International Journal of Molecular Sciences

1323 KiB

Open AccessArticle

Nutritional Strategies for the Individualized Treatment of Non-Alcoholic Fatty Liver Disease (NAFLD) Based on the Nutrient-Induced Insulin Output Ratio (NIOR)

by Ewa Stachowska, Karina Ryterska, Dominika Maciejewska, Marcin Banaszczak, Piotr Milkiewicz, Małgorzata Milkiewicz, Izabela Gutowska, Piotr Ossowski, Małgorzata Kaczorowska, Dominika Jamioł-Milc, Anna Sabinicz, Małgorzata Napierała, Lidia Wądołowska and Joanna Raszeja-Wyszomirska

Int. J. Mol. Sci. 2016, 17(7), 1192; https://doi.org/10.3390/ijms17071192 - 22 Jul 2016

Cited by 24 | Viewed by 6992

Abstract

Nutrients play a fundamental role as regulators of the activity of enzymes involved in liver metabolism. In the general population, the action of nutrients may be affected by gene polymorphisms. Therefore, individualization of a diet for individuals with fatty liver seems to be [...] Read more.

Nutrients play a fundamental role as regulators of the activity of enzymes involved in liver metabolism. In the general population, the action of nutrients may be affected by gene polymorphisms. Therefore, individualization of a diet for individuals with fatty liver seems to be a fundamental step in nutritional strategies. In this study, we tested the nutrient-induced insulin output ratio (NIOR), which is used to identify the correlation between the variants of genes and insulin resistance. We enrolled 171 patients, Caucasian men (n = 104) and women (n = 67), diagnosed with non-alcoholic fatty liver disease (NAFLD). From the pool of genes sensitive to nutrient content, we selected genes characterized by a strong response to the NIOR. The polymorphisms included Adrenergic receptor (b3AR), Tumor necrosis factor (TNFα), Apolipoprotein C (Apo C III). Uncoupling Protein type I (UCP-1), Peroxisome proliferator activated receptor γ2 (PPAR-2) and Apolipoprotein E (APOEs). We performed three dietary interventions: a diet consistent with the results of genotyping (NIOR (+)); typical dietary recommendations for NAFLD (Cust (+)), and a diet opposite to the genotyping results (NIOR (−) and Cust (−)). We administered the diet for six months. The most beneficial changes were observed among fat-sensitive patients who were treated with the NIOR (+) diet. These changes included improvements in body mass and insulin sensitivity and normalization of blood lipids. In people sensitive to fat, the NIOR seems to be a useful tool for determining specific strategies for the treatment of NAFLD. Full article

(This article belongs to the Special Issue Non-Alcoholic Fatty Liver Disease Research 2016)

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2057 KiB

Open AccessArticle

Revisiting the Lamotrigine-Mediated Effect on Hippocampal GABAergic Transmission

by Yu-Yin Huang, Yu-Chao Liu, Cheng-Ta Lee, Yen-Chu Lin, Mong-Lien Wang, Yi-Ping Yang, Kaung-Yi Chang and Shih-Hwa Chiou

Int. J. Mol. Sci. 2016, 17(7), 1191; https://doi.org/10.3390/ijms17071191 - 22 Jul 2016

Cited by 13 | Viewed by 5850

Abstract

Lamotrigine (LTG) is generally considered as a voltage-gated sodium (Na_v) channel blocker. However, recent studies suggest that LTG can also serve as a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel enhancer and can increase the excitability of GABAergic interneurons (INs). Perisomatic inhibitory INs, [...] Read more.

Lamotrigine (LTG) is generally considered as a voltage-gated sodium (Na_v) channel blocker. However, recent studies suggest that LTG can also serve as a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel enhancer and can increase the excitability of GABAergic interneurons (INs). Perisomatic inhibitory INs, predominantly fast-spiking basket cells (BCs), powerfully inhibit granule cells (GCs) in the hippocampal dentate gyrus. Notably, BCs express abundant Na_v channels and HCN channels, both of which are able to support sustained action potential generation. Using whole-cell recording in rat hippocampal slices, we investigated the net LTG effect on BC output. We showed that bath application of LTG significantly decreased the amplitude of evoked compound inhibitory postsynaptic currents (IPSCs) in GCs. In contrast, simultaneous paired recordings from BCs to GCs showed that LTG had no effect on both the amplitude and the paired-pulse ratio of the unitary IPSCs, suggesting that LTG did not affect GABA release, though it suppressed cell excitability. In line with this, LTG decreased spontaneous IPSC (sIPSC) frequency, but not miniature IPSC frequency. When re-examining the LTG effect on GABAergic transmission in the cornus ammonis region 1 (CA1) area, we found that LTG markedly inhibits both the excitability of dendrite-targeting INs in the stratum oriens and the concurrent sIPSCs recorded on their targeting pyramidal cells (PCs) without significant hyperpolarization-activated current (I_h) enhancement. In summary, LTG has no effect on augmenting I_h in GABAergic INs and does not promote GABAergic inhibitory output. The antiepileptic effect of LTG is likely through Na_v channel inhibition and the suppression of global neuronal network activity. Full article

(This article belongs to the Section Biochemistry)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Suppression of somatic GABAergic transmission onto GCs by LTG. (A) Schematic of experiment configuration: A stimulating electrode placed at a distance of 100–200 µm from a recorded GC within the GCL. ML, molecular layer; GCL, granule cell layer; (B) (Top) Exemplar average compound IPSCs (cIPSCs) (15–20 sweeps) recorded in the control, in LTG (100 µM), after LTG washout and after the addition of Gabazine (SR95531); (Bottom) Plot of the peak amplitudes of cIPSC against time; (C) Plot of the mean peak amplitude of cIPSC (n = 10) against time. Data were normalized to the baseline before LTG application. Symbols indicate the mean; error bars indicate SEM; (D) Dose-response relationship of cIPSC inhibition by LTG (1, 10, 30, 100 and 1000 µM). Data fitted to a single Hill equation with IC50 = 121.2 µM and Hill coefficient = 1.51. Each point represents the average from 5–14 experiments, as given in parentheses; error bars indicate SEM. Full article ">Figure 2
LTG had little effect on GABA release at BC-GC synapses. (A) Schematic diagram showing the BC-GC paired recording configuration. OML, outer molecular layer; IML, inner moleucular layer; (B) The 25-Hz bursts of five presynaptic APs (red) and postsynaptic unitary IPSC (uIPSC) traces (black, average of 25–30 sweeps) in the control (Ctrl) and after bath perfusion of LTG (100 µM); (C) Summary of the normalized uIPSC1 mean peak amplitude from five BC-GC pairs against time. Symbols indicate the mean; error bars indicate SEM; (D) Mean ratio of uIPSCn/uIPSC1 plotted against the number within the train (n). Full article ">Figure 3
LTG decreased the frequency of spontaneous IPSCs (sIPSCs), but not miniature IPSCs (mIPSCs) in GCs. (A1) (Top) Recording configuration: Whole-cell voltage-clamp recordings from a GC at −70 mV in the DG. CA1, cornus ammonis region 1; (Middle and bottom) Traces of sIPSCs recorded before (black) and after (gray) LTG (100 µM) application; (A2,A3) Cumulative distributions of sIPSC inter-event intervals (A2) and amplitudes (A3) from the control (black; n = 10) and LTG (gray; n = 10). Insets show the bar graph summaries of the averages. ** p < 0.01; (B1) (Top) Traces of mIPSCs recorded at −70 mV in the control (black); (Bottom) Events in the presence of LTG (100 µM) application (gray); (B2,B3) Cumulative distributions of mIPSC inter-event intervals (B2) and amplitude (B3) in the control (black, n = 10) and LTG (gray, n = 10). Insets show the bar graph summaries of the averages. Full article ">Figure 4
LTG suppressed BC excitability. (A) Reconstruction of a biocytin-filled BC whose axon arborized in the GCL. Soma and dendrites are shown in red, and axons are in black; (B) Exemplar traces of APs evoked by 1-s depolarizing current pulses (400, 800, 1200 pA) in the presence of synaptic blockers. Black traces, control; gray traces, LTG. Cells were held at −70 mV by holding the current adjustment throughout the experiment; (C) Mean AP frequency plotted against the injected current (n = 10). ** p < 0.01; (D) Exemplar voltage traces recorded during 1-s hyperpolarizing current pulses (−100 pA and −300 pA, respectively) under whole-cell current-clamp before (black traces) and after 100 µM LTG (gray traces) application; (E,F) Summary bar graphs of the effect of 100 µM LTG on (E) input resistance and (F) the sag ratio (voltage change at the end of the 1-s pulse/maximal voltage change or −300 pA current injection) with no significant difference under control and LTG conditions (n = 9). ns, no significance; (G) Summary of the plot of the spontaneous spike frequency against time illustrating the effect of 100 µM LTG (n = 10). BCs were slightly depolarized to fire persistent APs by sustained somatic current injection (100–200 pA); (H) (Top) A stimulating electrode (monopolar glass pipette) placed in the GCL at a distance of 100–300 µm from the recorded BC; (Bottom) 10 consecutive spikes of APs in the control (stimulus intensity 25 µA) (left), in the presence of 100 µM LTG at the same stimulus intensity (middle) and in the presence of 100 µM LTG after the increase in stimulation intensity (right, 29 µA); (I) Spike probability plotted against stimulus intensity in the control (black) and after LTG (gray). The dashed line indicates that LTG increases the threshold for spike initiation (current leading to 50% successes); (J) Summary of the LTG effect on the AP threshold. Data were normalized to the control from five BCs. * p < 0.05. Full article ">Figure 5
LTG decreased sIPSC frequency in CA1 PCs without affecting mIPSC frequency. (A1) (Top) Whole-cell voltage-clamp recordings from a CA1 PC; (Middle and bottom) Traces of sIPSCs recorded at −70 mV in the control (black) and LTG (100 µM) (gray); (A2,A3) Cumulative distributions of sIPSC inter-event intervals (A2) and amplitudes (A3) from the control (black) and LTG (gray). Insets show the bar graph summaries of the cell averages (n = 8) for the frequency (A2) and amplitude (A3). ** p < 0.01; (B1) (Top) Traces of mIPSCs recorded at −70 mV in control conditions (black); (Bottom) Events in the presence of LTG (100 µM) application (gray); (B2,B3) Cumulative distributions of mIPSC inter-event intervals (B2) and amplitudes (B3) from the control (black) and LTG (gray). Insets show the bar graph summaries of the ell averages (n = 6) for the frequency (B2) and amplitude (B3), respectively. * p < 0.05. Full article ">Figure 6
LTG suppressed oriens-lacunosum moleculare (O-LM) IN excitability without affecting Ih. (A) Reconstruction of a biocytin-filled O-LM IN. Soma and dendrite are shown in light blue, and axons are in black. SLM, stratum lacunosum moleculare; SR, stratum radiatum; SP, stratum pyramidal; SO, stratum oriens; (B) Exemplar AP traces from whole-cell current-clamp recordings of control (black) and LTG (gray) in an O-LM IN evoked by 1-s depolarizing current steps in the presence of synaptic blockers; (C) The AP frequency was plotted against the stepwise stimulus intensity (100~600 pA) and revealed significant inhibition by LTG (n = 8). ** p < 0.01; (D) Voltage responses to 1-s hyperpolarizing (−100 pA and −300 pA) current pulses from the same cell were recorded; (E) Summary of the input resistance. ns, no significance; (F) Summary of the sag ratio (voltage change at the end of the 1-s pulse/maximal voltage change or −300 pA current injection) with no significant difference under the control and LTG condition; (G) (Top) In a resting membrane potential around −60 mV, an O-LM IN generated spontaneous firing and was abolished by LTG (100 µM); (Bottom) Plot of normalized induced spontaneous spike frequency against time illustrating the effect of 100 µM LTG (n = 8); (H) Currents activated by hyperpolarizing pulse from a holding potential of −50 mV−120 mV with an increment of 10 mV under voltage clamp mode from a representative O-LM IN in the control condition, with LTG (100 µM) and 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidiniumchloride (ZD7288) (50 µM); (I) Plotting the ZD7288-sensitive current (current of the control or LTG with digital subtraction of the ZD current) against hyperpolarizing voltage steps before and after LTG application demonstrated that LTG had no significant effect on Ih (n = 6); (J) The same protocol as (H) was made in CA1 PC; (K) Plotting the ZD7288-sensitive current against hyperpolarizing voltage steps before and after LTG application demonstrated that LTG had no significant effect on Ih (n = 6). Full article ">

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Open AccessArticle

Neuroprotective Effect of Salvianolic Acids against Cerebral Ischemia/Reperfusion Injury

by Shuai Hou, Ming-Ming Zhao, Ping-Ping Shen, Xiu-Ping Liu, Yuan Sun and Jia-Chun Feng

Int. J. Mol. Sci. 2016, 17(7), 1190; https://doi.org/10.3390/ijms17071190 - 22 Jul 2016

Cited by 59 | Viewed by 6622

Abstract

This study investigated the neuroprotective effect of salvianolic acids (SA) against ischemia/reperfusion (I/R) injury, and explored whether the neuroprotection was dependent on mitochondrial connexin43 (mtCx43) via the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. In vitro, we measured astrocyte apoptosis, mitochondrial membrane potential, and [...] Read more.

