuronal death. AIMP2: aminoacyl tRNA synthetase complex-interacting multifunctional protein 2; FBP: far up stream element binding protein.</p> Full article ">
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Review
Neuroprotection, Growth Factors and BDNF-TrkB Signalling in Retinal Degeneration
by Atsuko Kimura, Kazuhiko Namekata, Xiaoli Guo, Chikako Harada and Takayuki Harada
Int. J. Mol. Sci. 2016, 17(9), 1584; https://doi.org/10.3390/ijms17091584 - 20 Sep 2016
Cited by 148 | Viewed by 12632
Abstract
Neurotrophic factors play key roles in the development and survival of neurons. The potent neuroprotective effects of neurotrophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor (GDNF) and nerve growth factor (NGF), suggest that they are [...] Read more.
Neurotrophic factors play key roles in the development and survival of neurons. The potent neuroprotective effects of neurotrophic factors, including brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derived neurotrophic factor (GDNF) and nerve growth factor (NGF), suggest that they are good therapeutic candidates for neurodegenerative diseases. Glaucoma is a neurodegenerative disease of the eye that causes irreversible blindness. It is characterized by damage to the optic nerve, usually due to high intraocular pressure (IOP), and progressive degeneration of retinal neurons called retinal ganglion cells (RGCs). Current therapy for glaucoma focuses on reduction of IOP, but neuroprotection may also be beneficial. BDNF is a powerful neuroprotective agent especially for RGCs. Exogenous application of BDNF to the retina and increased BDNF expression in retinal neurons using viral vector systems are both effective in protecting RGCs from damage. Furthermore, induction of BDNF expression by agents such as valproic acid has also been beneficial in promoting RGC survival. In this review, we discuss the therapeutic potential of neurotrophic factors in retinal diseases and focus on the differential roles of glial and neuronal TrkB in neuroprotection. We also discuss the role of neurotrophic factors in neuroregeneration. Full article
(This article belongs to the Special Issue Neurotrophic Factors—Historical Perspective and New Directions)
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<p>Dock3 enhances BDNF-mediated axonal elongation. (<b>A</b>) Retinal ganglion cells were cultured in the presence or absence of BDNF for 3 days. When Dock3 is overexpressed, the BDNF-induced neurite outgrowth is remarkably enhanced (Dock3). Scale bar, 20 μm; GFP, green fluorescent protein; (<b>B</b>) Quantification of (<b>A</b>). <span class="html-italic">n</span> = 50 for each experimental condition. Data are mean ± SEM of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01. Modified from Namekata et al., <span class="html-italic">Proc. Natl. Acad. Sci. USA</span>, 2010, <span class="html-italic">107</span>, 7586–7591, Figure S3, ref. [<a href="#B97-ijms-17-01584" class="html-bibr">97</a>].</p> Full article ">Figure 2
<p>The Dock3 signalling pathways in optic nerve regeneration. Reproduced from Namekata et al., <span class="html-italic">Prog. Retin. Eye Res.</span> 2014, <span class="html-italic">43</span>, 1–16, ref. [<a href="#B95-ijms-17-01584" class="html-bibr">95</a>]. In the RhoG-Elmo pathway, Dock3 is recruited to the plasma membrane by the formation of a RhoG-Elmo-Dock3 complex, leading to translocation of WAVE and Rac1 activation. This signalling pathway stimulates actin dynamics (<b>left</b>); In the TrkB-Fyn pathway, Dock3 promotes both actin dynamics and microtubule dynamics. Recruitment of Dock3 to the plasma membrane by BDNF stimulation induces activation of Rac1/WAVE signalling and promotes actin dynamics (<b>middle</b>); GSK-3β is translocated to the plasma membrane by Dock3, where it is inactivated, leading to stimulation of microtubule dynamics via CRMP-2 and APC (<b>right</b>).</p> Full article ">Figure 3
<p>A schematic diagram of the retina with cell type-specific deletion of TrkB. In the TrkB<sup>GFAP</sup> KO retina, TrkB from Müller glia are selectively deleted. In the TrkB<sup>c−kit</sup> KO mice, TrkB from retinal ganglion cells and amacrine cells are selectively deleted. WT, wild-type.</p> Full article ">Figure 4
<p>Neuroprotective effects of glial and neuronal TrkB signalling in the retina. BDNF exerts neuroprotective effects directly through TrkB expressed in retinal ganglion cells (RGCs) (blue arrow), and/or indirectly through TrkB expressed in Müller glia (red arrow). Stimulation of glial TrkB by BDNF upregulates various neurotrophic factors including BDNF, GDNF, FGF, CNTF and NGF (black arrows). These in turn increase neurotrophic factor production in an autocrine manner (pink arrow) and act on RGCs to promote survival in a paracrine manner (red arrow) [<a href="#B112-ijms-17-01584" class="html-bibr">112</a>,<a href="#B117-ijms-17-01584" class="html-bibr">117</a>]. In addition, stimulation of Müller glia by valproic acid (VPA) upregulates BDNF and NGF, which act on RGCs leading to neuroprotection.</p> Full article ">
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Article
Alterations in Serum Polyunsaturated Fatty Acids and Eicosanoids in Patients with Mild to Moderate Chronic Obstructive Pulmonary Disease (COPD)
by Bjoern Titz, Karsta Luettich, Patrice Leroy, Stephanie Boue, Gregory Vuillaume, Terhi Vihervaara, Kim Ekroos, Florian Martin, Manuel C. Peitsch and Julia Hoeng
Int. J. Mol. Sci. 2016, 17(9), 1583; https://doi.org/10.3390/ijms17091583 - 20 Sep 2016
Cited by 30 | Viewed by 7307
Abstract
Smoking is a major risk factor for several diseases including chronic obstructive pulmonary disease (COPD). To better understand the systemic effects of cigarette smoke exposure and mild to moderate COPD—and to support future biomarker development—we profiled the serum lipidomes of healthy smokers, smokers [...] Read more.
Smoking is a major risk factor for several diseases including chronic obstructive pulmonary disease (COPD). To better understand the systemic effects of cigarette smoke exposure and mild to moderate COPD—and to support future biomarker development—we profiled the serum lipidomes of healthy smokers, smokers with mild to moderate COPD (GOLD stages 1 and 2), former smokers, and never-smokers (n = 40 per group) (ClinicalTrials.gov registration: NCT01780298). Serum lipidome profiling was conducted with untargeted and targeted mass spectrometry-based lipidomics. Guided by weighted lipid co-expression network analysis, we identified three main trends comparing smokers, especially those with COPD, with non-smokers: a general increase in glycero(phospho)lipids, including triglycerols; changes in fatty acid desaturation (decrease in ω-3 polyunsaturated fatty acids, and an increase in monounsaturated fatty acids); and an imbalance in eicosanoids (increase in 11,12- and 14,15-DHETs (dihydroxyeicosatrienoic acids), and a decrease in 9- and 13-HODEs (hydroxyoctadecadienoic acids)). The lipidome profiles supported classification of study subjects as smokers or non-smokers, but were not sufficient to distinguish between smokers with and without COPD. Overall, our study yielded further insights into the complex interplay between smoke exposure, lung disease, and systemic alterations in serum lipid profiles. Full article
(This article belongs to the Section Molecular Toxicology)
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<p>The serum lipidome reflects smoking and chronic obstructive pulmonary disease (COPD) status. (<b>A</b>) Measured lipid classes and their median concentrations; (<b>B</b>) differentially abundant lipid classes. The heatmap shows lipid classes (<span class="html-italic">y</span>-axis) with significant difference in abundance in any of the group comparisons (<span class="html-italic">x</span>-axis). The log 2-fold change is color-coded (see color key). Statistical significance is marked: ×, FDR adjusted <span class="html-italic">p</span>-value &lt;0.20, *, FDR adjusted <span class="html-italic">p</span>-value &lt;0.05. Sex, age group, and body mass index were used as covariates in the statistical model; (<b>C</b>) differentially abundant lipid species. Heatmap as in <a href="#ijms-17-01583-f001" class="html-fig">Figure 1</a><b>B</b>, but for individual lipids. The lipids are grouped by class (color bar on right). See the abbreviations section for lipid nomenclature. Triacylglycerols (TAGs) are summarized by their total composition, e.g., TAG 54:3 total (total fatty acid chain length: number of double bonds; the possible individual fatty acid compositions are in parentheses).</p> Full article ">Figure 2
<p>Lipidomics data allow for classification of the study groups. (<b>A</b>) Performance characteristics of elastic net logistic regressions for five classification tasks: all smokers (S) vs. all non-smokers (N), CS vs. NS, COPD vs. NS, COPD vs. CS, and FS vs. NS. The classifier was assessed by repeated (<span class="html-italic">n</span> = 100) three-fold cross-validation and four performance metrics are shown (mean ± SEM): Matthew’s correlation coefficient (MCC), area under the receiver operator curve (ROC), sensitivity (Sens), and specificity (Spec); (<b>B</b>) average classification predictions of the S vs. N classifier for each individual sample of the four study groups. The cross-validation (CV) predictions for each sample were averaged over the repeats (<span class="html-italic">n</span> = 100) and represented as a boxplot for each study group (black line = median). By default, a sample with a predicted score &gt;0.5 (red, dashed line) was classified as a smoker (S), otherwise as a non-smoker (N); (<b>C</b>) final classification prediction scores for the full S vs. N model. Other details are as in <a href="#ijms-17-01583-f002" class="html-fig">Figure 2</a><b>B</b>; (<b>D</b>) lipids and their coefficients in the S vs. N classifier. An increase in lipids with a negative coefficient (red) tilted the classification of a sample toward smoker; an increase in lipids with a positive coefficient (blue) tilted the classification of a sample toward non-smoker. See abbreviations section for lipid nomenclature.</p> Full article ">Figure 3
<p>Weighted lipid co-expression network reveals structural and functional lipid modules. (<b>A</b>) Lipid modules detected in the weighted lipid co-expression network. Cluster dendrogram shows the hierarchical clustering of the topological overlap metric. Identified modules are color coded and numbered from 0 to 13. The module features are summarized for the main clusters discussed in the text. See <a href="#app1-ijms-17-01583" class="html-app">Figure S1</a> for the complete annotation of the modules; (<b>B</b>) associations between the lipid modules (M0–M13) and the fatty acid composition and lipid classes of their members. See abbreviations section for lipid nomenclature.</p> Full article ">Figure 4
<p>Lipid modules show differences among the study groups. (<b>A</b>) Association of the lipid clusters with study group comparisons. Note that this analysis is based on the module eigenlipids (MEs), which are numbered according to their respective modules; (<b>B</b>) lipids with 20:5 and 22:6 fatty acids were significantly less abundant in the COPD group. The heatmap is as in <a href="#ijms-17-01583-f001" class="html-fig">Figure 1</a>B, but for the concentrations of the different (conjugated) fatty acids.</p> Full article ">Figure 5
<p>Lipid modules correlate with clinical parameters, blood markers, and sputum protein profiles. (<b>A</b>) Correlation between module eigenlipids and clinical measurements: body mass index (BMI), glucose, triacylglycerols, cholesterol measured in blood by clinical chemistry, lipid modifier use, and the lung function parameters forced FEV1 % Pred., FEV1/FVC best ratio, and transfer factor for carbon monoxide (T<sub>L</sub>CO) % Pred. Pearson correlation coefficients are shown (red: positive, blue: negative). Significant correlations with a FDR adjusted <span class="html-italic">p</span>-value &lt;0.05 are marked (“*”); (<b>B</b>) correlation between module eigenlipids and plasma markers. The Pearson correlation coefficients are color-coded and statistically significant correlations with FDR adjusted <span class="html-italic">p</span>-values &lt;0.05 are marked. Only markers with at least one significant correlation are included. For the correlation, the measured marker values/concentrations were log-transformed. See <a href="#app1-ijms-17-01583" class="html-app">Table S3</a> for the group comparison results for these markers. C3, complement factor C3; CRP, C-reactive protein; VDBP, vitamin D-binding protein; FRTN, ferritin; AAT, alpha-1-antitrypsin; (<b>C</b>) Correlation between module eigenlipids and protein expression profiles in induced sputum [<a href="#B8-ijms-17-01583" class="html-bibr">8</a>]. Protein labels are the official symbols of the respective genes (<a href="http://www.genenames.org" target="_blank">www.genenames.org</a>) [<a href="#B30-ijms-17-01583" class="html-bibr">30</a>]. The Pearson correlation coefficients are color-coded and statistically significant correlations with FDR adjusted <span class="html-italic">p</span>-values &lt;0.05 are marked (“*”). Only module eigenlipids with at least one significant correlation are included.</p> Full article ">
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Article
Salinity-Induced Variation in Biochemical Markers Provides Insight into the Mechanisms of Salt Tolerance in Common (Phaseolus vulgaris) and Runner (P. coccineus) Beans
by Mohamad Al Hassan, Mihaela Morosan, María Del Pilar López-Gresa, Jaime Prohens, Oscar Vicente and Monica Boscaiu
Int. J. Mol. Sci. 2016, 17(9), 1582; https://doi.org/10.3390/ijms17091582 - 20 Sep 2016
Cited by 52 | Viewed by 7107
Abstract
The evaluation of biochemical markers is important for the understanding of the mechanisms of tolerance to salinity of Phaseolus beans. We have evaluated several growth parameters in young plants of three Phaseolus vulgaris cultivars subjected to four salinity levels (0, 50, 100, and [...] Read more.