This study investigated the neuroprotective effect of salvianolic acids (SA) against ischemia/reperfusion (I/R) injury, and explored whether the neuroprotection was dependent on mitochondrial connexin43 (mtCx43) via the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. In vitro, we measured astrocyte apoptosis, mitochondrial membrane potential, and also evaluated the morphology of astrocyte mitochondria with transmission electron microscopy. In vivo, we determined the cerebral infarction volume and measured superoxide dismutase (SOD) activity and malondialdehyde (MDA) content. Additionally, mtCx43, p-mtCx43, AKT, and p-AKT levels were determined. In vitro, we found that I/R injury induced apoptosis, decreased cell mitochondrial membrane potential (MMP), and damaged mitochondrial morphology in astrocytes. In vivo, we found that I/R injury resulted in a large cerebral infarction, decreased SOD activity, and increased MDA expression. Additionally, I/R injury reduced both the p-mtCx43/mtCx43 and p-AKT/AKT ratios. We reported that both in vivo and in vitro, SA ameliorated the detrimental outcomes of the I/R. Interestingly, co-administering an inhibitor of the PI3K/AKT pathway blunted the effects of SA. SA represents a potential treatment option for cerebral infarction by up-regulating mtCx43 through the PI3K/AKT pathway. Full article

(This article belongs to the Special Issue Neuroprotective Strategies 2016)

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Figure 1
The effects of SA and LY on cell apoptosis and structure following OGD: (A) representative images of cell apoptosis by TUNEL staining; (B) analysis of the apoptotic index; (C) representative images of mitochondrial ultrastructure by TEM, where areas surrounded by red boxes in the above images correspond to the magnified images below; and (D) analysis of mitochondrial injury using Flameng scores. n = 3 in each group. a p ≤ 0.01 vs. Sham; b p ≤ 0.05 vs. OGD; c p ≤ 0.05 vs. SA. SA: salvianolic acids; LY: LY 294002; OGD: oxygen glucose deprivation; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling. Full article ">Figure 2
The effects of SA and LY on astrocyte MMP following OGD: (A) representative flow cytometry images of MMP observed with the Rh 123 probe; and (B) MMP analysis is calculated as a percent relative to the mean fluorescence intensity of the sham group. n = 3 in each group. a p ≤ 0.05 vs. Sham; b p ≤ 0.05 vs. OGD; c p ≤ 0.05 vs. SA. SA: salvianolic acids; LY: LY 294002; OGD: oxygen glucose deprivation; MMP: mitochondrial membrane potential; Rh 123: Rhodamine 123. Full article ">Figure 3
The effects of SA and LY on infarction volume and neurological deficits in rats following MCAO: (A) representative images of cerebral infarction after 2 h of MCAO and a 12 h reperfusion in rat brains by 2,3,5-triphenyltetrazolium chloride (TTC) staining; (B) analysis of infarct volume, which is calculated as a percent relative to total cerebral volume (n = 3 in each group. a p ≤ 0.01 vs. Sham; b p ≤ 0.05 vs. IR; c p ≤ 0.05 vs. SA); and (C) neurological deficits analysis (n = 10 in each group. a p ≤ 0.01 vs. Sham; b p > 0.05 vs. IR; c p > 0.05 vs. SA). SA: salvianolic acids; MCAO: middle cerebral artery occlusion; LY: LY 294002; TTC: 2,3,5-triphenyltetrazolium chloride; IR: ischemia-reperfusion. Additionally, we investigated neurological deficits following MCAO, as shown in <a href="#ijms-17-01190-f003" class="html-fig">Figure 3</a>C. All rats in the sham group were scored zero. However, there was a significant deterioration in neurological functioning in the I/R group, compared to the sham group (p ≤ 0.01). No improvement was noted in the scores in the SA groups compared to the I/R group after surgery. Full article ">Figure 4
The effects of SA on the expression of mtCx43, p-mtCx43, AKT, and p-AKT in the rat cerebral cortex after I/R injury: (A) Western blotting showed mtCx43, p-Cx43 and p-mtCx43/p-Cx43 were up-regulated in SA group after MCAO. These effects were reduced by LY; (B) Western blotting showed p-AKT and p-AKT/AKT were up-regulated in SA group after MCAO. These effects were reduced by LY. n = 3 in each group, a p ≤ 0.01 vs. Sham; b p ≤ 0.05 vs. IR; c p ≤ 0.05 vs. SA. There was no difference in AKT level. I/R: ischemia-reperfusion; SA: salvianolic acids; mtCx43: mitochondrial connexin 43; Cx43: connexin 43; p-: phosphorylated; MCAO: middle cerebral artery occlusion; AKT: protein kinase B; VDAC-1: voltage-dependent anion channel; LY: LY 294002. Full article ">

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Open AccessArticle

Orofacial Manifestations and Temporomandibular Disorders of Systemic Scleroderma: An Observational Study

by Vito Crincoli, Laura Fatone, Margherita Fanelli, Rossana Patricia Rotolo, Angela Chialà, Gianfranco Favia and Giovanni Lapadula

Int. J. Mol. Sci. 2016, 17(7), 1189; https://doi.org/10.3390/ijms17071189 - 22 Jul 2016

Cited by 69 | Viewed by 15957

Abstract

Scleroderma is a disorder involving oral and facial tissues, with skin hardening, thin lips, deep wrinkles, xerostomia, tongue rigidity, and microstomia. The aim of this study was to investigate the prevalence of oral manifestations and temporomandibular disorders (TMD) in Systemic Sclerosis (SSc) patients [...] Read more.

Scleroderma is a disorder involving oral and facial tissues, with skin hardening, thin lips, deep wrinkles, xerostomia, tongue rigidity, and microstomia. The aim of this study was to investigate the prevalence of oral manifestations and temporomandibular disorders (TMD) in Systemic Sclerosis (SSc) patients compared with healthy people. Eighty patients (6 men, 74 women) fulfilling ACR/EULAR SSc Criteria were enrolled. A randomly selected group of 80 patients, matched by sex and age served as control group. The examination for TMD signs and symptoms was based on the standardized Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) through a questionnaire and clinical examination. SSc patients complained more frequently (78.8%) of oral symptoms (Xerostomia, dysgeusia, dysphagia and stomatodynia) than controls (28.7%) (χ² = 40.23 p = 0.001). TMD symptoms (muscle pain on chewing, difficulty in mouth opening, headaches) were complained by 92.5% of SSc patients and by 76.2% of controls (χ² = 8.012 p = 0.005). At the clinical examination, 85% of SSc patients showed restricted opening versus 20.0% of controls (χ² = 67.77 p = 0.001), 81.2% of SSc showed reduced right lateral excursion versus 50% of controls (χ² = 17.316 p = 0.001); 73.8% of SSc showed limited left lateral excursion versus 53.8% of controls (χ² = 6.924 p = 0.009); and 73.8% of SSc had narrow protrusion versus 56.2% of controls (χ² = 5.385 p = 0.02). Full article

(This article belongs to the Special Issue Inflammatory Skin Conditions)

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3762 KiB

Open AccessArticle

High Expression of XRCC6 Promotes Human Osteosarcoma Cell Proliferation through the β-Catenin/Wnt Signaling Pathway and Is Associated with Poor Prognosis

by Bin Zhu, Dongdong Cheng, Shijie Li, Shumin Zhou and Qingcheng Yang

Int. J. Mol. Sci. 2016, 17(7), 1188; https://doi.org/10.3390/ijms17071188 - 22 Jul 2016

Cited by 17 | Viewed by 7450

Abstract

Increasing evidences show that XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) was upregulated and involved in tumor growth in several tumor types. However, the correlation of XRCC6 and human osteosarcoma (OS) is still unknown. This study was conducted with [...] Read more.

Increasing evidences show that XRCC6 (X-ray repair complementing defective repair in Chinese hamster cells 6) was upregulated and involved in tumor growth in several tumor types. However, the correlation of XRCC6 and human osteosarcoma (OS) is still unknown. This study was conducted with the aim to reveal the expression and biological function of XRCC6 in OS and elucidate the potential mechanism. The mRNA expression level of XRCC6 was measured in osteosarcoma cells and OS samples by quantitative transcription-PCR (qRT-PCR). The expression of XRCC6 protein was measured using Western blot and immunohistochemical staining in osteosarcoma cell lines and patient samples. Cell Counting Kit 8 (CCK8), colony-forming and cell cycle assays were used to test cell survival capacity. We found that XRCC6 was overexpressed in OS cells and OS samples compared with the adjacent non-tumorous samples. High expression of XRCC6 was correlated with clinical stage and tumor size in OS. Reduced expression of XRCC6 inhibits OS cell proliferation through G2/M phase arrest. Most importantly, further experiments demonstrated that XRCC6 might regulate OS growth through the β-catenin/Wnt signaling pathway. In conclusion, these findings indicate that XRCC6 exerts tumor-promoting effects for OS through β-catenin/Wnt signaling pathway. XRCC6 may serve as a novel therapeutic target for OS patients. Full article

(This article belongs to the Special Issue Molecular Classification of Human Cancer: Diagnosis and Treatment 2016)

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Graphical abstract

Graphical abstract
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XRCC6 expression is up-regulated in osteosarcoma (OS) clinical samples and cell lines. (A–C) The mRNA and protein expression level of XRCC6 were measured by TaqMan real-time PCR and Western blot assays in OS cell lines (including MNNG/HOS, MG63 and U2OS) and human osteoblastic cell line (hFOB); (D,E) relative expression of XRCC6 was detected using qRT-PCR in 20 pairs of OS samples and their corresponding noncancerous samples. The expression of XRCC6 was overexpressed in OS tissues compared with the noncancerous tissues. Statistical analysis was performed using paired t test (D). Full article ">Figure 2
Knockdown of XRCC6 inhibited cell proliferation through G2/M phase arrest. (A,B) The expression of XRCC6 was downregulated by a targeted siRNA; (C,D) a CCK8 assay was used to detect the proliferation of MNNG/HOS cells and U2OS cells after transfection with targeted siRNA. Diagrams showing the results of a CCK-8 assay that MNNG/HOS and U2OS proliferation were inhibited by downregulating XRCC6 expression; (E,G) cell cycle profiles determined by propidium iodide (PI) staining and flow cytometry assays of MNNG/HOS transfected with si-XRCC6 or si-NC; (F,H) cell cycle profiles determined by propidium iodide (PI) staining and flow cytometry assays of U2OS transfected with si-XRCC6 or si-NC. The data are representative of three independent experiments. Error bars represent SD (Standard Deviation). * p < 0.05, ** p < 0.01 by Student’s t test. Full article ">Figure 3
Decreased XRCC6 impaired OS cell colony-forming capacity. (A,C) Colony formation assay of MNNG/HOS cells and U2OS cells transfected with targeted siRNA or si-NC. After two weeks, cells in each well were fixed and counted. Representative photo micrographs of MNNG/HOS cells (A); and U2OS cells (C), colonies in culture plates; (B,D) significant reduction in the colony-forming efficacy in MNNG/HOS cells (B), and U2OS cells (D). Following XRCC6 knockdown. Data are expressed as mean ± SD. of three independent experiments. ** p < 0.01, by student’s t test. (Magnification: 1×). Full article ">Figure 4
XRCC6 regulates the β-catenin/Wnt signaling pathway. (A) Western blot analysis of β-catenin and the downstream protein level of this pathway including c-MYC, Cyclin D1 in both MNNG/HOS and U2OS cells transfected with targeted siRNA or si-NC. Knockdown of XRCC6 expression by targeted siRNA led to reduced expression of β-catenin and the downstream protein level of this pathway in both MNNG/HOS and U2OS cells; (B) a densitometric analysis of the Western blotting bands was performed. * p < 0.05 by Student’s t test. Full article ">Figure 5
High expression of XRCC6 correlates to OS clinical stage and tumor size. Representative images of histological inspection of an OS tissue or noncancerous tissue micrographs were shown as labeled. XRCC6 was overexpressed in OS tissues compared with corresponding noncancerous tissues. Full article ">

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Open AccessReview

Non-Invasive Methods to Monitor Mechanisms of Resistance to Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer: Where Do We Stand?

by Paola Ulivi

Int. J. Mol. Sci. 2016, 17(7), 1186; https://doi.org/10.3390/ijms17071186 - 22 Jul 2016

Cited by 20 | Viewed by 6390

Abstract

The induction of resistance mechanisms represents an important problem for the targeted therapy of patients with non-small-cell lung cancer (NSCLC). The best-known resistance mechanism induced during treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is EGFR T790M mutation for which specific [...] Read more.

The induction of resistance mechanisms represents an important problem for the targeted therapy of patients with non-small-cell lung cancer (NSCLC). The best-known resistance mechanism induced during treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) is EGFR T790M mutation for which specific drugs are have been developed. However, other molecular alterations have also been reported as induced resistance mechanisms to EGFR-TKIs. Similarly, there is growing evidence of acquired resistance mechanisms to anaplastic lymphoma kinase (ALK)-TKI treatment. A better understanding of these acquired resistance mechanisms is essential in clinical practice as patients could be treated with specific drugs that are active against the induced alterations. The use of free circulating tumor nucleic acids or circulating tumor cells (CTCs) enables resistance mechanisms to be characterized in a non-invasive manner and reduces the need for tumor re-biopsy. This review discusses the main resistance mechanisms to TKIs and provides a comprehensive overview of innovative strategies to evaluate known resistance mechanisms in free circulating nucleic acids or CTCs and potential future orientations for these non-invasive approaches. Full article

(This article belongs to the Special Issue Liquid Biopsy for Clinical Application)

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1839 KiB

Open AccessArticle

Mutant LRP6 Impairs Endothelial Cell Functions Associated with Familial Normolipidemic Coronary Artery Disease

by Jian Guo, Yang Li, Yi-Hong Ren, Zhijun Sun, Jie Dong, Han Yan, Yujun Xu, Dao Wen Wang, Gu-Yan Zheng, Jie Du and Xiao-Li Tian

Int. J. Mol. Sci. 2016, 17(7), 1173; https://doi.org/10.3390/ijms17071173 - 22 Jul 2016

Cited by 12 | Viewed by 5429

Abstract

Mutations in the genes low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) and myocyte enhancer factor 2A (MEF2A) were reported in families with coronary artery disease (CAD). We intend to determine the mutational spectrum of these genes among hyperlipidemic and normolipidemic [...] Read more.

Mutations in the genes low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) and myocyte enhancer factor 2A (MEF2A) were reported in families with coronary artery disease (CAD). We intend to determine the mutational spectrum of these genes among hyperlipidemic and normolipidemic CAD families. Forty probands with early-onset CAD were recruited from 19 hyperlipidemic and 21 normolipidemic Chinese families. We sequenced all exons and intron-exon boundaries of LRP6 and MEF2A, and found a novel heterozygous variant in LRP6 from a proband with normolipidemic CAD. This variant led to a substitution of histidine to tyrosine (Y418H) in an evolutionarily conserved domain YWTD in exon 6 and was not found in 1025 unrelated healthy individuals. Co-segregated with CAD in the affected family, LRP6_Y418H significantly debilitated the Wnt3a-associated signaling pathway, suppressed endothelial cell proliferation and migration, and decreased anti-apoptotic ability. However, it exhibited no influences on low-density lipoprotein cholesterol uptake. Thus, mutation Y418H in LRP6 likely contributes to normolipidemic familial CAD via impairing endothelial cell functions and weakening the Wnt3a signaling pathway. Full article

(This article belongs to the Special Issue Atherosclerosis and Vascular Imaging 2016)

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Figure 1
Novel mutation of LRP6 identified in a CAD pedigree. (A) Pedigree of the family with the LRP6 Y418H mutation. Numbered individuals correspond to those in <a href="#ijms-17-01173-t001" class="html-table">Table 1</a>. Circles represent females; Squares represent males; Proband is indicated by the arrow; Individuals with CAD are indicated by black symbols; Individuals without CAD are shown as unfilled symbols; Presymptomatic carriers are shown by symbols by gray symbols; Symbols with a slash through them indicate deceased subjects; Genotypes of the LRP6 mutation were shown below the symbols who were willing to participate in the study; Filled stars indicates that the genotype of subject I2 was speculated; Individuals who were not available for studied are indicated with question mark; (B) Coronary angiogram of the proband. The red arrows indicates the stenosis; (C) DNA sequence analysis for a segment of LRP6 exon 6 from a healthy control (top) and the proband (below). A red arrow points out a single base mutation in the proband, and it results in the substitution of histidine for tyrosine at codon 418; (D) Conservatism analysis by interspecies alignments. The mutation position is indicated with a red frame. Full article ">Figure 2
Effect of LRP6Y418H and LRP6R611C on Wnt signal transduction. (A) RT-PCR showed that there was no significant difference between the over-expression levels of LRP6WT/LRP6R611C/LRP6Y418H; (B) Western blot showed no significant difference between total expression levels or membrane location of wild-type and mutant LRP6. Results were replicated three times and a representative figure was shown; (C,D) Statistical result of (B); (E) Luciferase assay was performed with different amount of Wnt3a. RLU: relative light units. Results were obtained with four independent transfections. Error bars, standard deviation. * indicate p-value for one-way ANOVA plus post-hoc test <0.05. NS, not significant in one-way ANOVA plus post-hoc test. Full article ">Figure 3
Effect of LRP6Y418H and LRP6R611C on endothelial cell functions; (A) Comparison of endothelial cells’ proliferation. The same amount of cells was over-expressed with wild-type LRP6 or mutant. After 48 h, cell number was counted for each group. Results were calculated using eight random fields from four independent biological replications; (B) Comparison of endothelial cell migration. Results were calculated using six views from three independent replications of Boyden chamber assay; (C) Electrophorogram for DNA ladder assay. SD, serum depletion; (D) Scatter diagram from flow cytometry assay. Data were presented in 2D diagrams plotting PI against Annexin-APC. Compensation for background fluorescence was performed by measuring target signals of single color controls and negative controls. Two quadrants in the right-side diagram represent apoptotic cells; (E) Statistical result for proportion of apoptotic cells in each group. Three biological repeats were taken into calculation; (F–J) Relative mRNA level of markers for endothelial cell activation (IL6, SELE, and ICAM) and senescence (P21 and P16). Three biological repeats were taken into calculation. Error bars, standard deviation. * indicate p-value for one-way ANOVA plus post-hoc test <0.05. NS, not significant. Full article ">Figure 4
Effect of LRP6Y418H and LRP6R611C on LDL uptake in HUVEC. (A) An overview of the Dil-LDL uptake in HUVEC; Left, bright field; Right, Fluorescent field; (B) Quantitative results were calculated using six random views from three independent replications. Error bars, standard deviation. * indicate p-value for one-way ANOVA plus post-hoc test <0.05. NS, not significant; (C) Enlarged views for single cells. Top, GFP translated by IRES following LRP6; Middle, Dil-LDL; Bottom, merged data. Full article ">

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Open AccessArticle

De Novo Assembly and Characterization of the Transcriptome of Grasshopper Shirakiacris shirakii

by Zhongying Qiu, Fei Liu, Huimeng Lu, Hao Yuan, Qin Zhang and Yuan Huang

Int. J. Mol. Sci. 2016, 17(7), 1110; https://doi.org/10.3390/ijms17071110 - 22 Jul 2016

Cited by 10 | Viewed by 5762

Abstract

Background: The grasshopper Shirakiacris shirakii is an important agricultural pest and feeds mainly on gramineous plants, thereby causing economic damage to a wide range of crops. However, genomic information on this species is extremely limited thus far, and transcriptome data relevant to insecticide [...] Read more.

Background: The grasshopper Shirakiacris shirakii is an important agricultural pest and feeds mainly on gramineous plants, thereby causing economic damage to a wide range of crops. However, genomic information on this species is extremely limited thus far, and transcriptome data relevant to insecticide resistance and pest control are also not available. Methods: The transcriptome of S. shirakii was sequenced using the Illumina HiSeq platform, and we de novo assembled the transcriptome. Results: Its sequencing produced a total of 105,408,878 clean reads, and the de novo assembly revealed 74,657 unigenes with an average length of 680 bp and N50 of 1057 bp. A total of 28,173 unigenes were annotated for the NCBI non-redundant protein sequences (Nr), NCBI non-redundant nucleotide sequences (Nt), a manually-annotated and reviewed protein sequence database (Swiss-Prot), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Based on the Nr annotation results, we manually identified 79 unigenes encoding cytochrome P450 monooxygenases (P450s), 36 unigenes encoding carboxylesterases (CarEs) and 36 unigenes encoding glutathione S-transferases (GSTs) in S. shirakii. Core RNAi components relevant to miroRNA, siRNA and piRNA pathways, including Pasha, Loquacious, Argonaute-1, Argonaute-2, Argonaute-3, Zucchini, Aubergine, enhanced RNAi-1 and Piwi, were expressed in S. shirakii. We also identified five unigenes that were homologous to the Sid-1 gene. In addition, the analysis of differential gene expressions revealed that a total of 19,764 unigenes were up-regulated and 4185 unigenes were down-regulated in larvae. In total, we predicted 7504 simple sequence repeats (SSRs) from 74,657 unigenes. Conclusions: The comprehensive de novo transcriptomic data of S. shirakii will offer a series of valuable molecular resources for better studying insecticide resistance, RNAi and molecular marker discovery in the transcriptome. Full article

(This article belongs to the Section Biochemistry)