The evaluation of biochemical markers is important for the understanding of the mechanisms of tolerance to salinity of Phaseolus beans. We have evaluated several growth parameters in young plants of three Phaseolus vulgaris cultivars subjected to four salinity levels (0, 50, 100, and 150 mM NaCl); one cultivar of P. coccineus, a closely related species reported as more salt tolerant than common bean, was included as external reference. Biochemical parameters evaluated in leaves of young plants included the concentrations of ions (Na+, K+, and Cl−), osmolytes (proline, glycine betaine, and total soluble sugars), and individual soluble carbohydrates. Considerable differences were found among cultivars, salinity levels, and in their interaction for most traits. In general, the linear component of the salinity factor for the growth parameters and biochemical markers was the most important. Large differences in the salinity response were found, with P. vulgaris cultivars “The Prince” and “Maxidor” being, respectively, the most susceptible and tolerant ones. Our results support that salt stress tolerance in beans is mostly based on restriction of Na+ (and, to a lesser extent, also of Cl−) transport to shoots, and on the accumulation of myo-inositol for osmotic adjustment. These responses to stress during vegetative growth appear to be more efficient in the tolerant P. vulgaris cultivar “Maxidor”. Proline accumulation is a reliable marker of the level of salt stress affecting Phaseolus plants, but does not seem to be directly related to stress tolerance mechanisms. These results provide useful information on the responses to salinity of Phaseolus. Full article
(This article belongs to the Special Issue Pulses)
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<p>Salt stress-induced changes in growth parameters of 5-week-old <span class="html-italic">Phaseolus</span> plants. Salt stress-induced changes in: (<b>a</b>) stem length (%), with the mean stem lengths of control, non-treated plants (<span class="html-italic">Phaseolus vulgaris</span>, cv. “The Prince”: 60.00 cm; cv. “Judía de Franco”: 174.61 cm; cv. “Maxidor”: 44.16 cm; <span class="html-italic">Phaseolus coccineus</span>: 219.00 cm) considered as 100% for each cultivar; (<b>b</b>) number of leaves; (<b>c</b>) fresh weight (%), with the mean fresh weight of control plants (<span class="html-italic">Phaseolus vulgaris</span>, cv. “The Prince”: 31.54 g; cv. “Judía de Franco”: 34.87 g; cv. “Maxidor”: 17.17 g; <span class="html-italic">Phaseolus coccineus</span>: 30.26 g) considered as 100% for each cultivar; (<b>d</b>) water content (%). Measurements were performed after three weeks of treatment. The values shown are means with SD (<span class="html-italic">n</span> = 5). For each cultivar, different lowercase letters indicate significant differences between treatments according to the Tukey test (α = 0.05).</p> Full article ">Figure 2
<p>Salt stress-induced changes in ions levels (Na<sup>+</sup>, Cl<sup>−</sup>, and K<sup>+</sup>) of 5-week-old <span class="html-italic">Phaseolus</span> plants. Salt stress-induced changes in: (<b>a</b>) sodium, (<b>b</b>) chloride, and (<b>c</b>) potassium contents in leaves of <span class="html-italic">Phaseolus</span> plants of the studied cultivars. Measurements were performed after three weeks of treatment. The values shown are means with SD (<span class="html-italic">n</span> = 5). For each cultivar, different lowercase letters indicate significant differences between treatments according to the Tukey test (α = 0.05).</p> Full article ">Figure 3
<p>Salt stress-induced changes in the levels of osmolytes of 5-week-old <span class="html-italic">Phaseolus</span> plants. Salt stress-induced changes in the levels of: (<b>a</b>) proline (Pro); (<b>b</b>) glycine betaine (GB); and (<b>c</b>) total soluble sugars (TSS) in the same samples as in <a href="#ijms-17-01582-f002" class="html-fig">Figure 2</a>. Measurements were performed after three weeks of treatment. The values shown are means with SD (<span class="html-italic">n</span> = 5). For each cultivar, different lowercase letters indicate significant differences between treatments according to the Tukey test (α = 0.05).</p> Full article ">Figure 3 Cont.
<p>Salt stress-induced changes in the levels of osmolytes of 5-week-old <span class="html-italic">Phaseolus</span> plants. Salt stress-induced changes in the levels of: (<b>a</b>) proline (Pro); (<b>b</b>) glycine betaine (GB); and (<b>c</b>) total soluble sugars (TSS) in the same samples as in <a href="#ijms-17-01582-f002" class="html-fig">Figure 2</a>. Measurements were performed after three weeks of treatment. The values shown are means with SD (<span class="html-italic">n</span> = 5). For each cultivar, different lowercase letters indicate significant differences between treatments according to the Tukey test (α = 0.05).</p> Full article ">Figure 4
<p>Salt stress-induced changes in the levels of major soluble carbohydrates of 5-week-old <span class="html-italic">Phaseolus</span> plants. Salt stress-induced changes in the levels of: (<b>a</b>) fructose; (<b>b</b>) sucrose; and (<b>c</b>) <span class="html-italic">myo</span>-inositol, separated by HPLC, in the same samples as in <a href="#ijms-17-01582-f002" class="html-fig">Figure 2</a>. Measurements were performed after three weeks of treatment. The values shown are means with SD (<span class="html-italic">n</span> = 5). For each cultivar, different lowercase letters indicate significant differences between treatments according to the Tukey test (α = 0.05).</p> Full article ">Figure 4 Cont.
<p>Salt stress-induced changes in the levels of major soluble carbohydrates of 5-week-old <span class="html-italic">Phaseolus</span> plants. Salt stress-induced changes in the levels of: (<b>a</b>) fructose; (<b>b</b>) sucrose; and (<b>c</b>) <span class="html-italic">myo</span>-inositol, separated by HPLC, in the same samples as in <a href="#ijms-17-01582-f002" class="html-fig">Figure 2</a>. Measurements were performed after three weeks of treatment. The values shown are means with SD (<span class="html-italic">n</span> = 5). For each cultivar, different lowercase letters indicate significant differences between treatments according to the Tukey test (α = 0.05).</p> Full article ">
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Article
Candida antarctica Lipase B Immobilized onto Chitin Conjugated with POSS® Compounds: Useful Tool for Rapeseed Oil Conversion
by Jakub Zdarta, Marcin Wysokowski, Małgorzata Norman, Agnieszka Kołodziejczak-Radzimska, Dariusz Moszyński, Hieronim Maciejewski, Hermann Ehrlich and Teofil Jesionowski
Int. J. Mol. Sci. 2016, 17(9), 1581; https://doi.org/10.3390/ijms17091581 - 20 Sep 2016
Cited by 15 | Viewed by 6330
Abstract
A new method is proposed for the production of a novel chitin-polyhedral oligomeric silsesquioxanes (POSS) enzyme support. Analysis by such techniques as X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy confirmed the effective functionalization of the chitin surface. The resulting hybrid carriers were used [...] Read more.
A new method is proposed for the production of a novel chitin-polyhedral oligomeric silsesquioxanes (POSS) enzyme support. Analysis by such techniques as X-ray photoelectron spectroscopy (XPS) and Raman spectroscopy confirmed the effective functionalization of the chitin surface. The resulting hybrid carriers were used in the process of immobilization of the lipase type b from Candida antarctica (CALB). Fourier transform infrared spectroscopy (FTIR) confirmed the effective immobilization of the enzyme. The tests of the catalytic activity showed that the resulting support-biocatalyst systems remain hydrolytically active (retention of the hydrolytic activity up to 87% for the chitin + Methacryl POSS® cage mixture (MPOSS) + CALB after 24 h of the immobilization), as well as represents good thermal and operational stability, and retain over 80% of its activity in a wide range of temperatures (30–60 °C) and pH (6–9). Chitin-POSS-lipase systems were used in the transesterification processes of rapeseed oil at various reaction conditions. Produced systems allowed the total conversion of the oil to fatty acid methyl esters (FAME) and glycerol after 24 h of the process at pH 10 and a temperature 40 °C, while the Methacryl POSS® cage mixture (MPOSS) was used as a chitin-modifying agent. Full article
(This article belongs to the Special Issue Frontiers of Marine Biomaterials)
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<p>(<b>a</b>) C 1s spectrum for chitin and chitin-vinyl POSS hybrid; (<b>b</b>) Si 2p spectrum for chitin-vinyl POSS hybrid.</p> Full article ">Figure 2
<p>Raman spectra of α-chitin, POSS compounds and the resulting hybrid materials: (<b>a</b>) chitin-APOSS; (<b>b</b>) chitin-EPOSS; (<b>c</b>) chitin-MPOSS; and (<b>d</b>) chitin-VPOSS.</p> Full article ">Figure 3
<p>Thermogravimetric (TG) curves for α-chitin and for the products obtained by functionalization with various POSS compounds.</p> Full article ">Figure 4
<p>Results of FTIR analysis of the native lipase B from <span class="html-italic">Candida antarctica</span> (CALB), chitin, chitin-POSS hybrid materials, and products after enzyme immobilization: (<b>a</b>) chitin + MPOSS + CALB; (<b>b</b>) chitin + VPOSS + CALB; and (<b>c</b>) chitin + CALB.</p> Full article ">Figure 5
<p>(<b>a</b>) Thermal stability; (<b>b</b>) effect of the pH; (<b>c</b>) reusability; and (<b>d</b>) storage stability of the immobilized lipase B from <span class="html-italic">Candida antarctica</span>.</p> Full article ">Figure 6
<p>Proposed mechanism for the formation of: (<b>a</b>) chitin + VPOSS + CALB; and (<b>b</b>) chitin + MPOSS + CALB systems. Hydrogen bonds are marked by red dotted line.</p> Full article ">
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Review
Clinical Application of Circulating Tumour Cells in Prostate Cancer: From Bench to Bedside and Back
by Luis León-Mateos, María Vieito, Urbano Anido, Rafael López López and Laura Muinelo Romay
Int. J. Mol. Sci. 2016, 17(9), 1580; https://doi.org/10.3390/ijms17091580 - 20 Sep 2016
Cited by 10 | Viewed by 7280
Abstract
Prostate cancer is the most common cancer in men worldwide. To improve future drug development and patient management, surrogate biomarkers associated with relevant outcomes are required. Circulating tumour cells (CTCs) are tumour cells that can enter the circulatory system, and are principally responsible [...] Read more.
Prostate cancer is the most common cancer in men worldwide. To improve future drug development and patient management, surrogate biomarkers associated with relevant outcomes are required. Circulating tumour cells (CTCs) are tumour cells that can enter the circulatory system, and are principally responsible for the development of metastasis at distant sites. In recent years, interest in detecting CTCs as a surrogate biomarker has ghiiukjrown. Clinical studies have revealed that high levels of CTCs in the blood correlate with disease progression in patients with prostate cancer; however, their predictive value for monitoring therapeutic response is less clear. Despite the important progress in CTC clinical development, there are critical requirements for the implementation of their analysis as a routine oncology tool. The goal of the present review is to provide an update on the advances in the clinical validation of CTCs as a surrogate biomarker and to discuss the principal obstacles and main challenges to their inclusion in clinical practice. Full article
(This article belongs to the Special Issue Circulating Tumor Cells)
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<p>Schematic representing the value of CTCs for achieving precision oncology in patients with prostate cancer.</p> Full article ">
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Communication
Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples
by Gladys Arreaza, Ping Qiu, Ling Pang, Andrew Albright, Lewis Z. Hong, Matthew J. Marton and Diane Levitan
Int. J. Mol. Sci. 2016, 17(9), 1579; https://doi.org/10.3390/ijms17091579 - 20 Sep 2016
Cited by 63 | Viewed by 8724
Abstract
In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability [...] Read more.
In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC), circulating tumor DNA (ctDNA), etc.), tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC), in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.). Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research. Full article
(This article belongs to the Special Issue Next-Generation Sequencing for Clinical Application)
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<p>(<b>A</b>) DNA yield (ng/mm<sup>2</sup> tissue) from different labs. QIAamp FFPE kit (QIAGEN Cat# 56404, Hilden, Germany) was used by Labs A, C, D. RecoverAll (ThermoFisher Cat# AM1975, Waltham, MA, USA) was used by Lab B; (<b>B</b>) DNA amplifiability from different labs. Amplifiability values have been converted to the same log 2 scale (<span class="html-italic">C</span><sub>t</sub> value). Values across labs are not comparable due to lack of common reference DNA. Values from the same lab correlate with the quality of FFPE.</p> Full article ">Figure 2
<p>Amplifiability of DNA extracted by different labs measured by Asuragen Quantitative Functional Index (QFI) (<b>A</b>) and RNase P (<b>B</b>) assays. S12 from Lab A was run out and quality control (QC) data is not available. Missing data for S11 is due to poor FFPE block quality and high fat content of the breast tissue. CRC: colorectal cancer.</p> Full article ">
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Review
Recent Advances in Antimicrobial Polymers: A Mini-Review
by Keng-Shiang Huang, Chih-Hui Yang, Shu-Ling Huang, Cheng-You Chen, Yuan-Yi Lu and Yung-Sheng Lin
Int. J. Mol. Sci. 2016, 17(9), 1578; https://doi.org/10.3390/ijms17091578 - 20 Sep 2016
Cited by 231 | Viewed by 12701
Abstract
Human safety and well-being is threatened by microbes causing numerous infectious diseases resulting in a large number of deaths every year. Despite substantial progress in antimicrobial drugs, many infectious diseases remain difficult to treat. Antimicrobial polymers offer a promising antimicrobial strategy for fighting [...] Read more.