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Figure 1
Homology analysis of unigenes for S. shirakii. (A) E-value distribution of the top BLASTX hits against the Nr database for S. shirakii unigenes with an E-value cutoff of 10−5; (B) Similarity distribution of the top BLAST hits for each sequence; (C) Species distribution of the top S. shirakii sequence BLASTX hits. BLAST analysis against the non-redundant database was performed with an E-value cutoff of 10−5. Full article ">Figure 2
Gene Ontology. Classification of S. shirakii unigenes based on Gene Ontology (GO). Full article ">Figure 3
Clusters of orthologous group (COG) function classification of the S. shirakii unigenes. Full article ">Figure 4
Comparison of unigene expression between adults and larvae of Shirakiacris shirakii. The differentially-expressed unigenes (DEUs) are shown in red and green, while blue indicates unigenes that were not differentially-expressed between the adults and larvae of S. shirakii. SsA represents the adults of S. shirakii; SsL represents the larvae of S. shirakii. Full article ">Figure 5
The phylogenetic analysis of sequences encoding P450s from S. shirakii and D. melanogaster. Branch numbers represent bootstrap values (1000 replicates). The 79 S. shirakii unigenes encoding P450s are marked with filled red circles. The sequences used to reconstruct the maximum likelihood (ML) tree are available as S1 Data. Full article ">Figure 6
The phylogenetic analysis of sequences encoding CarEs from S. shirakii and L. migratoria. Branch numbers represent bootstrap values (1000 replicates). The 36 S. shirakii unigenes encoding CarEs are marked with filled red circles. The sequences used to reconstruct the maximum likelihood (ML) tree are available as S2 Data. Full article ">Figure 7
Statistics of simple sequence repeat (SSR) nucleotide classes found in the transcriptome of S. shirakii. Full article ">Figure 8
Differential expression of DEUs in S. shirakii. (A) Gene expression data obtained by qRT-PCR analysis. Expression ratios of seven genes in SsL compared to SsA; (B) The fold changes of the genes were calculated as the SsL/SsA comparison and are shown on the y-axis. SsA represents the adults of S. shirakii; SsL represents the larvae of S. shirakii; (C) Correlation between the expression fold change level of DEUs between adults and larvae. Full article ">Figure 8 Cont.
Differential expression of DEUs in S. shirakii. (A) Gene expression data obtained by qRT-PCR analysis. Expression ratios of seven genes in SsL compared to SsA; (B) The fold changes of the genes were calculated as the SsL/SsA comparison and are shown on the y-axis. SsA represents the adults of S. shirakii; SsL represents the larvae of S. shirakii; (C) Correlation between the expression fold change level of DEUs between adults and larvae. Full article ">

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Open AccessArticle

Prolonged Starvation Causes Up-Regulation of AQP1 in Adipose Tissue Capillaries of AQP7 Knock-Out Mice

by Mariusz T. Skowronski, Agnieszka Skowronska, Aleksandra Rojek, Michal K. Oklinski and Søren Nielsen

Int. J. Mol. Sci. 2016, 17(7), 1101; https://doi.org/10.3390/ijms17071101 - 22 Jul 2016

Cited by 9 | Viewed by 5554

Abstract

Aquaporins (AQPs) are membrane proteins involved in the regulation of cellular transport and the balance of water and glycerol and cell volume in the white adipose tissue (WAT). In our previous study, we found the co-expression of the AQP1 water channel and AQP7 [...] Read more.

Aquaporins (AQPs) are membrane proteins involved in the regulation of cellular transport and the balance of water and glycerol and cell volume in the white adipose tissue (WAT). In our previous study, we found the co-expression of the AQP1 water channel and AQP7 in the mouse WAT. In our present study, we aimed to find out whether prolonged starvation influences the AQP1 expression of AQP7 knock-out mice (AQP7 KO) in the WAT. To resolve this hypothesis, immunoperoxidase, immunoblot and immunogold microscopy were used. AQP1 expression was found with the use of immunohistochemistry and was confirmed by immunogold microscopy in the vessels of mouse WAT of all studied groups. Semi-quantitative immunoblot and quantitative immunogold microscopy showed a significant increase (by 2.5- to 3-fold) in the abundance of AQP1 protein expression in WAT in the 72 h starved AQP7 KO mice as compared to AQP7+/+ (p < 0.05) and AQP7−/− (p < 0.01) controls, respectively. In conclusion, the AQP1 water channel located in the vessels of WAT is up-regulated in response to prolonged starvation in the WAT of AQP7 KO mice. The present data suggest that an interaction of different AQP isoforms is required for maintaining proper water homeostasis within the mice WAT. Full article

(This article belongs to the Special Issue Aquaporin)

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Figure 1
AQP1 expression in capillary endothelia of mouse WAT. Immunohistochemical staining of AQP1 in paraffin-embedded sections of the WAT from control and fasted mice (control: AQP7−/− (A); AQP7+/+ (B); fasted: AQP7−/− (C); AQP7+/+ (D)). Anti-AQP1 antibody labels small capillaries of WAT in control (A,B) and fasted (C,D) mice. Arrows indicate localization of AQP1 in mouse WAT. A bar indicates 50 µm. Full article ">Figure 2
Western blot analysis of AQP1 protein expression in capillary endothelia of WAT from control and fasted mice (control: AQP7−/−, AQP7+/+; fasted: AQP7−/−, AQP7+/+). Anti-AQP1 antibody recognizes a ~29 kDa band in membrane fractions from mouse WAT (A). Densitometric analysis of AQP1 was performed and normalized against total protein amount (B). Full article ">Figure 3
Immunoelectron-microscopic localization of AQP1 in an ultrathin Lowicryl section of peri-epididymal WAT capillaries from control and fasted mice (control: AQP7−/− (A); AQP7+/+ (B); fasted: AQP7−/− (C); AQP7+/+ (D)). Both apical and basal plasma membranes exhibit labeling (arrows, A–D). Magnification; ×28,000; L; lumen. Full article ">Figure 4
Numbers of gold particles/µm2 in capillary endothelia of WAT from control and fasted mice (control: AQP7−/−, AQP7+/+; fasted: AQP7−/−, AQP7+/+). Quantitation of the electron micrograph results was performed using the iTEM, FIVE Digital Imaging Solutions, based on the analySIS Platform (Olympus, Tokyo, Japan), number of areas counted (n = 20) for each mean ± SD. Full article ">

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Open AccessReview

The Clinical Value of High-Intensity Signals on the Coronary Atherosclerotic Plaques: Noncontrast T1-Weighted Magnetic Resonance Imaging

by Shoichi Ehara, Kenji Matsumoto and Kenei Shimada

Int. J. Mol. Sci. 2016, 17(7), 1187; https://doi.org/10.3390/ijms17071187 - 21 Jul 2016

Cited by 6 | Viewed by 5159

Abstract

Over the past several decades, significant progress has been made in the pathohistological assessment of vulnerable plaques and in invasive intravascular imaging techniques. However, the assessment of plaque morphology by invasive modalities is of limited value for the detection of subclinical coronary atherosclerosis [...] Read more.

Over the past several decades, significant progress has been made in the pathohistological assessment of vulnerable plaques and in invasive intravascular imaging techniques. However, the assessment of plaque morphology by invasive modalities is of limited value for the detection of subclinical coronary atherosclerosis and the subsequent prediction or prevention of acute cardiovascular events. Recently, magnetic resonance (MR) imaging technology has reached a sufficient level of spatial resolution, which allowed the plaque visualization of large and static arteries such as the carotids and aorta. However, coronary wall imaging by MR is still challenging due to the small size of coronary arteries, cardiac and respiratory motion, and the low contrast-to-noise ratio between the coronary artery wall and the surrounding structures. Following the introduction of carotid plaque imaging with noncontrast T1-weighted imaging (T1WI), some investigators have reported that coronary artery high-intensity signals on T1WI are associated with vulnerable plaque morphology and an increased risk of future cardiac events. Although there are several limitations and issues that need to be resolved, this novel MR technique for coronary plaque imaging could influence treatment strategies for atherothrombotic disease and may be useful for understanding the pathophysiological mechanisms of atherothrombotic plaque formation. Full article

(This article belongs to the Special Issue Atherosclerosis and Vascular Imaging 2016)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
A representative case of a HIS lesion on T1WI associated with an intraluminal thrombus. (A) Coronary angiography revealing an intracoronary thrombus identified by the presence of intraluminal filling defects surrounded by contrast agents in the distal right coronary artery (RCA) and an ulceration in the proximal RCA; (B) The OCT examination showing a plaque rupture without a thrombus in the proximal RCA; (C) In contrast, the culprit lesion in the distal RCA with a plaque rupture with a large intracoronary thrombus; (D) Thrombus aspiration and plain old balloon angioplasty (POBA) performed on the culprit lesion; (E) Two days later after POBA, whole-heart coronary MR angiography revealing no significant stenosis in the distal RCA; (F) Coronary T1WI demonstrating intraluminal HIS on the culprit lesion (circle). However, there is no HIS at the ulceration of the proximal RCA; (G) Fused image showing intraluminal HIS in the area corresponding to the culprit lesion (circle). R in panels E–G indicates right side. Full article ">Figure 2
A representative case of an intrawall HIS lesion on T1WI compared with plaque morphology on CT, IVUS and OCT. (A,B) Coronary angiography revealing severe coronary stenosis (circle) in the distal right coronary artery (RCA); (C) Coronary CT angiography showing the napkin-ring sign and positive arterial remodeling; (D) The IVUS image showing a low attenuation plaque; (E) The OCT examination showing a signal-poor region with irregular high- or low-backscattering borders without thrombus; (F) Whole-heart coronary MR angiography showing significant stenosis in the distal RCA (circle); (G) Coronary T1WI demonstrating intrawall HIS (circle); (H) Fused image showing intrawall HIS (circle) in the area corresponding to the severe stenosis. Full article ">Figure 3
The culprit lesion in the proximal left anterior descending coronary artery (LAD). (A) Coronary angiography revealing severe coronary stenosis in the proximal LAD (square) and no significant stenosis in the left main coronary artery (LMCA) (circle); (B) The OCT examination showing intracoronary thrombus in the proximal LAD; (C) There is a lipid-rich plaque in the LMCA (circle); (D) Coronary T1WI demonstrating HIS in the area corresponding to the culprit lesion of the proximal LAD (square). However, no HIS in the LMCA with the lipid-rich plaque is found (circle). Full article ">

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Open AccessArticle

Assessment of Dextran Antigenicity of Intravenous Iron Preparations with Enzyme-Linked Immunosorbent Assay (ELISA)

by Susann Neiser, Taija S. Koskenkorva, Katrin Schwarz, Maria Wilhelm and Susanna Burckhardt

Int. J. Mol. Sci. 2016, 17(7), 1185; https://doi.org/10.3390/ijms17071185 - 21 Jul 2016

Cited by 21 | Viewed by 8483

Abstract

Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran [...] Read more.

Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran can cause severe, antibody-mediated dextran-induced anaphylactic reactions (DIAR), the purpose of this study was to explore the potential of various intravenous iron preparations, non-dextran-based or dextran/dextran-based, to induce these reactions. An IgG-isotype mouse monoclonal anti-dextran antibody (5E7H3) and an enzyme-linked immunosorbent assay (ELISA) were developed to investigate the dextran antigenicity of low molecular weight iron dextran, ferumoxytol, iron isomaltoside 1000, ferric gluconate, iron sucrose and ferric carboxymaltose, as well as isomaltoside 1000, the isolated carbohydrate component of iron isomaltoside 1000. Low molecular weight iron dextran, as well as dextran-based ferumoxytol and iron isomaltoside 1000, reacted with 5E7H3, whereas ferric carboxymaltose, iron sucrose, sodium ferric gluconate, and isolated isomaltoside 1000 did not. Consistent results were obtained with reverse single radial immunodiffusion assay. The results strongly support the hypothesis that, while the carbohydrate alone (isomaltoside 1000) does not form immune complexes with anti-dextran antibodies, iron isomaltoside 1000 complex reacts with anti-dextran antibodies by forming multivalent immune complexes. Moreover, non-dextran based preparations, such as iron sucrose and ferric carboxymaltose, do not react with anti-dextran antibodies. This assay allows to assess the theoretical possibility of a substance to induce antibody-mediated DIARs. Nevertheless, as this is only one possible mechanism that may cause a hypersensitivity reaction, a broader set of assays will be required to get an understanding of the mechanisms that may lead to intravenous iron-induced hypersensitivity reactions. Full article

(This article belongs to the Special Issue Recent Advances in Metal Based Drugs)

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Figure 1
Reactivity of different IV iron preparations with 5E7H3 by reverse single radial immunodiffusion. Positive antigen/antibody reaction, indicated by circular turbidity around the well, is observed in the upper row for the positive control (dextran 5000) and the dextran/dextran-based preparations (LMWID, FMX, and IIM). In the lower row, the negative control (dextran 1000) as well as the non-dextran-based preparations (SFG, IS, and FCM) do not show any reaction. FCM, ferric carboxymaltose; FMX, ferumoxytol; IIM, iron isomaltoside 1000; IS, iron sucrose; LMWID, low molecular weight iron dextran; SFG, sodium ferric gluconate. Full article ">Figure 2
Schematic illustration of immune complex formation and inhibition. (A) Immune complex formation with high molecular weight (HMW) dextran and anti-dextran antibody. A large insoluble complex is generated, which may trigger DIARs; (B) Inhibition of immune complex formation by dextran 1000, i.e., hapten prophylaxis. Dextran 1000 binds to the anti-dextran antibody without formation of an immune complex and thus does not induce any immune response. The anti-dextran antibody is no longer available for binding to the HMW dextran, which thus is less likely to induce DIARs; (C) Proposed mechanism of the observed in vitro multivalent immune complex formation with iron isomaltoside 1000 (IIM) and anti-dextran antibody. In IIM, a number of isomaltoside 1000 (reduced dextran 1000) units are attached to the polynuclear iron core. The presented in vitro results suggest that IIM can act as a polyvalent higher molecular weight dextran and lead to immune complex formation. As in (A), a large insoluble complex is formed, which may trigger DIARs. (A,B) Based on Richter et al. [<a href="#B40-ijms-17-01185" class="html-bibr">40</a>]. Full article ">

408 KiB

Open AccessArticle

Psychopathological Variables and Sleep Quality in Psoriatic Patients

by Maria Luca, Antonina Luca, Maria Letizia Musumeci, Federica Fiorentini, Giuseppe Micali and Carmela Calandra

Int. J. Mol. Sci. 2016, 17(7), 1184; https://doi.org/10.3390/ijms17071184 - 21 Jul 2016

Cited by 17 | Viewed by 5756

Abstract

Psoriasis is an inflammatory disease frequently associated with psychiatric disturbances and sleep disorders. The aim of the study was to assess the prevalence of depression, interaction anxiety, audience anxiety, and sleep quality in psoriatic patients. One hundred and two psoriatic patients were enrolled [...] Read more.

Psoriasis is an inflammatory disease frequently associated with psychiatric disturbances and sleep disorders. The aim of the study was to assess the prevalence of depression, interaction anxiety, audience anxiety, and sleep quality in psoriatic patients. One hundred and two psoriatic patients were enrolled and underwent the following questionnaires: Zung Self-Rating Depression Scale (SDS), Interaction Anxiousness Scale (IAS), Audience Anxiousness Scale (AAS), Pittsburgh Sleep Quality Index (PSQI). The severity of skin lesions was assessed by Psoriasis Area Severity Index (PASI). The presence of a link between clinical variables and with demographic data has been investigated. Psoriasis was linked to depression, interaction and audience anxiety, as well as to poor sleep quality; 37.5% of patients were depressed, 46.1% scored above 37 at the IAS, 47.1% scored above 33 at the AAS. Thirty-nine subjects (38.2%) presented a PSQI ≥ 5. An association between interaction anxiety and lower limbs psoriasis-related erythema as well as between PSQI and head psoriasis-related erythema was found, particularly among male patients. Hence, psoriatic patients should be assessed from a holistic point of view, in order to identify associated disorders that could benefit from targeted treatments. Full article

(This article belongs to the Special Issue Sleep, Circadian Rhythm and Skin)

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3220 KiB

Open AccessArticle

The Effects of Aquaporin-1 in Pulmonary Edema Induced by Fat Embolism Syndrome

by Yiwei Zhang, Kun Tian, Yan Wang, Rong Zhang, Jiawei Shang, Wei Jiang and Aizhong Wang

Int. J. Mol. Sci. 2016, 17(7), 1183; https://doi.org/10.3390/ijms17071183 - 21 Jul 2016

Cited by 17 | Viewed by 6265

Abstract

This study was designed to investigate the role of aquaporin1 (AQP1) in the pathologic process of pulmonary edema induced by fat embolism syndrome (FES) and the effects of a free fatty acid (FFA) mixture on AQP1 expression in pulmonary microvascular endothelial cells (PMVECs). [...] Read more.

This study was designed to investigate the role of aquaporin1 (AQP1) in the pathologic process of pulmonary edema induced by fat embolism syndrome (FES) and the effects of a free fatty acid (FFA) mixture on AQP1 expression in pulmonary microvascular endothelial cells (PMVECs). In vivo, edema was more serious in FES mice compared with the control group. The expression of AQP1 and the wet-to-dry lung weight ratio (W/D) in the FES group were significantly increased compared with the control group. At the same time, inhibition of AQP1 decreased the pathological damage resulting from pulmonary edema. Then we performed a study in vitro to investigate whether AQP1 was induced by FFA release in FES. The mRNA and protein level of AQP1 were increased by FFAs in a dose- and time-dependent manner in PMVECs. In addition, the up-regulation of AQP1 was blocked by the inhibitor of p38 kinase, implicating the p38 MAPK pathway as involved in the FFA-induced AQP1 up-regulation in PMVECs. Our results demonstrate that AQP1 may play important roles in pulmonary edema induced by FES and can be regarded as a new therapy target for treatment of pulmonary edema induced by FES. Full article

(This article belongs to the Special Issue Aquaporin)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Pulmonary edema occurs in FES mice. (A) Gross morphology of the lungs in the control and FES (fat embolism syndrome) groups. Blue arrow, infarction tissues; (B) Lung sections from the control and FES groups were stained with Oil Red O; (C) Lung sections from the FES group at different time points after fat injection were stained with H & E. Blue arrow, ruptured alveolar wall; (D) W/D ratio in the FES group at different time points after fat injection. * p < 0.05, the statistics were made by comparing with Ctrl group, respectively. Full article ">Figure 2
AQP1 is increased in lung of FES mice and inhibition of AQP1 reverses pulmonary edema in FES mice. (A) Western blot and (B) immunohistochemical analyses of AQP1 expression in the FES group at different time points after fat injection. Staining score was shown on the right; (C) Lung sections from the control, FES and FES + AQP1 inhibitors (bumetanideand acetazolamide, respectively) groups were stained with H&E. Blue arrow, ruptured alveolar wall, infiltration of red blood cells, and widened alveolar septa; (D) W/D ratio of the control, FES, FES + bumetanide and FES + acetazolamide groups. * p < 0.05; ** p < 0.001, the statistics were made by comparing with Ctrl group, respectively. # p < 0.05, the statistic was made by comparing with FES group. Full article ">Figure 3
FFA induced up-regulation of AQP1 expression in PMVECs. (A) Primary cultured PMVECs obtained from normal rats. The PMVECs were polygonal or fusiform with a uniform size and displayed a similar and typical cobblestone-like morphology. Magnification 200×; (B) Fluorescence microscopy showed that the PMVECs exhibited green fluorescence after staining with FITC-BSI. The nuclei were stained blue by DAPI. Magnification 200×; (C,D) The protein and mRNA levels of AQP1 in PMVECs stimulated by 500 μM FFAs for different times; (E,F) The protein and mRNA levels of AQP1 in PMVECs stimulated by different concentrations of FFAs for 6 h. ** p < 0.01, the statistics were made by comparing with Ctrl group, respectively. Full article ">Figure 4
Activation of phosphorylation of MAPK by FFAs and effects of p38 inhibition on FFA-induced up-regulation of AQP1 in PMVECs. (A) Western blot analysis of p-ERK, p-p38 kinase, p-JNK expression in PMVECs stimulated by FFAs. P-p38 kinase was significantly activated; (B) SB203580 was confirmed to inhibit the expression of p-p38 kinase in PMVECs stimulated by FFAs; (C) Expression of AQP1 was reduced by SB203080 in PMVECs stimulated by FFAs. * p < 0.05, the statistics were made by comparing with Ctrl group, respectively. # p < 0.05, the statistics were made by comparing with FES group. Full article ">

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Open AccessArticle

High Morphologic Plasticity of Microglia/Macrophages Following Experimental Intracerebral Hemorrhage in Rats

by Shu-Sheng Yang, Li Lin, Yue Liu, Jie Wang, Jiang Chu, Teng Zhang, Lin-Na Ning, Yan Shi, Ying-Yan Fang, Peng Zeng, Jian-Zhi Wang, Ming-Yi Qiu and Qing Tian

Int. J. Mol. Sci. 2016, 17(7), 1181; https://doi.org/10.3390/ijms17071181 - 21 Jul 2016

Cited by 16 | Viewed by 6845

Abstract

As current efforts have limited effects on the clinical outcome of intracerebral hemorrhage (ICH), the mechanisms including microglia/macrophages that involved inflammation need further investigation. Here, 0.4 units of collagenase VII were injected into the left caudate putamen (CPu) to duplicate ICH rat models. [...] Read more.

As current efforts have limited effects on the clinical outcome of intracerebral hemorrhage (ICH), the mechanisms including microglia/macrophages that involved inflammation need further investigation. Here, 0.4 units of collagenase VII were injected into the left caudate putamen (CPu) to duplicate ICH rat models. In the brains of ICH rats, microglia/macrophages, the nearest cells to the hemorrhagic center, were observed as ameboid and Prussian-blue positive. Furthermore, the ameboid microglia/macrophages were differentiation (CD) 68 and interleukin-1β (IL-1β) positive, and neither CD206 nor chitinase3-like 3 (Ym1) positive, suggesting their strong abilities of phagocytosis and secretion of IL-1β. According to the distance to the hemorrhagic center, we selected four areas—I, II, III, and IV—to analyze the morphology of microglia/macrophages. The processes decreased successively from region I to region IV. Microglia/macrophages in region IV had no processes. The processes in region I were radially distributed, however, they showed obvious directivity towards the hemorrhagic center in regions II and III. Region III had the largest density of compactly arrayed microglia/macrophages. All these in vivo results present the high morphologic plasticity of microglia/macrophages and their functions in the pathogenesis of ICHs. Full article

(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)

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Figure 1
Increased and accumulated microglia/macrophages and astrocytes in the hemorrhagic caudate putamen (CPu) of rats. 0.4 U of collagenase VII (2 μL) was injected into the left CPu of three-month-old male Sprague–Dawley (SD) rats (A, ICH). Sham rats (Sham) received 2 μL 0.9% NaCl injection. Beam balance test (B, n = 5/group), elevated body swing test (C, n = 5/group), and brain magnetic resonance imaging (MRI) test (D, n = 4/group) were performed to evaluate the model. By immunohistochemistry staining on brain slices (20 μm), Iba-1 (marker of microglia/macrophage) positive cells and GFAP (marker of astrocyte) positive cells were shown in the collagenase VII injected CPu (E, Bar = 50 μm). By double-label immunofluorescence staining, microglia/macrophages (green) were found much closer to the hemorrhagic center (HC) than astrocytes (red) (F, Bar = 50 μm). The data were expressed as means ± standard error of the mean (SEM).* p < 0.05, ** p < 0.01, *** p < 0.001, vs. Sham. Full article ">Figure 2
Ameboid microglia/macrophages in the hemorrhagic center presented Prussian blue positive. Brain slices (20 μm) of intracerebral hemorrhage (ICH) rats were immunohistochemically stained by Iba-1 (A, n = 5) or further double stained with Prussian blue (B,C, n = 5). The optical densities of Prussian-blue staining were quantitatively analyzed in D (n = 5). Microglia/macrophages in the brain slices taken at 7, 14 and 28 days had stronger blue staining than those in three-day brain slices (C,D). Bar = 25 μm. Data were expressed as means ± SEM. * p < 0.05, ** p < 0.01, vs. ICH 3 d. Δ p < 0.05, vs. ICH 7 d. Full article ">Figure 3
Ameboid microglia/macrophages were mainly differentiation (CD)68 positive in the hemorrhagic center. Brain slices (20 μm) were taken from ICH rats at three days after collagenase VII injection. By double-label immunofluorescence staining, ameboid Iba1 positive (red, A) or CD11b positive (green, B,C) microglia/macrophages in the hemorrhagic center were CD68 positive (green, A, n = 3), neither CD206 nor chitinase 3-like 3 (Ym1) positive (red, B,C) (B and C, n = 3). Bar = 30 μm. Full article ">Figure 4
Increased IL-1β in the hemorrhagic CPu was detected at three and seven days after collagenase VII injection. IL-1β in the CPu was studied by immunohistochemistry (A,B, Bar = 100 μm) on brain slices (20 μm) at one, three, and seven days after collagenase VII injection (ICH 1 d, 3 d and 7 d, n = 3). Control CPu (Sham) was from 2 μL 0.9% NaCl injected rats (n = 3). Data were expressed as means ± SEM. *** p < 0.001, vs. Sham. ΔΔΔ p < 0.001 vs. ICH 1 d. To study the cytokine expressions of activated microglia in the hemorrhagic center, brain slices were double-stained with antibodies specific to pro-inflammatory cytokines interleukin (IL)-1β (C, green) ortumor necrosis factor-α (TNF-α) (D, green) and CD68 (red in C,D, n = 3). Bar = 50 μm. Full article ">Figure 5
Microglia/macrophages showed high morphologic plasticity. Brain slices (20 μm) were taken three days after operation from Sham rats (A) and ICH rats (B,C). By immunohistochemistry, Iba-1 marked microglia/macrophages in the left CPu were observed (n = 3, Bar = 200 μm). According to the distance to the hemorrhagic center (HC), we selected four areas (B,C) to analyze the morphologic features of activated microglia/macrophages, and the left CPu of sham rats was used as control (Sham, A,D,E). The process numbers (D) and the densities (E) of microglia/macrophages were studied. To evaluate the process directivity of a microglia/macrophage, the cell body was set as the origin, and the y-axis was set pointing to the hemorrhagic center (F). The directivity was calculated as the process number in quadrants I and IV deducted the number in quadrants II and III, and the difference was divided by the total process number (G). The morphologic characteristics of activated microglia/macrophages after ICH was summarized in (H). Data were expressed as means ± SEM. * p < 0.05, *** p < 0.001, vs. Sham. ΔΔ p < 0.01, ΔΔΔ p < 0.001, vs. region I. ## p < 0.01, ### p < 0.001, vs. region II. ▲▲▲ p < 0.001, vs. region III. Full article ">

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Open AccessArticle

Aluminum Toxicity-Induced Alterations of Leaf Proteome in Two Citrus Species Differing in Aluminum Tolerance

by Huan Li, Lin-Tong Yang, Yi-Ping Qi, Peng Guo, Yi-Bin Lu and Li-Song Chen

Int. J. Mol. Sci. 2016, 17(7), 1180; https://doi.org/10.3390/ijms17071180 - 21 Jul 2016

Cited by 41 | Viewed by 7276

Abstract

Seedlings of aluminum-tolerant ‘Xuegan’ (Citrus sinensis) and Al-intolerant ‘sour pummelo’ (Citrus grandis) were fertigated for 18 weeks with nutrient solution containing 0 and 1.2 mM AlCl₃·6H₂O. Al toxicity-induced inhibition of photosynthesis and the decrease of [...] Read more.

Seedlings of aluminum-tolerant ‘Xuegan’ (Citrus sinensis) and Al-intolerant ‘sour pummelo’ (Citrus grandis) were fertigated for 18 weeks with nutrient solution containing 0 and 1.2 mM AlCl₃·6H₂O. Al toxicity-induced inhibition of photosynthesis and the decrease of total soluble protein only occurred in C. grandis leaves, demonstrating that C. sinensis had higher Al tolerance than C. grandis. Using isobaric tags for relative and absolute quantification (iTRAQ), we obtained more Al toxicity-responsive proteins from C. sinensis than from C. grandis leaves, which might be responsible for the higher Al tolerance of C. sinensis. The following aspects might contribute to the Al tolerance of C. sinensis: (a) better maintenance of photosynthesis and energy balance via inducing photosynthesis and energy-related proteins; (b) less increased requirement for the detoxification of reactive oxygen species and other toxic compounds, such as aldehydes, and great improvement of the total ability of detoxification; and (c) upregulation of low-phosphorus-responsive proteins. Al toxicity-responsive proteins related to RNA regulation, protein metabolism, cellular transport and signal transduction might also play key roles in the higher Al tolerance of C. sinensis. We present the global picture of Al toxicity-induced alterations of protein profiles in citrus leaves, and identify some new Al toxicity-responsive proteins related to various biological processes. Our results provide some novel clues about plant Al tolerance. Full article

(This article belongs to the Special Issue Plant Proteomic Research)

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Effects of Al toxicity on leaf CO2 assimilation (A); stomatal conductance (B); intercellular CO2 concentration (C); Al (D) and total soluble protein (E) concentrations. Bars represent the means ± standard error SE (n = 5). DW: dry weight; FW: fresh weight. Differences among four treatment combinations (two species × two Al) were analyzed by two-way analysis of variance. Means were separated by Duncan’s new multiple range test. Different letters above the bars indicate a significant difference at p < 0.05. Full article ">Figure 2
Classification of Al toxicity-responsive proteins in C. sinensis (A) and C. grandis (B) leaves; and Venn diagram analysis of Al toxicity-responsive proteins (C). Full article ">Figure 3
Effects of Al toxicity on the activities of superoxide dismutase (SOD) (A); ascorbate peroxidase (APX) (B); catalase (CAT) (C); monodehydroascorbate reductase (MDAR) (D) and lipoxygenase (LOX) (E) in C. sinensis leaves. Bars represent the means ± SE (n = 4). Significance tests for two means (control and Al toxicity) were carried out by the unpaired t-test at the p < 0.05 level. Different letters above the bars indicate a significant difference at p < 0.05. Full article ">

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Open AccessReview

Matrix Metalloproteinases in Non-Neoplastic Disorders

by Akinori Tokito and Michihisa Jougasaki

Int. J. Mol. Sci. 2016, 17(7), 1178; https://doi.org/10.3390/ijms17071178 - 21 Jul 2016

Cited by 65 | Viewed by 12714

Abstract

The matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases belonging to the metzincin superfamily. There are at least 23 members of MMPs ever reported in human, and they and their substrates are widely expressed in many tissues. Recent growing evidence has established that MMP not [...] Read more.

The matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases belonging to the metzincin superfamily. There are at least 23 members of MMPs ever reported in human, and they and their substrates are widely expressed in many tissues. Recent growing evidence has established that MMP not only can degrade a variety of components of extracellular matrix, but also can cleave and activate various non-matrix proteins, including cytokines, chemokines and growth factors, contributing to both physiological and pathological processes. In normal conditions, MMP expression and activity are tightly regulated via interactions between their activators and inhibitors. Imbalance among these factors, however, results in dysregulated MMP activity, which causes tissue destruction and functional alteration or local inflammation, leading to the development of diverse diseases, such as cardiovascular disease, arthritis, neurodegenerative disease, as well as cancer. This article focuses on the accumulated evidence supporting a wide range of roles of MMPs in various non-neoplastic diseases and provides an outlook on the therapeutic potential of inhibiting MMP action. Full article

(This article belongs to the Special Issue Metalloproteins)

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6466 KiB

Open AccessReview

Molecular Approaches to Genetically Improve the Accumulation of Health-Promoting Secondary Metabolites in Staple Crops—A Case Study: The Lipoxygenase-B1 Genes and Regulation of the Carotenoid Content in Pasta Products

by Grazia M. Borrelli and Daniela Trono

Int. J. Mol. Sci. 2016, 17(7), 1177; https://doi.org/10.3390/ijms17071177 - 21 Jul 2016

Cited by 22 | Viewed by 8348

Abstract

Secondary metabolites, also known as phytochemicals, represent a large subset of plant molecules that include compounds with health-promoting effects. Indeed, a number of epidemiological studies have shown that, when taken regularly and in adequate amounts, these molecules can have long-term beneficial effects on [...] Read more.

Secondary metabolites, also known as phytochemicals, represent a large subset of plant molecules that include compounds with health-promoting effects. Indeed, a number of epidemiological studies have shown that, when taken regularly and in adequate amounts, these molecules can have long-term beneficial effects on human health, through reduction of the incidence of degenerative diseases, such as cardiovascular diseases, obesity, diabetes, and cancer. As the dietary intake of these phytochemicals is often inadequate, various strategies are in use to improve their content in staple crops, and the end-products thereof. One of the most effective strategies is crop improvement through genetic approaches, as this is the only way to generate new cultivars in which the high accumulation of a given phytochemical is stably fixed. Efforts to genetically improve quality traits are rapidly evolving, from classical breeding to molecular-assisted approaches; these require sound understanding of the molecular bases underlying the traits, to identify the genes/alleles that control them. This can be achieved through global analysis of the metabolic pathway responsible for phytochemical accumulation, to identify the link between phytochemical content and the activities of key enzymes that regulate the metabolic pathway, and between the key enzymes and their encoding genes/alleles. Once these have been identified, they can be used as markers for selection of new improved genotypes through biotechnological approaches. This review provides an overview of the major health-promoting properties shown to be associated with the dietary intake of phytochemicals, and describes how molecular approaches provide means for improving the health quality of edible crops. Finally, a case study is illustrated, of the identification in durum wheat of the Lipoxygenase-B1 genes that control the final carotenoid content in semolina-based foods, such as pasta products. Full article

(This article belongs to the Section Molecular Plant Sciences)