Human safety and well-being is threatened by microbes causing numerous infectious diseases resulting in a large number of deaths every year. Despite substantial progress in antimicrobial drugs, many infectious diseases remain difficult to treat. Antimicrobial polymers offer a promising antimicrobial strategy for fighting pathogens and have received considerable attention in both academic and industrial research. This mini-review presents the advances made in antimicrobial polymers since 2013. Antimicrobial mechanisms exhibiting either passive or active action and polymer material types containing bound or leaching antimicrobials are introduced. This article also addresses the applications of these antimicrobial polymers in the medical, food, and textile industries. Full article
(This article belongs to the Special Issue Antimicrobial Polymers 2016)
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<p>The schematic reaction mechanisms of passive and active action of the antimicrobial polymers.</p> Full article ">
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Review
Reactivities of Quinone Methides versus o-Quinones in Catecholamine Metabolism and Eumelanin Biosynthesis
by Manickam Sugumaran
Int. J. Mol. Sci. 2016, 17(9), 1576; https://doi.org/10.3390/ijms17091576 - 20 Sep 2016
Cited by 68 | Viewed by 15186
Abstract
Melanin is an important biopolymeric pigment produced in a vast majority of organisms. Tyrosine and its hydroxylated product, dopa, form the starting material for melanin biosynthesis. Earlier studies by Raper and Mason resulted in the identification of dopachrome and dihydroxyindoles as important intermediates [...] Read more.
Melanin is an important biopolymeric pigment produced in a vast majority of organisms. Tyrosine and its hydroxylated product, dopa, form the starting material for melanin biosynthesis. Earlier studies by Raper and Mason resulted in the identification of dopachrome and dihydroxyindoles as important intermediates and paved way for the establishment of well-known Raper–Mason pathway for the biogenesis of brown to black eumelanins. Tyrosinase catalyzes the oxidation of tyrosine as well as dopa to dopaquinone. Dopaquinone thus formed, undergoes intramolecular cyclization to form leucochrome, which is further oxidized to dopachrome. Dopachrome is either converted into 5,6-dihydroxyindole by decarboxylative aromatization or isomerized into 5,6-dihydroxyindole-2-carboxylic acid. Oxidative polymerization of these two dihydroxyindoles eventually produces eumelanin pigments via melanochrome. While the role of quinones in the biosynthetic pathway is very well acknowledged, that of isomeric quinone methides, however, remained marginalized. This review article summarizes the key role of quinone methides during the oxidative transformation of a vast array of catecholamine derivatives and brings out the importance of these transient reactive species during the melanogenic process. In addition, possible reactions of quinone methides at various stages of melanogenesis are discussed. Full article
(This article belongs to the Special Issue Biochemistry and Mechanisms of Melanogenesis)
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<p>Raper–Mason Pathway for the biosynthesis of eumelanin. Tyrosinase converts tyrosine to dopaquinone via dopa However, dopa is not formed directly from tyrosine but indirectly by the reduction of dopaquinone. Dopaquinone undergoes intrmolecular cyclization producing leucochrome nonenzymatically, which is further oxidized to dopachrome by dopaquinone. Dopachrome is converted to 5,6-dihydroxyindole (DHI) as the major product and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) as the minor product. Further oxidation of DHI to melanochrome and its eventual polymerization leads to melanin pigment.</p> Full article ">Figure 2
<p>Formation of hydroxylated catechols and their further reactions. <span class="html-italic">o</span>-Quinone formed from catechol by two-electron oxidation can undergo addition with water forming hydroxyl catechol, which will readily undergo oxidation to hydroxy-<span class="html-italic">p</span>-quinone as well as semiquinone by reaction with molecular oxygen. Eventually, these quinonoid compounds will exhibit polymerization.</p> Full article ">Figure 3
<p>Nucleophilic addition of internal carboxyl group on quinone. Both carboxymethyl-<span class="html-italic">o</span>-quinone and carboxyethyl-<span class="html-italic">o</span>-quinone exhibit rapid intramolecular cyclization due to the presence of suitably substituted internal carboxyl group yielding cyclic products.</p> Full article ">Figure 4
<p>Reactions of quinone with internal amines. Dopamine quinone formed by the two-electron oxidation of dopamine reacts rapidly with the internal amino group forming leucochrome, which is converted readily to dopaminechrome similar to the dopachrome conversion reaction shown in <a href="#ijms-17-01576-f001" class="html-fig">Figure 1</a>. Interestingly dopaminechrome is also generated during the oxidation of 6-hydroxydopamine as a condensation of product of 6-hydroxydopaminequinone.</p> Full article ">Figure 5
<p>Reactions of quinone with external amines. Quinones formed by the oxidation of catechols react with external amino group forming 4-substituted catechols. These catechols are unstable and form further adducts via substituted quinones.</p> Full article ">Figure 6
<p>Rapid reaction of <span class="html-italic">p</span>-quinone methide. The simplest <span class="html-italic">p</span>-quinone methide arises from the two-electron oxidation of <span class="html-italic">p</span>-cresol. It is extremely unstable and reacts instantaneously with water forming the Michael-1,6-adduct, 4-hydroxybenzyl alcohol.</p> Full article ">Figure 7
<p>Mechanism of dopamine hydroxylation reaction. Initially, oxidation of dopamine to a quinone methide and hydration of the resultant quinone methide was proposed to be the route for the biosynthesis of norepinephrine. However, studies with labeled oxygen revealed that the hydroxylation is accompanied by the incorporation of one atom of molecular oxygen into dopamine by a specific dopamine-β-hydroxylase reaction.</p> Full article ">Figure 8
<p>Initial observation on the quinone methide formation in insect cuticle. Incubation of 4-alkylcatechols with intact cuticle resulted in the covalent binding of catechols through their side chain to the cuticle and generation of side chain hydroxylated products.</p> Full article ">Figure 9
<p>Laccase catalyzed oxidation of catechol. Laccase oxidizes catechols to the semiquinone. Two molecules of semiquinone undergo rapid disproportionation generating the starting material and quinone product.</p> Full article ">Figure 10
<p>Laccase catalyzed quinone methide production from 4-alkyl phenols. Laccase produces quinone methide as the final product in some cases. For example, it oxidizes certain phenolic substrates such as syringaldazine and 2,6-dimethoxy-4-allylphenol to their corresponding semiquinones that undergo disproportionation to quinone methides and starting compounds.</p> Full article ">Figure 11
<p>Mechanism of quinone methide production in insect cuticle. Quinone methide production from 4-alkylcatechols is accomplished in insect cuticle by a two-enzyme system consisting of phenoloxidase, which oxidizes the catechols to <span class="html-italic">o</span>-quinones and quinone isomerase that converts phenoloxidase generated 4-alkylquinones to <span class="html-italic">p</span>-quinone methides (R = alkyl substitution).</p> Full article ">Figure 12
<p>Oxidation chemistry of 3,4-dihydroxybenzylamine and 3,4-dihydroxybenzyl alcohol. 3,4-Dihydroxybenzylamine is easily oxidized to its quinone, which undergoes rapid nonenzymatic isomerization to quinone methide. Quinone methide reacts with water to form carbinolamine and then looses ammonia generating 3,4-dihydroxybenzaldehyde as the final product. A similar conversion of 3,4-dihydroxybenzyl alcohol to 3,4-dihydroxybenzaldehyde also occurs through a quinone methide intermediate.</p> Full article ">Figure 13
<p>Oxidative transformation of 3,4-dihydroxybenzyl cyanide. 3,4-Dihydroxybenzyl cyanide upon oxidation to its quinone undergoes rapid isomerization to quinone methide, which readily polymerizes to uncharacterized products.</p> Full article ">Figure 14
<p>Multiple routes for the oxidative transformation of 3,4-dihydroxyphenylacetic acid. 3,4-Dihydroxyphenylacetic acid (<b>1</b>) is readily oxidized by tyrosinase to its quinone (<b>2</b>). This quinone exhibits three different reactions. It can undergo decarboxylation to the quinone methide (<b>12</b>), which reacts with water producing 3,4-dihydroxybenylalcohol (<b>13</b>). 3,4-dihydroxybenylalcohol is also oxidized by tyrosinase to its quinone (<b>14</b>) and nonenzymatically converted to 3,4-dihydroxybenzaldehyde (<b>11</b>) through quinone methide intermediate (<b>10</b>). Carboxymethyl-<span class="html-italic">o</span>-quinone (<b>2</b>) also isomerizes to quinone methide (<b>7</b>), which generates 3,4-dihydroxymandelic acid (<b>8</b>) by 1,6-addtion of water. Tyrosinase catalyzed oxidation of 3,4-dihydroxy mandelic acid produces the mandeloquinone (<b>9</b>), that is nonenzymatically decarboxylated to the same quinone methide (<b>10</b>) produced by 3,4-dihydroxybenzyl alcohol, thus yielding the same final product, 3,4-dihydroxy benzaldehyde (<b>11</b>). Finally, quinone (<b>2</b>) undergoes rapid intramolecular cyclization to form 2,5,6-trihydroxy benzofuran (<b>4</b>) via furanone (<b>3</b>). This furan is initially believed to form the quinone (<b>5</b>) before getting converted to quinone methide (<b>6</b>), and eventual polymerization.</p> Full article ">Figure 15
<p>Oxidation of transformations of 3,4-dihydroxyphenethyl alcohol. Tyrosinase (<b>A</b>) catalyzes the oxidation of 3,4-dihydroxyphenethyl alcohol to its quinone, which can undergo either enzyme catalyzed isomerization or nonenzymatic isomerization (<b>B</b>) to its quinone methide. Water addition to this quinone methide and reoxidation of 3,4-dihydroxyphenyl glycol and yet another isomerization produces a transient quinone methide that readily yields 2-hydroxy-3,4-dihydroxy acetophenone as the final product.</p> Full article ">Figure 16
<p>Oxidative transformation of dihydrocaffeate derivatives. Dihydrocaffeate derivatives (R” = NHCH<sub>3</sub> or OCH<sub>3</sub>) upon oxidation produce the unstable quinones that exhibit rapid tautomerization to quinone methides. These quinone methides suffer another nonenzymatic isomerization producing more stable caffeic acid derivatives. This reaction also occurs with <span class="html-italic">N</span>-acetyldopa esters (R = NHCOCH<sub>3</sub>; R” = OCH<sub>3</sub> or OCH<sub>2</sub>CH<sub>3</sub>).</p> Full article ">Figure 17
<p>First demonstration of quinone methide production in a dopachrome derivative. α-Methyl dopa methyl ester upon oxidation produces its dopachrome derivative, which rapidly isomerizes to quinone methide under physiological conditions.</p> Full article ">Figure 18
<p>Reaction catalyzed by dopachrome converting enzymes. A base on the enzyme abstracts the β-proton on dopachrome causing isomerization of dopachrome to quinone methide. Quinone methide can readily exhibit another isomerization generating DHICA in the case of mammalian DCT. Since quinone methide is a β,γ-unsaturated acid, it can also readily exhibit decarboxylation. With insect DCDT, the reaction accompanies decarboxylation-coupled aromatization resulting in the formation of DHI.</p> Full article ">Figure 19
<p>Formation of quinone methide adducts of dopamine during its oxidation. Dopamine quinone formed by the oxidation of dopamine undergoes intramolecular cyclization producing the leucochrome and then dopaminechrome. Isomerization of dopaminechrome to its quinone methide and aromatization of the catecholic ring produces DHI. A quinone methide adduct of DHI in its oxidative stage has been characterized from the reaction mixture.</p> Full article ">Figure 20
<p>Oxidation of epinephrine and norepinephrine. Oxidation of epinephrine (R = methyl group) and norepinephrine (R = H) results in their corresponding quinones. These quinones also cyclize and get oxidized to form iminochromes. The iminochromes upon conversion to quinone methide easily isomerize and produce adrenolutin/noradrenolutin, which will also form melanin pigment eventually.</p> Full article ">Figure 21
<p>Possible reactive intermediates generated from DHI. DHI can undergo easily oxidation with molecular oxygen producing reactive semiquinones. In addition it is also susceptible to two-electron oxidation generating quinone, quinone methide and iminochrome.</p> Full article ">Figure 22
<p>Structure of DHI dimers. Oxidative polymerization of DHI yields 2,2′, 2,4′ and 2,7′ coupled DHI dimers as the major products.</p> Full article ">Figure 23
<p>Two-electron oxidation products of DHICA. Two electron oxidation of DHICA will produce imino quinone, quinone methide and iminochrome products.</p> Full article ">Figure 24
<p>Structure of DHICA dimers. Oxidative polymerization of DHIDA yields 4,4′, 4,7′ and 7,7′ coupled DHICA dimers as the major products and 3,4′ and 3,7′ coupled dimers as the minor products.</p> Full article ">Figure 25
<p>Oxidative transformations of dehydro NADA. Dehydro NADA upon oxidation produces directly a highly reactive quinone methide, which crosslinks proteins and chitin (not shown in figure). It also reacts with the starting compound forming a benzodioxan dimer. The accumulated dimer reacts with another molecule of quinone methide forming trimer. Oligomers up to hexamer have been characterized. One-electron oxidation of dehydro NADA generates the semiquinone, which is in equilibrium with the quinone methide radical. Radical coupling in this case produces the same dimer but not the oligomers.</p> Full article ">Figure 26
<p>Oxidative dimerization of 1,2-dehydro-<span class="html-italic">N</span>-acetyldopa methyl ester. Oxidation of 1,2-Dehydro-<span class="html-italic">N</span>-acetyldopa methyl ester by tyrosinase results in the production of conventional quinone product and not the quinone methide analog. This quinone also dimerizes and produces the same benzodioxan type adduct as that produced by dehydro NADA but by an ionic Diels–Alder type reaction.</p> Full article ">Figure 27
<p>Modified Raper–Mason Pathway for eumelanin biosynthesis. Tyrosinase (<b>A</b>) converts tyrosine to dopaquinone and oxidizes dopa to dopaquinone. External addition of cysteine to dopaquinone and the oxidative polymerization of cysteinyldopa result in the production of pheomelanin pigments (not shown in Figure). Intramolecular cyclization of dopaquinone produces leucochrome, which is further oxidized to dopachrome by nonenzymatic reactions (<b>B</b>). Dopachrome is isomerized to transient quinone methide (1) that is converted to either DHICA by mammalian DCT (<b>C</b>) or DHI by insect DCDT (<b>C′</b>). Oxidation of these two indoles by tyrosinase or nonenzymatic reactions or by DHICA oxidase (<b>D</b>) produces among other reactive species, quinone methides, which will have a major role in eventual polymerization of these monomeric compounds (R = H for DHI derivative; and COOH for DHICA derivative).</p> Full article ">
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Review
Molecular Pathogenesis of NASH
by Alessandra Caligiuri, Alessandra Gentilini and Fabio Marra
Int. J. Mol. Sci. 2016, 17(9), 1575; https://doi.org/10.3390/ijms17091575 - 20 Sep 2016
Cited by 159 | Viewed by 15765
Abstract
Nonalcoholic steatohepatitis (NASH) is the main cause of chronic liver disease in the Western world and a major health problem, owing to its close association with obesity, diabetes, and the metabolic syndrome. NASH progression results from numerous events originating within the liver, as [...] Read more.
Nonalcoholic steatohepatitis (NASH) is the main cause of chronic liver disease in the Western world and a major health problem, owing to its close association with obesity, diabetes, and the metabolic syndrome. NASH progression results from numerous events originating within the liver, as well as from signals derived from the adipose tissue and the gastrointestinal tract. In a fraction of NASH patients, disease may progress, eventually leading to advanced fibrosis, cirrhosis and hepatocellular carcinoma. Understanding the mechanisms leading to NASH and its evolution to cirrhosis is critical to identifying effective approaches for the treatment of this condition. In this review, we focus on some of the most recent data reported on the pathogenesis of NASH and its fibrogenic progression, highlighting potential targets for treatment or identification of biomarkers of disease progression. Full article
(This article belongs to the Special Issue Non-Alcoholic Fatty Liver Disease Research 2016)
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<p>Outline of the pathogenesis of NASH. Signals generated inside the liver as a consequence of increased lipid accumulation, together with signals derived from extrahepatic organs cooperate to induce inflammation and fibrosis. FFA, free fatty acids; PAMPs, pathogen-associated molecular patterns; ER, endoplasmic reticulum; ROS, reactive oxygen species; HSC, hepatic stellate cell.</p> Full article ">Figure 2
<p>Epigenetic pathways implicated in the pathogenesis of NASH. The major pathways and their main effectors are depicted.</p> Full article ">Figure 3
<p>Inflammasomes and the liver. In steatosis, hepatic damage leads to generation of damage-associated molecular pattern (DAMPs), while alterations in microbiota lead to increased availability of pathogen-associated molecular patterns (PAMPs). DAMPs and PAMPs act on receptors localized on liver cells leading to activation of different inflammasomes and release of cytokines implicated in NASH. NLRP3: NOD-like receptor family, pyrin domain containing 3; AIM2: Abscent in melanoma 2.</p> Full article ">
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Article
Alliin Attenuated RANKL-Induced Osteoclastogenesis by Scavenging Reactive Oxygen Species through Inhibiting Nox1
by Yueqi Chen, Jingjing Sun, Ce Dou, Nan Li, Fei Kang, Yuan Wang, Zhen Cao, Xiaochao Yang and Shiwu Dong
Int. J. Mol. Sci. 2016, 17(9), 1516; https://doi.org/10.3390/ijms17091516 - 20 Sep 2016
Cited by 38 | Viewed by 6267
Abstract
The healthy skeleton requires a perfect coordination of the formation and degradation of bone. Metabolic bone disease like osteoporosis is resulted from the imbalance of bone formation and/or bone resorption. Osteoporosis also reflects lower level of bone matrix, which is contributed by up-regulated [...] Read more.
The healthy skeleton requires a perfect coordination of the formation and degradation of bone. Metabolic bone disease like osteoporosis is resulted from the imbalance of bone formation and/or bone resorption. Osteoporosis also reflects lower level of bone matrix, which is contributed by up-regulated osteoclast-mediated bone resorption. It is reported that monocytes/macrophage progenitor cells or either hematopoietic stem cells (HSCs) gave rise to multinucleated osteoclasts. Thus, inhibition of osteoclastic bone resorption generally seems to be a predominant therapy for treating osteoporosis. Recently, more and more natural compounds have been discovered, which have the ability of inhibiting osteoclast differentiation and fusion. Alliin (S-allyl-l-cysteine sulfoxides, SACSO) is the major component of aged garlic extract (AGE), bearing broad-spectrum natural antioxidant properties. However, its effects on bone health have not yet been explored. Hence, we designed the current study to explore its effects and role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast fusion and differentiation. It was revealed that alliin had an inhibitory effect in osteoclasteogenesis with a dose-dependent manner via blocking the c-Fos-NFATc1 signaling pathway. In addition, alliin decreased the generation of reactive oxygen species (ROS) and down-regulated the expression of NADPH oxidase 1 (Nox1). The overall results revealed that alliin could be a potential therapeutic agent in the treatment of osteoporosis. Full article
(This article belongs to the Special Issue The Mechanism of Action of Food Components in Disease Prevention)
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<p>Alliin had no cytotoxicity during RANKL-induced osteoclastogenesis. (<b>A</b>) The chemical formula of alliin; (<b>B</b>) CCK-8 analysis of cell viability of RAW264.7 cells treated with various concentrations of alliin for 48 h; and (<b>C</b>) CCK-8 analysis of cell viability of RAW264.7 cells treated with various concentrations of alliin for 72 h. Data in the figures represent the mean ± SD. N.S. represents the difference was not statistically significant. It was based on one way ANOVA.</p> Full article ">Figure 2
<p>Alliin had an inhibitory effect on TRAP-positive cell formation. (<b>A</b>) Representative light microscope images of RAW264.7 cells cultured in a 96-well plate in the presence of RANKL (50 ng/mL) and M-CSF (50 ng/mL) with different concentration of alliin for 72 h were stained for TRAP (<b>red</b>). Scale bar represents 200 µm. The black arrows pointed out the representative TRAP (+) cells; and (<b>B</b>) quantitative analysis shows the number of TRAP (+) cells with more than three nuclei in each well (96-well plate). Data in the figures represent the mean ± SD. ** (<span class="html-italic">p</span>-value &lt; 0.01) based on one way ANOVA.</p> Full article ">Figure 3
<p>Alliin inhibited RANKL-induced osteoclast fusion and differentiation in a dose-dependent manner. (<b>A</b>) RAW264.7 cells were pretreated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for about 72 h along with a range of alliin concentrations (0, 1, 10 µg/mL), following focal and adhesion staining, and finally photographed. The nuclei were stained for double immunofluorescence microscopy by DAPI (<b>blue</b>) and Vinculin monoclonal antibody (<b>red</b>). Each experiment was performed thrice. Scale bar was at 200 µm; (<b>B</b>) the quantitative test for the TRAP (+) cells having multiple nuclei in each well of 96-well plate; and (<b>C</b>) the total RNA extracted from RAW264.7 cells during RANKL-induced osteoclastogenesis treated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for 72 h with varying doses of alliin (0, 1, 10, 100 µg/mL). Relative mRNA expression levels of NFATc1, c-Fos, MMP-9, CD9, DC-STAMP, OC-STAMP, TRAP, and RANK against GAPDH are shown. Data in the figures represent the averages ± SD. * (<span class="html-italic">p</span>-value &lt; 0.05); ** (<span class="html-italic">p</span>-value &lt; 0.01) or *** (<span class="html-italic">p</span>-value &lt; 0.001) based on one way ANOVA.</p> Full article ">Figure 4
<p>Alliin inhibited osteoclastic RANKL-induced bone resorption. (<b>A</b>) RAW264.7 cells cultured on bovine slices, were pretreated with RANKL and M-CSF (50 ng/mL each) for five days, along with a range of alliin concentrations (0, 1, 10 µg/mL), following focal and adhesion staining, and finally photographed. Experiments were done in triplicate. Scale bar represents 400 µm; (<b>B</b>) quantification of the bone resorption area on the bone slices; (<b>C</b>) representative images of RAW264.7 cells cultured on osteo assay surface 96-well plates treated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for five days with varying concentrations of alliin (0, 1, 10 µg/mL) followed removal of osteoclasts. Each experiment was performed thrice. The resorption area can be measured by 400 µm bars; and (<b>D</b>) measurement of the bone resorption area at the osteo surface. Data represented here at respective places is the averages ± SD. ** (<span class="html-italic">p</span>-value &lt; 0.01) based on one way ANOVA.</p> Full article ">Figure 5
<p>Alliin scavenged ROS production in a dose-dependent way. (<b>A</b>) ROS detection by fluorescent probe DCFH-DA. Scale bar represents 200 µm; and (<b>B</b>) quantitative analysis of ROS (+) cells in each well (96-well plate). Data in the figures represent the mean ± SD. ** (<span class="html-italic">p</span>-value &lt; 0.01) and *** (<span class="html-italic">p</span>-value &lt; 0.001) based on one way ANOVA.</p> Full article ">Figure 6
<p>Alliin inhibited RANKL-induced osteoclast differentiation and fusion through inhibiting Nox1 activity and blocking the c-Fos-NFATc1 signaling pathway. Total protein was extracted from RAW264.7 cells during RANKL-induced osteoclastogenesis treated with RANKL (50 ng/mL) and M-CSF (50 ng/mL) for 72 h with varying doses of alliin (0 µg/mL, 1 µg/mL, 10 µg/mL). (<b>A</b>) Representative Western blot images of c-Fos, NFATc1, Nox1, and β-actin from RAW264.7 cells in different groups; (<b>B</b>) relative intensity of expression level of c-FOS, NFATc1, and Nox1 against β-actin; and (<b>C</b>) relative mRNA expression levels of Nox1 against GAPDH. Data in the figures represent the mean ± SD. * (<span class="html-italic">p</span>-value &lt; 0.05); ** (<span class="html-italic">p</span>-value &lt; 0.01) based on one way ANOVA.</p> Full article ">
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Article
Effect of Thyrotropin on Osteopontin, Integrin αvβ3, and VCAM-1 in the Endothelium via Activation of Akt
by Yumeng Yan, Fengwei Jiang, Yaxin Lai, Haoyu Wang, Aihua Liu, Chuyuan Wang, Yuanyuan Zhang, Weiping Teng and Zhongyan Shan
Int. J. Mol. Sci. 2016, 17(9), 1484; https://doi.org/10.3390/ijms17091484 - 20 Sep 2016
Cited by 7 | Viewed by 5649
Abstract
Numerous epidemiological studies have shown that subclinical hypothyroidism (SCH) can impair endothelial function and cause dyslipidemia. Studies have evaluated the effects of thyroid stimulating hormone (TSH) on endothelial cells, but the mechanism underlying the proatherosclerotic effect of increased TSH levels remains unclear. In [...] Read more.