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Schematic representation of the biosynthesis of secondary metabolites. E4P, erythrose 4-phosphate; G3P, glyceraldehyde 3-phosphate; PEP, phosphoenolpyruvate; PYR, pyruvate; DOX5P, deoxyxylulose 5-phosphate; IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; PP, pyrophosphate; GPP, geranyl pyrophosphate; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate. Full article ">Figure 2
General structures of the three most important classes of phenolic compounds for human health. Benzoic acids: p-hydroxybenzoic acid, R3=OH; vanillic acid, R2=OCH3 and R3=OH; gallic acid, R2=R3=R4=OH; syringic acid, R2=R4=OCH3 and R3=OH. Cinnamic acids: p-coumaric acid, R3=OH; caffeic acid, R3=R4=OH; ferulic acid, R2=OCH3 and R3=OH; sinapic acid, R2=R4=OCH3 and R3=OH. Flavonoids: R3–R6=H or other groups such as OH or oxo- groups. For most food flavonoids: R4’=R6=H and R5=OH. In biochanin A, R4’=OCH3; formononetin, R4’= OCH3 and R5=R6=H; glycetein, R5=H and R6=OH. Full article ">Figure 3
Structures of carotenes and xanthophylls that are important for human health. Full article ">Figure 4
Hydrolysis of glucosinolates catalyzed by myrosinase. Myrosinase catalyzes the hydrolysis of the thioglucoside linkage, thus leading to the formation of different products. R represents the amino-acid-derived side-chain. Full article ">Figure 5
Outline of how a functional approach can contribute to identification of a candidate gene associated with high levels of a health-promoting phytochemical in a staple crop. A germplasm collection is screened to determine the variability in nature of the trait. Genotypes characterized by contrasting metabolite accumulation are subjected to enzymatic analysis to identify key enzyme variants (i.e., allozymes) that are characterized by contrasting activities/efficiencies, and molecular analysis to identify alleles that encode the different allozymes and to evaluate their expression levels. Then, on the basis of both the enzymatic and molecular information, a candidate gene/allele can be identified that can then be efficiently used in advanced breeding programs that are aimed at the introgression of the trait of interest into an elite cultivar. Full article ">Figure 6
Reaction catalyzed by lipoxygenase (LOX) during pasta processing. LOX catalyzes hydroperoxidation of free linoleate (LH), to give the corresponding hydroperoxide (LOOH). During pasta processing the pentadienyl (L•) and peroxy radicals (LOO•) of linoleate oxidize the semolina carotenoid pigments, thus leading to carotenoid loss and discoloration of the end product. Full article ">Figure 7
Carotenoid loss during pasta processing. Cultivars: TRI, Trinakria; CAP, Capeiti 8; TRE, Tresor; PRI, Primadur; BRI, Brindur; SIM, Simeto; COS, Cosmodur. Data from [<a href="#B169-ijms-17-01177" class="html-bibr">169</a>]. Full article ">

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Open AccessArticle

Protective Effect of Salicornia europaea Extracts on High Salt Intake-Induced Vascular Dysfunction and Hypertension

by Nisha Panth, Sin-Hee Park, Hyun Jung Kim, Deuk-Hoi Kim and Min-Ho Oak

Int. J. Mol. Sci. 2016, 17(7), 1176; https://doi.org/10.3390/ijms17071176 - 21 Jul 2016

Cited by 32 | Viewed by 8352

Abstract

High salt intake causes and aggravates arterial hypertension and vascular dysfunction. We investigated the effect of Salicornia europaea extracts (SE) on vascular function and blood pressure. SE constituents were analyzed using high performance liquid chromatography, and SE’s effect on vascular function was evaluated [...] Read more.

High salt intake causes and aggravates arterial hypertension and vascular dysfunction. We investigated the effect of Salicornia europaea extracts (SE) on vascular function and blood pressure. SE constituents were analyzed using high performance liquid chromatography, and SE’s effect on vascular function was evaluated in isolated porcine coronary arteries. SE’s vascular protective effect was also evaluated in vivo using normotensive and spontaneous hypertensive rats (SHRs). SE mainly contained sodium chloride (55.6%), 5-(hydroxymethyl)furfural, p-coumaric acid, and trans-ferulic acid. High sodium (160 mmol/L) induced vascular dysfunction; however, SE containing the same quantity of sodium did not cause vascular dysfunction. Among the compounds in SE, trans-ferulic acid accounts for the vascular protective effect. Normotensive rats fed a high-salt diet showed significantly increased systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP), which decreased significantly in the SE-treated groups. In SHRs, high edible salt intake significantly increased SBP, DBP, and MAP, but SE intake was associated with a significantly lower MAP. Thus, SE did not induce vascular dysfunction, and trans-ferulic acid might be at least partly responsible for the vasoprotective effect of SE. Taken together, SE could be used as an alternative to purified salt to prevent and ameliorate hypertension. Full article

(This article belongs to the Section Bioactives and Nutraceuticals)

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HPLC profiles for Salicornia europaea extracts (SE) and three reference compounds: 5-(hydroxymethyl)furfural (1); p-coumaric acid (2); and trans-ferulic acid (3). Chromatograms were extracted at 305 nm. (A) chromatograms of SE; and (B–D) HPLC profile and UV spectra of 5-(hydroxymethyl)furfural (1); p-coumaric acid (2); and trans-ferulic acid (3). Full article ">Figure 2
Relaxant responses to bradykinin in porcine coronary arteries following incubation for 18 h with a medium containing a high sodium concentration (160 mmol/L) or Salicornia europaea extracts (SE) at the concentration equivalent to 160 mmol/L sodium. Relaxant responses are expressed as the percentage of relaxation of the initial tone induced by U46619. Values are expressed as mean ± SEM from n = 6 to 10 experiments. Concentration-response curves were compared using two-way ANOVA. “*” stands for p < 0.05 vs. control. Full article ">Figure 3
Dose-dependent vasorelaxant effects of 5-(hydroxymethyl)furfural, p-coumaric acid, and trans-ferulic acid in porcine coronary arteries (A) and vasorelaxant responses to bradykinin in porcine coronary arteries following incubation for 18 h with a medium containing a high sodium concentration (160 mmol/L) in presence or absence of 5-(hydroxymethyl)furfural (0.1 mmol/L), p-coumaric acid (0.1 mmol/L), and trans-ferulic acid (0.1 mmol/L) (B) and pre-treated with L-NAME (an inhibitor of eNOS, 0.1 mmol/L) (C). Relaxant responses are expressed as the percentage of relaxation of the initial tone induced by U46619. Values are expressed as mean ± SEM from n = 8 to 10 experiments. Concentration-response curves were compared using two-way ANOVA. “*” stands for p < 0.05 vs. NaCl. Full article ">Figure 4
Body weight (A,B) and food consumption (C,D) were examined in spontaneously hypertensive rats (B,D) and Sprague–Dawley (A,C) rats fed high edible salt and Salicornia europaea extracts (SE) for six weeks. CTR–SD, control SD rat; HS–SD, high edible salt-fed SD rat (800 mg/kg/day); SE–SD, SE-fed SD rat (1400 mg/kg/day); CTR–SHR, control SHR; HS–SHR, high edible salt-fed SHR (800 mg/kg/day); SE–SHR, SE-fed SHR (1400 mg/kg/day). Values are expressed as mean ± SEM. “***” stands for p < 0.001 vs. week 0. Full article ">Figure 5
Effect of Salicornia europaea extracts (SE) on the systolic blood pressure (A); diastolic blood pressure (B); and mean arterial pressure (C) in a normotensive SD rat group (CTR–SD) compared with the values in a high edible salt-fed group (HS–SD, 800 mg/kg/day) and a group fed SE (SE–SD, 1400 mg/kg/day). Values are expressed as mean ± SEM. “*”, “**” and “***” stand for p < 0.05, p < 0.01, and p < 0.001 vs. CTR–SD, respectively. “#” stands for p < 0.05 vs. HS–SD. Full article ">Figure 6
Effect of Salicornia europaea extracts (SE) on the systolic blood pressure (A); diastolic blood pressure (B); and mean arterial pressure (C) in the spontaneous hypertensive rat group (CTR–SHR) compared to the values in the high edible salt fed group (HS–SHR, 800 mg/kg/day) and the SE group (SE–SHR, 1400 mg/kg/day). Values are expressed as mean ± SEM. “***” stands for p < 0.001 vs. CTR-SD. “##” and “###” stands for p < 0.01 and p < 0.001 vs. CTR–SHR respectively. “$” stands for p < 0.05 vs. HS–SHR. Full article ">Figure 6 Cont.
Effect of Salicornia europaea extracts (SE) on the systolic blood pressure (A); diastolic blood pressure (B); and mean arterial pressure (C) in the spontaneous hypertensive rat group (CTR–SHR) compared to the values in the high edible salt fed group (HS–SHR, 800 mg/kg/day) and the SE group (SE–SHR, 1400 mg/kg/day). Values are expressed as mean ± SEM. “***” stands for p < 0.001 vs. CTR-SD. “##” and “###” stands for p < 0.01 and p < 0.001 vs. CTR–SHR respectively. “$” stands for p < 0.05 vs. HS–SHR. Full article ">

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Open AccessReview

Developments in Methods for Measuring the Intestinal Absorption of Nanoparticle-Bound Drugs

by Wei Liu, Hao Pan, Caiyun Zhang, Liling Zhao, Ruixia Zhao, Yongtao Zhu and Weisan Pan

Int. J. Mol. Sci. 2016, 17(7), 1171; https://doi.org/10.3390/ijms17071171 - 21 Jul 2016

Cited by 53 | Viewed by 12389

Abstract

With the rapid development of nanotechnology, novel drug delivery systems comprising orally administered nanoparticles (NPs) have been paid increasing attention in recent years. The bioavailability of orally administered drugs has significant influence on drug efficacy and therapeutic dosage, and it is therefore imperative [...] Read more.

With the rapid development of nanotechnology, novel drug delivery systems comprising orally administered nanoparticles (NPs) have been paid increasing attention in recent years. The bioavailability of orally administered drugs has significant influence on drug efficacy and therapeutic dosage, and it is therefore imperative that the intestinal absorption of oral NPs be investigated. This review examines the various literature on the oral absorption of polymeric NPs, and provides an overview of the intestinal absorption models that have been developed for the study of oral nanoparticles. Three major categories of models including a total of eight measurement methods are described in detail (in vitro: dialysis bag, rat gut sac, Ussing chamber, cell culture model; in situ: intestinal perfusion, intestinal loops, intestinal vascular cannulation; in vivo: the blood/urine drug concentration method), and the advantages and disadvantages of each method are contrasted and elucidated. In general, in vitro and in situ methods are relatively convenient but lack accuracy, while the in vivo method is troublesome but can provide a true reflection of drug absorption in vivo. This review summarizes the development of intestinal absorption experiments in recent years and provides a reference for the systematic study of the intestinal absorption of nanoparticle-bound drugs. Full article

(This article belongs to the Section Materials Science)

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Open AccessArticle

Enhancement of Anti-Hypoxic Activity and Differentiation of Cardiac Stem Cells by Supernatant Fluids from Cultured Macrophages that Phagocytized Dead Mesenchymal Stem Cells

by Liang Liu, Xian Jin, Zhong’e Zhou and Chengxing Shen

Int. J. Mol. Sci. 2016, 17(7), 1175; https://doi.org/10.3390/ijms17071175 - 20 Jul 2016

Cited by 1 | Viewed by 5129

Abstract

Background: Most mesenchymal stem cells (MSCs) die shortly after transplantation into a myocardial infarcted area. Dead MSCs (dMSCs) are phagocytized by macrophages (pMΦ) in vivo and in vitro; however, the effects of pMΦ on cardiac stem cells (CSCs) remain unknown. Methods: MSCs, CSCs, [...] Read more.