Numerous epidemiological studies have shown that subclinical hypothyroidism (SCH) can impair endothelial function and cause dyslipidemia. Studies have evaluated the effects of thyroid stimulating hormone (TSH) on endothelial cells, but the mechanism underlying the proatherosclerotic effect of increased TSH levels remains unclear. In the present study, SCH rat models were established in thyroidectomized Wistar rats that were given ʟ-T4 daily. The results showed that in vivo, the expression of osteopontin (OPN) vascular cell adhesion molecule (VCAM-1), and levels of integrin αvβ3 in the aortic tissue in SCH and Hypothyroidism (CH) groups was higher than in the control group. However, the effect in the SCH group was higher than in the CH group. In vitro, results showed that different concentration and time gradients of TSH stimulation could increase the expression of OPN, VCAM-1, and integrin αvβ3, and this was accompanied by extracellular signal regulated kinase 1/2 (Erk1/2) and Akt activation in human umbilical vein endothelial cells (HUVECs). TSH induced elevation of these proatherosclerotic factors was partially suppressed by a specific Akt inhibitor but not by a specific Erk inhibitor. Findings suggested that the endothelial dysfunction caused by SCH was related to increased proatherosclerotic factors induced by TSH via Akt activation. Full article
(This article belongs to the Section Biochemistry)
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<p>Osteopontin (OPN), integrin α<sub>v</sub>β<sub>3</sub>, and vascular cell adhesion molecule (VCAM-1) expression in aorta; tissues of control (CON), subclinical hypothyroidism (SCH), and hyoithyroidism (CH) rats. (<b>A</b>) The bands depict representative findings regarding protein expression levels of OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 protein expression in the aortic tissues in CON, SCH, and CH rats. These were evaluated by Western blotting using protein extracted from 20 mg of aorta tissues; (<b>B</b>) The bar graph shows the results of the semiquantitative measurements of OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1. Values are shown as the means ± SEM. * <span class="html-italic">p</span> &lt; 0.05 versus CON group; ** <span class="html-italic">p</span> &lt; 0.01 versus CON group.</p> Full article ">Figure 2
<p>OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 expression in the aorta endothelium from CON, SCH, and CH rats, as indicated by immunostaining. (<b>A</b>) Immunostaining of OPN, integrin α<sub>v</sub>β<sub>3</sub> and VCAM-1 in the endothelia of aortas from CON, SCH, and CH rats; (<b>B</b>) Semiquantitative analysis of the difference in OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 expression in the endothelia of the aortas from CON, SCH, and CH rats. Data were presented as the means ± SEM. * <span class="html-italic">p</span> &lt; 0.05 versus CON group; ** <span class="html-italic">p</span> &lt; 0.01 versus CON group. Scale bar = 50 µm.</p> Full article ">Figure 3
<p>The figure shows the endothelium in the CON, SCH, and CH groups. (<b>A</b>) Normal endothelial cell (E) in the CON group; (<b>B</b>) Dissolved endothelial cell membrane and abnormal nuclear (N) feature in the SCH group; (<b>C</b>) Dissolved endothelial cell membrane and heterogeneous chromatin edge accumulation in the CH group; (<b>D</b>) Endothelial cells shed and parts of the elastic membrane exposed in the CH group (→). Scale bar = 4 µm.</p> Full article ">Figure 4
<p>Effects of concentrations of TSH (0, 0.1, 1, 10, 100 mIU/mL) in HUVECs over 24 h. (<b>A</b>) The bands depict representative findings of OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 protein expression levels in HUVECs stimulated by different concentration of TSH; (<b>B</b>) The bar graphs showed the results of the semiquantitative measurements of OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1, respectively; (<b>C</b>) The bar graphs showed the results of the quantitative analysis of OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 from real-time PCR. The OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 mRNA levels were expressed as ratios relative to GAPDH. The results are shown as the means ± SEM of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 versus control; ** <span class="html-italic">p</span> &lt; 0.01 versus control.</p> Full article ">Figure 5
<p>The effects of 10 mIU/mL TSH for 0, 2, 6, 12, 24, and 48 h in HUVECs. (<b>A</b>) The bands depicted representative protein expression level findings for OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 in HUVECs stimulated by different durations of TSH; (<b>B</b>) The bar graphs show the results of the semiquantitative measurements of OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1; (<b>C</b>) The bar graphs show the results of the quantitative analysis of OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 from real-time PCR. The OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 mRNA levels are expressed as ratios relative to GAPDH. The results are shown as the means ± SEM of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 versus control; ** <span class="html-italic">p</span> &lt; 0.01 versus control.</p> Full article ">Figure 6
<p>Effects of specific inhibitors on TSH-induced OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 upregulation in HUVECs. HUVECs were pretreated with either 20 µmol/L LY294002 or 20 µmol/L PD98059 for 1 h and then treated with 10 mIU/mL TSH for 24 h. (<b>A</b>) The bands depicted representative findings for the protein expression levels of p-Akt, OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 in HUVECs as determined by Western blotting; (<b>B</b>) The bar graphs show the results of the semiquantitative measurements of of p-Akt, OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1; (<b>C</b>) The bands depicted representative findings for protein expression levels of p-Erk, OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1 in HUVECs, as determined by Western blotting; (<b>D</b>) The bar graphs showed the results of the semiquantitative measurements of p-Erk, OPN, integrin α<sub>v</sub>β<sub>3</sub>, and VCAM-1. The results are shown as the means ± SEM of three independent experiments. * <span class="html-italic">p</span> &lt; 0.05 versus control; ** <span class="html-italic">p</span> &lt; 0.01 versus control.</p> Full article ">
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Article
Metabolomics Analysis of the Larval Head of the Silkworm, Bombyx mori
by Yi Li, Xin Wang, Quanmei Chen, Yong Hou, Qingyou Xia and Ping Zhao
Int. J. Mol. Sci. 2016, 17(9), 1460; https://doi.org/10.3390/ijms17091460 - 20 Sep 2016
Cited by 19 | Viewed by 5960
Abstract
The head, which performs many biological functions, is the most complicated structure of an insect. Development, locomotor behavior, food intake, environmental sensing, and signal transduction are all controlled by the insect’s head. As a well-studied insect in Lepidoptera, the silkworm head has an [...] Read more.
The head, which performs many biological functions, is the most complicated structure of an insect. Development, locomotor behavior, food intake, environmental sensing, and signal transduction are all controlled by the insect’s head. As a well-studied insect in Lepidoptera, the silkworm head has an additional function of spinning silk fibers. To understand which molecules are involved in these physiological activities, we performed a metabolomics analysis of silkworm heads. By integrating GC-MS and LC-MS/MS, 90 metabolites were identified in the larval heads of silkworms. These were classified into 13 categories, including amino acids, sugars, organic acids, nucleotides, alcohols, and fatty acids. Informatics analysis revealed that these metabolites are involved in cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, organismal systems, and other pathways. The identified metabolites and pathways are involved in biological processes such as signal transduction, carbohydrate metabolism, endocrine activities, and sensory activities; reflecting the functions of various organs in silkworm heads. Thus, our findings provide references which elucidate the potential functions of the silkworm head and will be of great value for the metabolomics research of silkworms and other insects. Full article
(This article belongs to the Section Biochemistry)
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<p>Compounds that can be identified using GC-MS and LC-MS/MS.</p> Full article ">Figure 2
<p>Classification of the identified metabolites. (<b>A</b>) The numbers of metabolites identified by GC-MS or LC-MS/MS; (<b>B</b>) The percentage of metabolites identified by GC-MS and LC-MS/MS.</p> Full article ">Figure 3
<p>Monoisotopic mass distribution of the identified metabolites.</p> Full article ">Figure 4
<p>Categories of pathways in which the identified metabolites are involved.</p> Full article ">
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Correction
Correction: G. Bradley Schaefer. Clinical Genetic Aspects of ASD Spectrum Disorders. Int. J. Mol. Sci. 2016, 17, 180
by G. Bradley Schaefer
Int. J. Mol. Sci. 2016, 17(9), 1572; https://doi.org/10.3390/ijms17091572 - 19 Sep 2016
Cited by 15 | Viewed by 3278
Abstract
The author wishes to make a change to the published paper [1].[...] Full article
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Article
DNA Interaction Studies of Selected Polyamine Conjugates
by Marta Szumilak, Anna Merecz, Malgorzata Strek, Andrzej Stanczak, Tadeusz W. Inglot and Boleslaw T. Karwowski
Int. J. Mol. Sci. 2016, 17(9), 1560; https://doi.org/10.3390/ijms17091560 - 19 Sep 2016
Cited by 15 | Viewed by 5225
Abstract
The interaction of polyamine conjugates with DNA double helix has been studied. Binding properties were examined by ethidium bromide (EtBr) displacement and DNA unwinding/topoisomerase I/II (Topo I/II) activity assays, as well as dsDNA thermal stability studies and circular dichroism spectroscopy. Genotoxicity of the [...] Read more.
The interaction of polyamine conjugates with DNA double helix has been studied. Binding properties were examined by ethidium bromide (EtBr) displacement and DNA unwinding/topoisomerase I/II (Topo I/II) activity assays, as well as dsDNA thermal stability studies and circular dichroism spectroscopy. Genotoxicity of the compounds was estimated by a comet assay. It has been shown that only compound 2a can interact with dsDNA via an intercalative binding mode as it displaced EtBr from the dsDNA-dye complex, with Kapp = 4.26 × 106 M−1; caused an increase in melting temperature; changed the circular dichroism spectrum of dsDNA; converted relaxed plasmid DNA into a supercoiled molecule in the presence of Topo I and reduced the amount of short oligonucleotide fragments in the comet tail. Furthermore, preliminary theoretical study has shown that interaction of the discussed compounds with dsDNA depends on molecule linker length and charge distribution over terminal aromatic chromophores. Full article
(This article belongs to the Section Biochemistry)
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<p>Chemical structure of examined compounds.</p> Full article ">Figure 2
<p>Ethidium bromide (EtBr) displacement assay. Changes of EtBr-calf thymus DNA complex fluorescence observed after the addition of increasing amounts of tested compounds are displayed. (●) <b>1a</b>; (○) <b>1b</b>; (◊) 9-aminoacridine (<b>9AA</b>); (■) <b>2a</b>; (□) <b>2b</b>. Points represent means of three experiments ± Standard deviation (S.D).</p> Full article ">Figure 3
<p>Influence of compounds <b>1a</b> (<b>A</b>), <b>1b</b> (<b>B</b>), <b>2a</b> (<b>C</b>) and <b>2b</b> (<b>D</b>) on conversion of relaxed plasmid DNA to supercoiled molecule. Control reactions were carried out in the absence of Topo I (supercoiled plasmid, SC) (lane 1), with Topo I (relaxed plasmid) (lane 2), with Topo I and 0.1% DMSO (lane 2A). Plasmid conformation was analyzed in increasing concentrations of investigated compounds (lane 3–9, concentration: 0.5; 1; 5; 10; 15; 20 and 30 µM, respectively) with constant Topo I concentration. <b>9AA</b> (100 µM) was used as positive control (lane 10).</p> Full article ">Figure 4
<p>Influence of <b>2a</b> on Topo I activity. The rate at which relaxed plasmid DNA was converted to supercoiled molecule was monitored over a time course of 30 min. Lane 1—supercoiled plasmid, SC; lane 2—plasmid with Topo I after 30 min of incubation time; lane 3–7—plasmid with Topo I and in the presence of compound <b>2a</b> at the concentration of 15 μM after following incubation times: 1; 5; 10; 15; 30 min, respectively; lane 8–12—plasmid with Topo I and <b>9AA</b> at the concentration of 100 µM after following incubation times: 1; 5; 10; 15; 30 min, respectively (positive control).</p> Full article ">Figure 5
<p>Influence of <b>2a</b> on Topo II activity. The Topo II-mediated plasmid cleavage assay was carried out over constant enzyme concentration. Lane 1: supercoiled plasmid, SC; lane 2: plasmid with Topo II after 15 min of incubation time; lane 3: plasmid with Topo II and etoposide (50 µM) after 15 min of incubation time (positive control); lane 4–8: plasmid with Topo II and in the presence of compound <b>2a</b> at the concentration of 5, 10, 15, 20, 30 µM, respectively.</p> Full article ">Figure 6
<p>DNA damage in leukemia cells induced by <b>1a</b> (<b>A</b>), <b>1b</b> (<b>B</b>), <b>2a</b> (<b>C</b>) and <b>2b</b> (<b>D</b>) (60 min, 37 °C) at concentrations of 5, 10 and 15 µM with and without prior H<sub>2</sub>O<sub>2</sub> incubation (10 min, 4 °C) at a concentration of 15 µM with respect to the appropriate control (−H<sub>2</sub>O<sub>2</sub>/+H<sub>2</sub>O<sub>2</sub>). The values were measured as the average percentage of DNA in the comet tail ± SEM using alkaline version of comet assay.</p> Full article ">Figure 7
<p>The circular dichroism spectra of pure ct-DNA 100 µM (solid) and ct-DNA incubated with <b>2a</b> at concentration of 10 µM (dots).</p> Full article ">Figure 8
<p>The CD spectra of pure ds-oligonucleotide 2.5 µM (solid), ds-oligonucleotide with <b>2a</b> at a concentration of 2.5 µM added before hybridization (ON1—dots) and ds-oligonucleotide with <b>2a</b> at a concentration of 2.5 µM added after hybridization (ON2—dashes).</p> Full article ">
7183 KiB  
Article
Spider Silk-CBD-Cellulose Nanocrystal Composites: Mechanism of Assembly
by Sigal Meirovitch, Zvi Shtein, Tal Ben-Shalom, Shaul Lapidot, Carmen Tamburu, Xiao Hu, Jonathan A. Kluge, Uri Raviv, David L. Kaplan and Oded Shoseyov
Int. J. Mol. Sci. 2016, 17(9), 1573; https://doi.org/10.3390/ijms17091573 - 18 Sep 2016
Cited by 16 | Viewed by 9233
Abstract
The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of [...] Read more.
The fabrication of cellulose-spider silk bio-nanocomposites comprised of cellulose nanocrystals (CNCs) and recombinant spider silk protein fused to a cellulose binding domain (CBD) is described. Silk-CBD successfully binds cellulose, and unlike recombinant silk alone, silk-CBD self-assembles into microfibrils even in the absence of CNCs. Silk-CBD-CNC composite sponges and films show changes in internal structure and CNC alignment related to the addition of silk-CBD. The silk-CBD sponges exhibit improved thermal and structural characteristics in comparison to control recombinant spider silk sponges. The glass transition temperature (Tg) of the silk-CBD sponge was higher than the control silk sponge and similar to native dragline spider silk fibers. Gel filtration analysis, dynamic light scattering (DLS), small angle X-ray scattering (SAXS) and cryo-transmission electron microscopy (TEM) indicated that silk-CBD, but not the recombinant silk control, formed a nematic liquid crystalline phase similar to that observed in native spider silk during the silk spinning process. Silk-CBD microfibrils spontaneously formed in solution upon ultrasonication. We suggest a model for silk-CBD assembly that implicates CBD in the central role of driving the dimerization of spider silk monomers, a process essential to the molecular assembly of spider-silk nanofibers and silk-CNC composites. Full article
(This article belongs to the Special Issue Silk-Based Materials: From Production to Characterization)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Expression and purification of silk and silk-CBD. SDS-PAGE of soluble <span class="html-italic">E. coli</span> proteins, stained with Coomassie blue (<b>A</b>); and Western blot analysis (<b>B</b>) using an anti-HIS antibody. Lane 1, molecular weight marker; lane 2, total protein of the control bacteria (<span class="html-italic">E. coli</span> transformed with an empty vector); lane 3, total protein of silk expressing bacteria; lane 4, total protein of silk-CBD expressing bacteria; lane 5, control sample (empty vector) after Ni-NTA purification; lane 6, purified silk protein; and lane 7, purified silk-CBD fusion protein.</p> Full article ">Figure 2
<p>Adsorption/desorption isotherms. CBD (solid line), silk (dotted line), silk-CBD (dashed line), at different concentrations were allowed to adsorb to cellulose (Sigmacell 20) to the point of equilibrium. After equilibrium was reached, the highest protein concentration (1.2 mg/mL) to cellulose mixture was diluted to allow desorption.</p> Full article ">Figure 3
<p>SEM pictures of silk, silk-CBD, and composite silk-CBD-CNC sponges. (<b>A</b>,<b>B</b>) CNC sponge; (<b>C</b>) 100% silk sponge; (<b>D</b>) silk-CNC composite sponge (75% silk and 25% CNC); (<b>E</b>) 100% silk-CBD sponge; and (<b>F</b>–<b>H</b>) silk-CBD-CNC composite sponge (75% silk-CBD and 25% CNC) on a magnified scale.</p> Full article ">Figure 4
<p>DSC analysis of silk and silk-CBD sponges. Reverse heat flow vs. temperature during TMDSC scanning of silk and silk-CBD sponges at 2 °C/min. (<b>a</b>) 100% silk sponge; (<b>b</b>) 25% silk/75% CNC sponge; (<b>c</b>) 75% silk/25% CNC sponge; (<b>d</b>) 25% silk-CBD/75% CNC sponge; (<b>e</b>) 75% silk-CBD/25% CNC sponge; and (<b>f</b>) 100% silk-CBD sponge. The arrows indicate the glass transition temperatures.</p> Full article ">Figure 5
<p>SEM images of CNC film cross-sections (<b>A</b>,<b>D</b>); silk-CBD-CNC composite film with 1:10 weight ratio (<b>B</b>,<b>E</b>); and composite films with 1:5 weight ratio (<b>C</b>,<b>F</b>). The upper images are at 100,000× magnification and the bottom images at 200,000×. (Scale bar = 500 nm).</p> Full article ">Figure 6
<p>Top images present POM of CNC and silk-CBD-CNC composite films with increasing amounts of silk-CBD. Bottom images show the corresponding LC-PolScope analysis performed on the POM image. The color wheel in the corner presents the orientation of the sample alignment (20× magnification).</p> Full article ">Figure 7
<p>Qualitative comparison of transparency of CNC films compared to silk-CBD CNC 1:10 composite films. (<b>A</b>) 1:10 composite silk-CBD-CNC film; (<b>B</b>) CNC film.</p> Full article ">Figure 8
<p>Gel filtration and DLS of silk and silk-CBD samples before and after sonication (sonic.). Representative gel of silk (<b>A</b>); and silk-CBD (<b>B</b>) protein samples before (solid line) and after sonication (dashed line); SDS PAGE analysis of gel filtration fractions of sonicated silk (<b>C</b>); and silk-CBD (<b>D</b>) samples.</p> Full article ">Figure 9
<p>(<b>A</b>–<b>C</b>) Radially integrated solution small-angle X-ray scattering intensity from silk and silk-CBD non-concentrated and concentrated (conc.) samples before (<b>A</b>) and after sonication (<b>B</b>,<b>C</b>); (<b>D</b>,<b>E</b>) Schematic illustration of the silk-CBD hierarchical order in non-concentrated (<b>D1</b>,<b>E1</b>) and concentrated (<b>D2</b>,<b>E2</b>) samples, before (<b>D</b>) and after sonication (<b>E</b>). Models of the structures of self-assembled silk-CBD subunits before (<b>D3</b>) and after (<b>E3</b>) sonication. The orange units represent the CBD moiety and the green and blue units represent the crystalline domains and the less crystalline regions of the silk monomer, respectively.</p> Full article ">Figure 10
<p>Cryo-transmission electron microscopy (cryo-TEM) images of sonicated solutions of silk (<b>upper pictures</b>) and silk-CBD (<b>bottom pictures</b>). Scale bar is 60 nm.</p> Full article ">Figure 11
<p>Silk-CBD assembly model. (<b>A</b>) silk-CBD in solution; (<b>B</b>) Concentration and dialysis of the silk-CBD protein solution leads to protein dimer formation (<b>C</b>); and (<b>D</b>) Sonication of silk-CBD exposes hydrophobic domains, which allow for further interactions, creating higher molecular assemblies.</p> Full article ">Figure 12
<p>Schematic illustration of spider silk-CNC composite. CBD drives spider silk molecular ordering in two levels. (<b>A</b>) CBD’s ability to form dimers encourages spider silk alignment; and (<b>B</b>) CBD’s ability to bind CNCs drives directional alignment of silk-CBD-CNC composites.</p> Full article ">
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Article
Immunoglobulin Tau Heavy Chain (IgT) in Flounder, Paralichthys olivaceus: Molecular Cloning, Characterization, and Expression Analyses
by Yang Du, Xiaoqian Tang, Wenbin Zhan, Jing Xing and Xiuzhen Sheng
Int. J. Mol. Sci. 2016, 17(9), 1571; https://doi.org/10.3390/ijms17091571 - 17 Sep 2016
Cited by 38 | Viewed by 6035
Abstract
Immunoglobulin tau (IgT) is a new teleost immunoglobulin isotype, and its potential function in adaptive immunity is not very clear. In the present study, the membrane-bound and secreted IgT (mIgT and sIgT) heavy chain genes were cloned for the first time and characterized [...] Read more.
Immunoglobulin tau (IgT) is a new teleost immunoglobulin isotype, and its potential function in adaptive immunity is not very clear. In the present study, the membrane-bound and secreted IgT (mIgT and sIgT) heavy chain genes were cloned for the first time and characterized in flounder (Paralichthys olivaceus), and found the nucleic acid sequence were exactly same in the Cτ1–Cτ4 constant domains of mIgT and sIgT, but different in variable regions and the C-terminus. The amino acid sequence of mIgT shared higher similarity with Bovichtus diacanthus (51.2%) and Dicentrarchus labrax (45.0%). Amino acid of flounder IgT, IgM, and IgD heavy chain was compared and the highest similarity was found between IgT Cτ1 and IgM Cμ1 (38%). In healthy flounder, the transcript levels of IgT mRNA were the highest in gill, spleen, and liver, and higher in peripheral blood leucocytes, skin, and hindgut. After infection and vaccination with Edwardsiella tarda via intraperitoneal injection and immersion, the qRT-PCR analysis demonstrated that the IgT mRNA level was significantly upregulated in all tested tissues, with similar dynamic tendency that increased firstly and then decreased, and higher in gill, skin, hindgut, liver, and stomach in immersion than in the injection group, but no significant difference existed in spleen and head kidney between immersion and injection groups. These results revealed that IgT responses could be simultaneously induced in both mucosal and systemic tissues after infection/vaccination via injection and immersion route, but IgT might play a more important role in mucosal immunity than in systemic immunity. Full article
(This article belongs to the Section Biochemistry)
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Figure 1

Figure 1
<p>(<b>A</b>) Nucleotide and deduced amino acid sequences of the flounder membrane-bound IgT (mIgT, GenBank accession number KX174301). The sequence was divided into VH and four CH domains on the basis of sequence comparisons with the IgH chains of other teleosts. The cysteines (C) were denoted by green bold. The atypical polyadenylation signal (AATTAAA) in the 3′-UTR was shaded in red grey. The signal peptide sequence used hotlink and grey shadow. The four Ig like domains were shown in red arrows for the upstream and dark arrows for the downstream of each domain. The transmembrane region was shaded in yellow grey. Four putative <span class="html-italic">N</span>-glycosylation residues were marked with black bold and underline; (<b>B</b>) Nucleotide and deduced amino acid sequences of secreted tail part of the flounder secreted IgT (sIgT, GenBank accession number KX174302). The atypical polyadenylation signal (AATAAA) in the 3′-UTR was shaded in red grey.</p> Full article ">Figure 2
<p>Alignment of translated IgT/IgZ sequences in different fish species. The first alignment showed immunoglobulin constant domain (CH4-τ/ζ–CH4-τ/ζ) and a segment encoding the membrane proximal extracellular part, the transmembrane peptide, and a short cysteine tail. Cysteine and tryptophan residues were shaded green bold and purple bold, and underlined font putative predicted <span class="html-italic">N</span>-glycosylation sites were in black bold, respectively. In the second part of the alignment, the C-terminal part of the secreted IgT, which was encoded by the CH4 exon, the letters was shown in the gray shading. GenBank accession numbers for membrane and secreted forms of IgT/Z sequences were displayed: <span class="html-italic">Paralichthys olivaceus</span>: KX174301, KX174302; <span class="html-italic">Danio rerio</span>: AAT67444.1, AAT67446.1; <span class="html-italic">Oncorhynchus mykiss</span>: AAW66980.1, AAW66981.1; <span class="html-italic">Salmo salar</span>: ACX50290.1, ACX50293.1; <span class="html-italic">Dicentrarchus labrax</span>: AKK32392.1, AKK32388.1; <span class="html-italic">Bovichtus diacanthus</span>: AKA09866.1, AKA09828.1; <span class="html-italic">Ctenopharyngodon idella</span>: ABY76180.1, ADD82655.1; <span class="html-italic">Plecoglossus altivelis</span>: BAP75402.1, BAP75403.1.</p> Full article ">Figure 3
<p>Phylogenetic tree analysis of IgT family members from flounder and other fish species. The five-pointed star highlighted the position of <span class="html-italic">P. olivaceus</span>. The tree was constructed by the “neighbor-joining” method using MEGA 5.0 software. Node values represented the percent of bootstrap confidence derived from 1000 replicates. The accession number for each sequence followed the common species name.</p> Full article ">Figure 4
<p>Phylogenetic tree of the single heavy chain constant domains of IgT/Z from eight teleost species reported in <a href="#ijms-17-01571-f002" class="html-fig">Figure 2</a>. The triangle highlighted the position of <span class="html-italic">P.</span> <span class="html-italic">olivaceus</span>.</p> Full article ">Figure 5
<p>Tissue distribution pattern of IgT mRNA transcripts in healthy flounder. The gene expression profiles of IgT in thirteen tissues were determined by RT-PCR analysis. Individual tissues from three fish were equally pooled for RNA purification. Total RNA from three flounder (<span class="html-italic">n</span> = 3) were isolated and subjected to DNase I treatment and then transcribed into cDNA. β-Actin of flounder was employed as an internal reference gene. PBL: peripheral blood leucocytes; Gi: gill; Sk: skin; SL: spleen; HK: head kidney; TK: trunk kidney; Li: liver; Fg: foregut; Mg: midgut; Hg: hindgut; Mu: muscle; St: stomach; He: heart.</p> Full article ">Figure 6
<p>The gene expression profiles of IgT in different tissues ((<b>A</b>): gill, (<b>B</b>): skin, (<b>C</b>): spleen, (<b>D</b>): head kidney, (<b>E</b>): liver, (<b>F</b>): hindgut, (<b>G</b>): stomach, (<b>H</b>): muscle) of flounder were determined by qRT-PCR analysis post-infection by intraperitoneal (IP) injection with live <span class="html-italic">E.</span> <span class="html-italic">tarda</span> bacteria (0.2 mL; 1.0 × 10<sup>7</sup> CFU/mL per fish) and immersion (1.0 × 10<sup>8</sup> CFU/mL bath for 60 min), respectively. Individual tissue from three fish was equally pooled for RNA purification. 18sRNA of flounder was employed as an internal reference gene. Values were presented as means ± standard deviation (<span class="html-italic">n</span> = 3), and the asterisk (*) indicated the significant differences (<span class="html-italic">p</span> &lt; 0.05) between IP injection group and immersion group at the each time points after infection.</p> Full article ">Figure 7
<p>The dynamic changes of IgT mRNA transcripts expression in different tissues ((<b>A</b>): gill, (<b>B</b>): skin, (<b>C</b>): spleen, (<b>D</b>): head kidney, (<b>E</b>): liver, (<b>F</b>): hindgut, (<b>G</b>): stomach, (<b>H</b>): muscle) of flounder were determined by qRT-PCR assay post-immunization by IP injection with formalin-killed <span class="html-italic">E.</span> <span class="html-italic">tarda</span> bacteria (0.2 mL; 1.0 × 10<sup>8</sup> CFU/mL per fish) and bath immersion (1.0 × 10<sup>8</sup> CFU/mL for 60 min), respectively. Individual tissues from three fish were equally pooled for RNA purification. 18sRNA of flounder was employed as an internal reference gene. The identities of all PCR products were confirmed by sequencing. Values were presented as means ± standard deviation (<span class="html-italic">n</span> = 3), and the asterisk (*) indicated the significant differences (<span class="html-italic">p</span> &lt; 0.05) between IP injection group and immersion group at each time points after immunization.</p> Full article ">Figure 7 Cont.
<p>The dynamic changes of IgT mRNA transcripts expression in different tissues ((<b>A</b>): gill, (<b>B</b>): skin, (<b>C</b>): spleen, (<b>D</b>): head kidney, (<b>E</b>): liver, (<b>F</b>): hindgut, (<b>G</b>): stomach, (<b>H</b>): muscle) of flounder were determined by qRT-PCR assay post-immunization by IP injection with formalin-killed <span class="html-italic">E.</span> <span class="html-italic">tarda</span> bacteria (0.2 mL; 1.0 × 10<sup>8</sup> CFU/mL per fish) and bath immersion (1.0 × 10<sup>8</sup> CFU/mL for 60 min), respectively. Individual tissues from three fish were equally pooled for RNA purification. 18sRNA of flounder was employed as an internal reference gene. The identities of all PCR products were confirmed by sequencing. Values were presented as means ± standard deviation (<span class="html-italic">n</span> = 3), and the asterisk (*) indicated the significant differences (<span class="html-italic">p</span> &lt; 0.05) between IP injection group and immersion group at each time points after immunization.</p> Full article ">
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Review
Biology, Pest Status, Microbiome and Control of Kudzu Bug (Hemiptera: Heteroptera: Plataspidae): A New Invasive Pest in the U.S.
by Anirudh Dhammi, Jaap B. Van Krestchmar, Loganathan Ponnusamy, Jack S. Bacheler, Dominic D. Reisig, Ames Herbert, Alejandro I. Del Pozo-Valdivia and R. Michael Roe
Int. J. Mol. Sci. 2016, 17(9), 1570; https://doi.org/10.3390/ijms17091570 - 16 Sep 2016
Cited by 20 | Viewed by 9207
Abstract
Soybean is an important food crop, and insect integrated pest management (IPM) is critical to the sustainability of this production system. In recent years, the introduction into the United States of the kudzu bug currently identified as Megacopta cribraria (F.), poses a threat [...] Read more.
Soybean is an important food crop, and insect integrated pest management (IPM) is critical to the sustainability of this production system. In recent years, the introduction into the United States of the kudzu bug currently identified as Megacopta cribraria (F.), poses a threat to soybean production. The kudzu bug was first discovered in the state of Georgia, U.S. in 2009 and since then has spread to most of the southeastern states. Because it was not found in the North American subcontinent before this time, much of our knowledge of this insect comes from research done in its native habitat. However, since the U.S. introduction, studies have been undertaken to improve our understanding of the kudzu bug basic biology, microbiome, migration patterns, host selection and management in its expanding new range. Researchers are not only looking at developing IPM strategies for the kudzu bug in soybean, but also at its unique relationship with symbiotic bacteria. Adult females deposit bacterial packets with their eggs, and the neonates feed on these packets to acquire the bacteria, Candidatus Ishikawaella capsulata. The kudzu bug should be an informative model to study the co-evolution of insect function and behavior with that of a single bacteria species. We review kudzu bug trapping and survey methods, the development of bioassays for insecticide susceptibility, insecticide efficacy, host preferences, impact of the pest on urban environments, population expansion, and the occurrence of natural enemies. The identity of the kudzu bug in the U.S. is not clear. We propose that the kudzu bug currently accepted as M. cribraria in the U.S. is actually Megacopta punctatissima, with more work needed to confirm this hypothesis. Full article
(This article belongs to the Special Issue Plant-Insect Interactions)
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Full article ">Figure 1
<p>Distribution of the kudzu bug <span class="html-italic">Megacopta cribraria</span> in the southeast U.S. from 2009–2015. Map is compiled by Wayne A. Gardner [<a href="#B6-ijms-17-01570" class="html-bibr">6</a>], University of Georgia (available at <a href="http://www.kudzubug.org/" target="_blank">http://www.kudzubug.org/</a>) (accessed on 20 November 2015).</p> Full article ">Figure 2
<p>Kudzu bug, <span class="html-italic">Megacopta cribraria,</span> adults (<b>left</b>) and immatures (<b>right</b>) on U.S. soybean. The high insect density demonstrates the high potential for host damage.</p> Full article ">Figure 3
<p>Typical life cycle and development stages of the kudzu bug, <span class="html-italic">Megacopta cribraria</span> (F.) (Hemiptera: Plataspidae), in the southeast United States.</p> Full article ">Figure 4
<p>Denaturing gradient gel electrophoresis (DGGE) of PCR products from the V3 region of 16S rDNA for bacteria found in the midgut and capsule of kudzu bugs collected in Raleigh, NC. MG, midgut; C, capsules; 1–3, replicates.</p> Full article ">Figure 5
<p>(<b>A</b>) Schematic diagram of trap used to collect kudzu bug by Horn and Hanula [<a href="#B31-ijms-17-01570" class="html-bibr">31</a>] which is based on Ulyshen and Hanula [<a href="#B59-ijms-17-01570" class="html-bibr">59</a>] with some modifications; and (<b>B</b>) flight capture trap used to study the emergence of kudzu bugs in Virginia.</p> Full article ">Figure 6
<p>Feeding disruption bioassay for insecticide susceptibility of kudzu bugs: (<b>A</b>) 16-well plate and exposure results for a 24 h assay. Wells marked with an “X”, contained a dead insect; (<b>B</b>) Close up of a single well. The blue disc in the center of the well is a hydratable caterpillar diet. At the start of the assay, the meal pad was hydrated with an aqueous concentration of the insecticide Capture, a single kudzu bug adult added to the well, and the well covered with a ventilated, transparent plastic (self-sealing) lid; and (<b>C</b>) Dose–kudzu bug mortality response for the assay shown in <b>A</b>. The concentration of the insecticide shown is that found in the hydration solution, not in the final hydrated diet. Mortality was defined as no movement when the insect was touched with a blunt probe and/or did not move when the plate was agitated. There was no control (no insecticide) mortality observed.</p> Full article ">Figure 7
<p><span class="html-italic">Ooencyrtus</span> sp. wasp discovered as an egg parasitoid of the kudzu bug in Virginia: (<b>A</b>) Kudzu bug egg mass with parasitoid present (red arrows indicate presence of the parasitoid); and (<b>B</b>) adult <span class="html-italic">Ooencyrtus</span> sp. wasp that emerged from the egg mass.</p> Full article ">
4990 KiB  
Article
Molecular Characterization and Growth Association of Two Apolipoprotein A-Ib Genes in Common Carp (Cyprinus carpio)
by Xinhua Wang, Xiaomu Yu and Jingou Tong
Int. J. Mol. Sci. 2016, 17(9), 1569; https://doi.org/10.3390/ijms17091569 - 16 Sep 2016
Cited by 9 | Viewed by 4375
Abstract
Apolipoprotein A-I (ApoA-I) is functionally involved in the transportation and metabolism of lipids in vertebrates. In this study, two isoforms of apoA-Ib in common carp (Cyprinus carpio L.) were characterized. Sequence comparison and phylogenetic analysis showed that C. carpio ApoA-Ib is [...] Read more.
Apolipoprotein A-I (ApoA-I) is functionally involved in the transportation and metabolism of lipids in vertebrates. In this study, two isoforms of apoA-Ib in common carp (Cyprinus carpio L.) were characterized. Sequence comparison and phylogenetic analysis showed that C. carpio ApoA-Ib is relatively conserved within cyprinid fishes. During embryonic development, C. carpio apoA-Ib was first expressed at the stage of multi-cells, and the highest mRNA level was observed at the stage of optic vesicle. A ubiquitous expression pattern was detected in various tissues with extreme predominance in the liver. Significantly different expression levels were observed between light and heavy body weight groups and also in the compensatory growth test. Seventeen and eight single-nucleotide polymorphisms (SNPs) were identified in matured mRNA of the C. carpio apoA-Ib.1 and apoA-Ib.2, respectively. Two of these SNPs (apoA-Ib.2-g.183A>T and apoA-Ib.2-g.1753C>T) were significantly associated with body weight and body length in two populations of common carp. These results indicate that apoA-Ib may play an important role in the modulation of growth and development in common carp. Full article
(This article belongs to the Section Biochemistry)
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<p>Comparison of deduced amino acid sequences of <span class="html-italic">C. carpio</span> ApoA-Ibs with several other species of vertebrates. Their accession numbers are as follows: Cc-ApoA-Ib.1 (<span class="html-italic">Cyprinus carpio</span>, KJ741859), Cc ApoA-Ib.2 (<span class="html-italic">Cyprinus carpio</span>, KJ741860), Cc ApoA-I (<span class="html-italic">Cyprinus carpio</span>, KF268349), Ar ApoA-I (<span class="html-italic">Hypophthalmichthys</span> <span class="html-italic">nobilis</span>, unpublished result), Hy ApoA-I (<span class="html-italic">Hypophthalmichthys molitrix</span>, ADF97611), Dr ApoA-Ib (<span class="html-italic">Danio rerio</span>, NP_001093614), Dr ApoA-Ia (<span class="html-italic">Danio rerio</span>, NP_571203), Om ApoA-I.1 (<span class="html-italic">Oncorhynchus mykiss</span>, NP_001117719), Om ApoA-I.2 (<span class="html-italic">Oncorhynchus mykiss</span>, NP_001117720), Gg ApoA-I (<span class="html-italic">Gallus gallus</span>, NP_990856), Mm ApoA-I (<span class="html-italic">Mus Musculus</span>, NP_033822), Hs ApoA-I (<span class="html-italic">Homo sapiens</span>, NP_000030). Boundaries of the signal peptide (signal), the propeptide (pro), the unrelated coding regions 1 and 2 (UCR1 and UCR2, respectively), the 33-codon block and the 11- or 22-residue repeats (4 to 13) are indicated above the sequence of Hs ApoA-I. Identity is covered with black and grey color.</p> Full article ">Figure 2
<p>Neighbor-joining phylogenetic tree based on 63 ApoA-I protein sequences of vertebrates. <span class="html-italic">Cyprinus</span> <span class="html-italic">carpio</span> ApoA-Ib.1 and ApoA-Ib.2 were marked by bold dot.</p> Full article ">Figure 3
<p>The relative expression levels of <span class="html-italic">apoA-Ib</span> in <span class="html-italic">Cyprinus carpio</span>. (<b>A</b>) The relative expression during embryonic development; (<b>B</b>) The relative expression in different tissues; (<b>C</b>) Comparative expression analysis of kidney, heart and liver between light and heavy body weight groups. Significant differences at <span class="html-italic">p</span> &lt; 0.05 and <span class="html-italic">p</span> &lt; 0.01 are labeled with * and **, respectively; (<b>D</b>) Body weight and expression analysis of the individuals participated in the compensatory growth test. Significant differences are labeled with different lowercase letters. Data are shown as the mean ± SEM (<span class="html-italic">n</span> = 5).</p> Full article ">Figure 3 Cont.
<p>The relative expression levels of <span class="html-italic">apoA-Ib</span> in <span class="html-italic">Cyprinus carpio</span>. (<b>A</b>) The relative expression during embryonic development; (<b>B</b>) The relative expression in different tissues; (<b>C</b>) Comparative expression analysis of kidney, heart and liver between light and heavy body weight groups. Significant differences at <span class="html-italic">p</span> &lt; 0.05 and <span class="html-italic">p</span> &lt; 0.01 are labeled with * and **, respectively; (<b>D</b>) Body weight and expression analysis of the individuals participated in the compensatory growth test. Significant differences are labeled with different lowercase letters. Data are shown as the mean ± SEM (<span class="html-italic">n</span> = 5).</p> Full article ">
3221 KiB  
Article
Advanced Glycation End-Products Induce Apoptosis of Vascular Smooth Muscle Cells: A Mechanism for Vascular Calcification
by Sayo Koike, Shozo Yano, Sayuri Tanaka, Abdullah M. Sheikh, Atsushi Nagai and Toshitsugu Sugimoto
Int. J. Mol. Sci. 2016, 17(9), 1567; https://doi.org/10.3390/ijms17091567 - 16 Sep 2016
Cited by 47 | Viewed by 9938
Abstract
Vascular calcification, especially medial artery calcification, is associated with cardiovascular death in patients with diabetes mellitus and chronic kidney disease (CKD). To determine the underlying mechanism of vascular calcification, we have demonstrated in our previous report that advanced glycation end-products (AGEs) stimulated calcium [...] Read more.
Vascular calcification, especially medial artery calcification, is associated with cardiovascular death in patients with diabetes mellitus and chronic kidney disease (CKD). To determine the underlying mechanism of vascular calcification, we have demonstrated in our previous report that advanced glycation end-products (AGEs) stimulated calcium deposition in vascular smooth muscle cells (VSMCs) through excessive oxidative stress and phenotypic transition into osteoblastic cells. Since AGEs can induce apoptosis, in this study we investigated its role on VSMC apoptosis, focusing mainly on the underlying mechanisms. A rat VSMC line (A7r5) was cultured, and treated with glycolaldehyde-derived AGE-bovine serum albumin (AGE3-BSA). Apoptotic cells were identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. To quantify apoptosis, an enzyme-linked immunosorbent assay (ELISA) for histone-complexed DNA fragments was employed. Real-time PCR was performed to determine the mRNA levels. Treatment of A7r5 cells with AGE3-BSA from 100 µg/mL concentration markedly increased apoptosis, which was suppressed by Nox inhibitors. AGE3-BSA significantly increased the mRNA expression of NAD(P)H oxidase components including Nox4 and p22phox, and these findings were confirmed by protein levels using immunofluorescence. Dihydroethidisum assay showed that compared with cBSA, AGE3-BSA increased reactive oxygen species level in A7r5 cells. Furthermore, AGE3-induced apoptosis was significantly inhibited by siRNA-mediated knockdown of Nox4 or p22phox. Double knockdown of Nox4 and p22phox showed a similar inhibitory effect on apoptosis as single gene silencing. Thus, our results demonstrated that NAD(P)H oxidase-derived oxidative stress are involved in AGEs-induced apoptosis of VSMCs. These findings might be important to understand the pathogenesis of vascular calcification in diabetes and CKD. Full article
(This article belongs to the Collection Programmed Cell Death and Apoptosis)
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<p>Glycolaldehyde-derived advanced glycation end-products-bovine serum albumin (AGE3-BSA) (100 µg/mL) increased calcium deposition in a rat vascular smooth muscle cell line and it was inhibited by caspase inhibitor. After reaching confluency, A7r5 cells were incubated with calcification medium containing control BSA (cBSA) or AGE3-BSA in the presence or absence of general caspase inhibitor Z-VAD-FMK (10 µM) or the control Z-FA-FMK (10 µM) for three days. Then, the calcium deposition was measured as described in the Method Section. To determine statistical significance, the results were analyzed by unpaired <span class="html-italic">t</span>-test, and the statistical significance was denoted as follows, ** <span class="html-italic">p &lt;</span> 0.001.</p> Full article ">Figure 2
<p>AGE3-BSA treatment induced apoptosis in A7r5 rat vascular smooth muscle cells. The cells were treated with cBSA, or indicated concentrations of AGE3-BSA, and the levels of apoptotic cells were measured using an ELISA-based method, as described in the Method section. Apoptosis was found to be increased by AGE3-BSA after treatment for five days. The results are presented here as averages ± SE of at least three independent experiments. The statistical significance of the results was analyzed by one-way ANOVA followed by LDS post-hoc test. Statistical significance was denoted as follows, ** <span class="html-italic">p &lt;</span> 0.001 vs. cBSA.</p> Full article ">Figure 3
<p>AGE3-BSA-induced apoptosis in A7r5 cells was mediated by NAD(P)H oxidase. (<b>a</b>) Cultured A7r5 cells were incubated in calcification medium containing cBSA, or AGE3-BSA (100 µg/mL) in the presence or absence of NAD(P)H oxidase inhibitors including GKT137831 (20 µM) or VAS2870 (10 µM) for three days. Apoptosis was evaluated by TUNEL assay, as described in the Method section. Cells in the culture were identified by nuclear staining with Hoechst, and evaluated under a fluorescence microscope; (<b>b</b>) For quantification, Hoechst and TUNEL double positive cells were counted in 10 random microscopic fields at 200× magnification, and expressed as percent TUNEL positive cells in a culture. Statistical significance of the results was analyzed by one-way ANOVA followed by LDS post-hoc test. Statistical significance was denoted as follows, ** <span class="html-italic">p &lt;</span> 0.001 vs. AGE3-BSA.</p> Full article ">Figure 4
<p>AGE3-BSA increased the mRNA expression of the components of NAD(P)H oxidase. A7r5 cells were treated with cBSA or AGE3-BSA (100 µg/mL) for three days. Total mRNA was isolated, and Nox1, Nox4 and p22<sup>phox</sup> mRNA levels were evaluated by real-time PCR, as described in the Method section. GAPDH mRNA was used as a loading control. The data presented here as averages ± SE of at least three experiments. The statistical significance of the results was analyzed by unpaired <span class="html-italic">t</span>-test. The statistical significance was denoted as follows, * <span class="html-italic">p &lt;</span> 0.05 vs. cBSA.</p> Full article ">Figure 5
<p>Effects of AGE3 on the protein expression of Nox4 and p22<sup>phox</sup>. A7r5 cells were treated with medium alone, cBSA or AGE3-BSA for three and five days. After treatment, Nox4 and p22<sup>phox</sup> proteins were analyzed by immunofluorescence staining, as described in Materials and Method. Representative photomicrographs of Nox4 immunofluorescence staining of Day 3 and 5 are shown in (<b>a</b>,<b>b</b>) respectively, and quantified fluorescence intensities in (<b>e</b>). 1–3; medium only (no treatment), 4–6; cBSA treatment, and 7–9; AGE3 treatment. Representative photomicrographs of p22<sup>phox</sup> immunofluorescence staining of Day 3 and 5 are shown in (<b>c</b>,<b>d</b>) respectively, and quantified fluorescence intensities in (<b>f</b>). Higher magnification photomicrographs are shown in the insets for localization of the expressed proteins. Cellular ROS levels were analyzed by DHE assay after treating the cells for five days. Representative photomicrographs of DHE fluorescence are shown in (<b>g</b>); and quantified fluorescence intensities in (<b>h</b>). Numerical data are presented here as average ± SE of at least four experiments. * <span class="html-italic">p &lt;</span> 0.05 vs. medium or cBSA of same time period. Scale bar = 100 µm.</p> Full article ">Figure 5 Cont.
<p>Effects of AGE3 on the protein expression of Nox4 and p22<sup>phox</sup>. A7r5 cells were treated with medium alone, cBSA or AGE3-BSA for three and five days. After treatment, Nox4 and p22<sup>phox</sup> proteins were analyzed by immunofluorescence staining, as described in Materials and Method. Representative photomicrographs of Nox4 immunofluorescence staining of Day 3 and 5 are shown in (<b>a</b>,<b>b</b>) respectively, and quantified fluorescence intensities in (<b>e</b>). 1–3; medium only (no treatment), 4–6; cBSA treatment, and 7–9; AGE3 treatment. Representative photomicrographs of p22<sup>phox</sup> immunofluorescence staining of Day 3 and 5 are shown in (<b>c</b>,<b>d</b>) respectively, and quantified fluorescence intensities in (<b>f</b>). Higher magnification photomicrographs are shown in the insets for localization of the expressed proteins. Cellular ROS levels were analyzed by DHE assay after treating the cells for five days. Representative photomicrographs of DHE fluorescence are shown in (<b>g</b>); and quantified fluorescence intensities in (<b>h</b>). Numerical data are presented here as average ± SE of at least four experiments. * <span class="html-italic">p &lt;</span> 0.05 vs. medium or cBSA of same time period. Scale bar = 100 µm.</p> Full article ">Figure 6
<p>AGE3-BSA-induced apoptosis is mediated through Nox4 or p22<sup>phox</sup>. (<b>a</b>) Nox4 or p22<sup>phox</sup> siRNA was transfected to A7r5 cells, total RNA was isolated three days after transfection, and Nox4 and p22<sup>phox</sup> mRNA levels were measured by real-time PCR, as described in the Method section. GAPDH mRNA was used as loading control. A scramble siRNA was used as a negative control (NC); (<b>b</b>) AGE3-BSA-induced apoptosis was inhibited by silencing of Nox4 and p22<sup>phox</sup> mRNA. Apoptosis was evaluated by cell death detection ELISA kit. The absorbance in cells treated with AGE3-BSA was compared with that in cells treated with cBSA. The ratio was demonstrated after correction with each cBSA. Results were analyzed by one-way ANOVA and LDS post-hoc test. ** <span class="html-italic">p &lt;</span> 0.001 vs. NC.</p> Full article ">
2564 KiB  
Review
The Role of Galectin-1 in Cancer Progression, and Synthetic Multivalent Systems for the Study of Galectin-1
by Jonathan M. Cousin and Mary J. Cloninger
Int. J. Mol. Sci. 2016, 17(9), 1566; https://doi.org/10.3390/ijms17091566 - 16 Sep 2016
Cited by 94 | Viewed by 11031
Abstract
This review discusses the role of galectin-1 in the tumor microenvironment. First, the structure and function of galectin-1 are discussed. Galectin-1, a member of the galectin family of lectins, is a functionally dimeric galactoside-binding protein. Although galectin-1 has both intracellular and extracellular functions, [...] Read more.
This review discusses the role of galectin-1 in the tumor microenvironment. First, the structure and function of galectin-1 are discussed. Galectin-1, a member of the galectin family of lectins, is a functionally dimeric galactoside-binding protein. Although galectin-1 has both intracellular and extracellular functions, the defining carbohydrate-binding role occurs extracellularly. In this review, the extracellular roles of galectin-1 in cancer processes are discussed. In particular, the importance of multivalent interactions in galectin-1 mediated cellular processes is reviewed. Multivalent interactions involving galectin-1 in cellular adhesion, mobility and invasion, tumor-induced angiogenesis, and apoptosis are presented. Although the mechanisms of action of galectin-1 in these processes are still not well understood, the overexpression of galectin-1 in cancer progression indicates that the role of galectin-1 is significant. To conclude this review, synthetic frameworks that have been used to modulate galectin-1 processes are reviewed. Small molecule oligomers of carbohydrates, carbohydrate-functionalized pseudopolyrotaxanes, cyclodextrins, calixarenes, and glycodendrimers are presented. These synthetic multivalent systems serve as important tools for studying galectin-1 mediated cancer cellular functions. Full article
(This article belongs to the Special Issue Glycan–Receptor Interaction)
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<p>Dimeric structure of galectin-1. Galectin-1 (<b>blue</b>) with lactose (<b>red</b>) bound in the apposing carbohydrate recognition domains. Reproduced with permission from Reference [<a href="#B52-ijms-17-01566" class="html-bibr">52</a>].</p> Full article ">Figure 2
<p>Galectin-1 mediates homotypic aggregation of cancer cells through multivalent interactions with cell-surface glycoproteins on adjacent cells and through reorganization of the cell surface, which exposes adhesion molecules.</p> Full article ">Figure 3
<p>Biphasic arbitration of cell–extracellular matrix (ECM) interactions by galectin-1: (<b>a</b>) galectin-1 mediated cross-linking of cell-surface glycoconjugates and ECM glycoproteins promotes adhesion; and (<b>b</b>) competitive binding to ECM glycoproteins by galectin-1 inhibits adhesion and promotes dissemination of tumor cells.</p> Full article ">Figure 4
<p>Galectin-1 competitively binds receptors involved in cell–ECM adhesion to promote migration and invasion.</p> Full article ">Figure 5
<p>Angiogenesis. Illustration of the angiogenesis cascade that involves: (1) pericyte detachment and basal membrane degradation in response to endothelial cell activation; (2) migration of endothelial tip cells in the direction of the growth factor gradient; (3) provision of support of endothelial tip cells by the underlying stalk cells; (4) continuation of this process to form luminized vessel sprouts; (5) fusion of sprouts; and (6) formation of a functional vessel which is further stabilized by deposition of a basal membrane and attraction of pericytes for structural support [<a href="#B3-ijms-17-01566" class="html-bibr">3</a>]. Figure reproduced with permission from Reference [<a href="#B3-ijms-17-01566" class="html-bibr">3</a>]. BM, basal membrane; EC, endothelial cell.</p> Full article ">Figure 6
<p>Provision of structural support for neovasculature by galectin-1.</p> Full article ">Figure 7
<p>Galectin-1 mediated T cell apoptosis. Galectin-1 induces segregation and clustering of CD45 in distinct microdomains from CD43/CD7 complexes. Green motif, galectin-1.</p> Full article ">Figure 8
<p>Lactulose amine dimers to target galectin-1.</p> Full article ">Figure 9
<p>Self-Assembled Pseudopolyrotaxanes, statistical binding mechanism for Galectin-1. Reprinted with permission from [<a href="#B136-ijms-17-01566" class="html-bibr">136</a>]. Red is the carbohydrate, blue is a bipyridinium segment, and green is the cyclodextrin.</p> Full article ">Figure 10
<p>(<b>a</b>) Lactose functionalized poly(amidoamine) (PAMAM) dendrimers used; and (<b>b</b>) PAMAM framework.</p> Full article ">
1531 KiB  
Communication
Plant-to-Plant Variability in Root Metabolite Profiles of 19 Arabidopsis thaliana Accessions Is Substance-Class-Dependent
by Susann Mönchgesang, Nadine Strehmel, Diana Trutschel, Lore Westphal, Steffen Neumann and Dierk Scheel
Int. J. Mol. Sci. 2016, 17(9), 1565; https://doi.org/10.3390/ijms17091565 - 16 Sep 2016
Cited by 20 | Viewed by 6401
Abstract
Natural variation of secondary metabolism between different accessions of Arabidopsis thaliana (A. thaliana) has been studied extensively. In this study, we extended the natural variation approach by including biological variability (plant-to-plant variability) and analysed root metabolic patterns as well as their [...] Read more.
Natural variation of secondary metabolism between different accessions of Arabidopsis thaliana (A. thaliana) has been studied extensively. In this study, we extended the natural variation approach by including biological variability (plant-to-plant variability) and analysed root metabolic patterns as well as their variability between plants and naturally occurring accessions. To screen 19 accessions of A. thaliana, comprehensive non-targeted metabolite profiling of single plant root extracts was performed using ultra performance liquid chromatography/electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC/ESI-QTOF-MS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS). Linear mixed models were applied to dissect the total observed variance. All metabolic profiles pointed towards a larger plant-to-plant variability than natural variation between accessions and variance of experimental batches. Ratios of plant-to-plant to total variability were high and distinct for certain secondary metabolites. None of the investigated accessions displayed a specifically high or low biological variability for these substance classes. This study provides recommendations for future natural variation analyses of glucosinolates, flavonoids, and phenylpropanoids and also reference data for additional substance classes. Full article
(This article belongs to the Special Issue Metabolomics in the Plant Sciences)
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<p>Nested experimental design with three levels. Each variance level had multiple replicates—to assess natural variation, 19 accessions of <span class="html-italic">Arabidopsis thaliana</span> (<span class="html-italic">A. thaliana</span>) were grown. Three independent biological experiments were performed to estimate non-biological variance derived from the experimental batch. To assess individual variability, four plants were harvested in each biological experiment for each accession. Single-plant root extracts were subjected to liquid chromatography-coupled mass spectrometry (LC/MS) and gas chromatography-coupled mass spectrometry (GC/MS) analysis.</p> Full article ">Figure 2
<p>Variance decomposition of LC/electrospray ionization (ESI)(−) MS data set. (<b>a</b>) Variances for plant, batch and accession were estimated with a linear mixed model (lmm), dot—variance of one feature, bar and number—mean variance over 2730 features; (<b>b</b>) cumulative intraclass correlation (ICC) distribution for all features (σ<sup>2</sup><sub>plant</sub>/σ<sup>2</sup><sub>total</sub>), dotted lines indicate 25%, 50% and 75% quantiles.</p> Full article ">Figure 3
<p>Biological variability of annotated secondary metabolites. (<b>a</b>) Variances for plant, batch and accession were estimated with a linear mixed model (lmm), dot—variance of one metabolite; (<b>b</b>) ICCs for glucosinolates (GSLs), flavonoids, and phenylpropanoids, dot—ICC of one metabolite, bar—mean ICC for substance class.</p> Full article ">
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