Background: Most mesenchymal stem cells (MSCs) die shortly after transplantation into a myocardial infarcted area. Dead MSCs (dMSCs) are phagocytized by macrophages (pMΦ) in vivo and in vitro; however, the effects of pMΦ on cardiac stem cells (CSCs) remain unknown. Methods: MSCs, CSCs, and macrophages were obtained from bone marrow, hearts, and peritoneal cavity of mice, respectively. dMSCs were harvested after hypoxia for 24 h, and incubated with macrophages (2:1) for another 2 days with or without lipopolysaccharide (LPS, 50 ng/mL) and sorted by flow cytometry to obtain pMΦ. Viability and apoptosis of CSCs were respectively evaluated with the cell counting kit-8 (CCk-8) assay and Annexin V-PE/7-AAD staining at 0, 6, 12, and 24 h of culture with supernatant fluids from macrophages (MΦ), LPS-stimulated macrophages (LPS-pMΦ), pMΦ, and MSCs. GATA-4 and c-TnI expression was measured by flow cytometry on the seventh day. Expression of inflammation and growth factors was assessed by real-time polymerase chain reaction (RT-PCR) in MΦ, LPS-pMΦ, and pMΦ cells. Results: pMΦ expressed higher levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β)and lower levels of tumor necrosis factor-α(TNF-α)and IL-6 than LPS-pMΦ, higher levels of growth factors and of GATA-4 and c-TnI at the 7th day, which were similar to those in MSCs. CSCs cultured with supernatant fluids of pMΦ exhibited higher proliferative, anti-hypoxic, and differentiation activities. Conclusion: The supernatant fluids of macrophages that had phagocytized dead MSCs encouraged changes in phenotype and growth factor expression, enhanced proliferation, differentiation, and anti-hypoxic activity of CSCs, which is relevant to understanding the persistent therapeutic effect of MSCs after their massive demise upon transplantation in myocardial infarction. Furthermore, some miRNAs or proteins which were extracted from the supernatant fluids may give us a new insight into the treatment of myocardial infarction in the future. Full article

(This article belongs to the Section Biochemistry)

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Figure 1
Dead mesenchymal stem cells (dMSCs) were phagocytosed by lipopolysaccharide (LPS)-stimulated macrophages. dMSC labeled with FITC-green fluorescent protein (GFP) and activated macrophages (2:1) stained with PE-F4/80 were co-cultured for 48 h, and then detected by fluorescence microscopy and flow cytometry (FCM). (A) MSCs were cultured with three conditions, including no hypoxia and 10% fetal bovine serum (FBS), 0.5% O2 for 6 h and phosphate buffered saline (PBS), 0.5% O2 for 24 h and PBS. The ratio of dMSCs was the greatest in third group; (B) Fluorescence microscopy was used to show the phenomenon of phagocytosis; (C,D) Phagocytosis ability of macrophage was evaluated by FCM. Significant difference was shown in two groups with or without LPS (p < 0.001), and the mean phagocytosis rate (%) of LPS-stimulated macrophage was 80%. The percentage of each group and each bar represents the mean value of triplicate results, which means three independent experiments were done. MΦ: macrophage. Full article ">Figure 2
Inflammatory factors secreted by the cell of MΦ, LPS + MΦ, and pMΦ. Anti-Inflammatory factors interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) had higher expression in pMΦ, and pro-Inflammatory factors IL-6 and tumor necrosis factor-α (TNF-α) had lower expression in pMΦ. *** p < 0.001 compared with MΦ. Each bar represents the mean value ± SD of triplicate results, which means three independent experiments were done. Full article ">Figure 3
Growth factors were secreted by the cell of MΦ, LPS+ MΦ, pMΦ, and MSCs. Compared to MΦ, all growth factors (Insulin like Growth Factor-1 (IGF-1), Phenyl Glycidyl Ether2 (PGE2), Keratinocyte Growth Factor (KGF), bFibroblast Growth Factor (bFGF)) showed significantly higher expression in pMΦ, and the results were similar or approaching that of the MSCs (** p < 0.01, *** p < 0.001). Each bar represents the mean value ±SD of triplicate results, which means three independent experiments were done. Full article ">Figure 4
Cell counting kit-8 (CCK-8) proliferation assay and Annexin V-PE/7-ADD detected by FCM were used to measure the proliferation and anti-hypoxic properties of CSCs. (A,B) CSCs which were co-cultured with the supernatants of MΦ, LPS-MΦ, pMΦ, and MSCs were harvested in 0, 6, 12, and 24 h in hypoxia (0.5% O2) conditions. Compared to the MΦ and LPS-MΦ, the proportion of apoptosis in CSCs was significantly lower in pMΦ and MSCs at each time point; (C) The proliferation of CSCs detected by CCK-8 was dramatically increased in pMΦ and MSCs groups.* p < 0.05; ** p < 0.01; and *** p < 0.001 compared with MΦ. The percentage of each group represents the mean value of triplicate results, which means three independent experiments were done. Full article ">Figure 5
The markers of cardiomyocytes were detected in CSCs. (A) CSCs were obtained from the hearts of mice and then GATA-4 and TnI were detected by FCM; (B,C) CSCs co-cultured with the supernatants of MΦ, LPS-MΦ, pMΦ, and MSCs were harvested after 7 days. FCM was used to detect cardiomyocytes markers. GATA-4 and TnI showed significantly higher expression in the pMΦ and MSC groups; ** p < 0.01; and *** p < 0.001, compared with MΦ. The percentage and the bar of each group represent the mean value of triplicate results, which means that three independent experiments were done. Full article ">

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Open AccessArticle

La Autoantigen Induces Ribosome Binding Protein 1 (RRBP1) Expression through Internal Ribosome Entry Site (IRES)-Mediated Translation during Cellular Stress Condition

by Wenqing Gao, Qi Li, Ruiyu Zhu and Jian Jin

Int. J. Mol. Sci. 2016, 17(7), 1174; https://doi.org/10.3390/ijms17071174 - 20 Jul 2016

Cited by 18 | Viewed by 6564

Abstract

The function of ribosome binding protein 1 (RRBP1) is regulating the transportation and secretion of some intracellular proteins in mammalian cells. Transcription of RRBP1 is induced by various cytokines. However, few studies focused on the process of RRPB1 mRNA translation. The RRBP1 mRNA [...] Read more.

The function of ribosome binding protein 1 (RRBP1) is regulating the transportation and secretion of some intracellular proteins in mammalian cells. Transcription of RRBP1 is induced by various cytokines. However, few studies focused on the process of RRPB1 mRNA translation. The RRBP1 mRNA has a long 5′ untranslated region that potentially formed a stable secondary structure. In this study, we show that the 5′ UTR of RRBP1 mRNA contains an internal ribosome entry site (IRES). Moreover, the RRBP1 expression is induced by chemotherapeutic drug paclitaxel or adriamycin in human hepatocellular carcinoma cells and accompanied with the increased expression of La autoantigen (La), which binds to RRBP1 IRES element and facilitates translation initiation. Interestingly, we found IRES-mediated RRBP1 translation is also activated during serum-starvation condition which can induce cytoplasmic localization of La. After mapping the entire RRBP1 5′ UTR, we determine the core IRES activity is located between nt-237 and -58. Furthermore, two apical GARR loops within the functional RRBP1 IRES elements may be important for La binding. These results strongly suggest an important role for IRES-dependent translation of RRBP1 mRNA in hepatocellular carcinoma cells during cellular stress conditions. Full article

(This article belongs to the Special Issue Post-Transcriptional Gene Regulation by Ribonucleoprotein Complexes)

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Open AccessReview

Aquaporins in the Colon as a New Therapeutic Target in Diarrhea and Constipation

by Nobutomo Ikarashi, Risako Kon and Kiyoshi Sugiyama

Int. J. Mol. Sci. 2016, 17(7), 1172; https://doi.org/10.3390/ijms17071172 - 20 Jul 2016

Cited by 113 | Viewed by 10360

Abstract

Aquaporins (AQPs) play important roles in the water transport system in the human body. There are currently 13 types of AQP, AQP0 through AQP12, which are expressed in various organs. Many members of the AQP family are expressed in the intestinal tract. AQP3 [...] Read more.

Aquaporins (AQPs) play important roles in the water transport system in the human body. There are currently 13 types of AQP, AQP0 through AQP12, which are expressed in various organs. Many members of the AQP family are expressed in the intestinal tract. AQP3 is predominantly expressed in the colon, ultimately controlling the water transport. Recently, it was clarified that several laxatives exhibit a laxative effect by changing the AQP3 expression level in the colon. In addition, it was revealed that morphine causes severe constipation by increasing the AQP3 expression level in the colon. These findings have shown that AQP3 is one of the most important functional molecules in water transport in the colon. This review will focus on the physiological and pathological roles of AQP3 in the colon, and discuss clinical applications of colon AQP3. Full article

(This article belongs to the Special Issue Aquaporin)

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Full article ">Figure 1
Distribution of aquaporin-3 (AQP3) expression in the rat colon. Full article ">Figure 2
Effect of magnesium sulfate on fecal water content (A); the mRNA expression level of sodium myo-inositol transporter, gene associated with osmotic pressure (B); and AQP3 protein expression level (C) in the rat colon. Dunnett’s test: * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. 0 h. Adapted with permission from Ikarashi et al. [<a href="#B29-ijms-17-01172" class="html-bibr">29</a>]. Copyright 2011 The Pharmaceutical Society of Japan. Full article ">Figure 3
Water transport in the colon after magnesium sulfate administration. Full article ">Figure 4
Effect of bisacodyl on fecal water content (A); and AQP3 protein expression level in the rat colon (B). Dunnett’s test: * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. 0 h. Adapted with permission from Ikarashi et al. [<a href="#B38-ijms-17-01172" class="html-bibr">38</a>]. Copyright 2011 The American Physiological Society. Full article ">Figure 5
Water transport in the colon after morphine administration. Full article ">Figure 6
Effect of combination of magnesium sulfate and bisacodyl on fecal water content (A); and AQP3 protein expression in the rat colon (B). Dunnett’s test: ** p < 0.01, and *** p < 0.001 vs. control group. Adapted with permission from Ikarashi et al. [<a href="#B60-ijms-17-01172" class="html-bibr">60</a>]. Copyright 2012 Elsevier. Full article ">

3029 KiB

Open AccessArticle

Structural Analysis of Hand Drawn Bumblebee Bombus terrestris Silk

by Andrea L. Woodhead, Tara D. Sutherland and Jeffrey S. Church

Int. J. Mol. Sci. 2016, 17(7), 1170; https://doi.org/10.3390/ijms17071170 - 20 Jul 2016

Cited by 5 | Viewed by 5696

Abstract

Bombus terrestris, commonly known as the buff-tailed bumblebee, is native to Europe, parts of Africa and Asia. It is commercially bred for use as a pollinator of greenhouse crops. Larvae pupate within a silken cocoon that they construct from proteins produced in [...] Read more.

Bombus terrestris, commonly known as the buff-tailed bumblebee, is native to Europe, parts of Africa and Asia. It is commercially bred for use as a pollinator of greenhouse crops. Larvae pupate within a silken cocoon that they construct from proteins produced in modified salivary glands. The amino acid composition and protein structure of hand drawn B. terrestris, silk fibres was investigated through the use of micro-Raman spectroscopy. Spectra were obtained from single fibres drawn from the larvae salivary gland at a rate of 0.14 cm/s. Raman spectroscopy enabled the identification of poly(alanine), poly(alanine-glycine), phenylalanine, tryptophan, and methionine, which is consistent with the results of amino acid analysis. The dominant protein conformation was found to be coiled coil (73%) while the β-sheet content of 10% is, as expected, lower than those reported for hornets and ants. Polarized Raman spectra revealed that the coiled coils were highly aligned along the fibre axis while the β-sheet and random coil components had their peptide carbonyl groups roughly perpendicular to the fibre axis. The protein orientation distribution is compared to those of other natural and recombinant silks. A structural model for the B. terrestris silk fibre is proposed based on these results. Full article

(This article belongs to the Special Issue Silk-Based Materials: From Production to Characterization)

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Full article ">Figure 1
Secondary electron images obtained from (a,b) section of a B. terrestris silk fibre hand drawn at 0.14 cm/s and (c) a fibre hand drawn at 0.10 cm/s. Full article ">Figure 2
Raman spectrum obtained from the hand drawn (0.14 cm/s) B. terrestris silk fibre. Full article ">Figure 3
The amino acid composition (molar percentage) determined for B. terrestris silk (blue) and determined from amino acid sequences of the four fibrous B. terrestris silk proteins; BterF1: ABW21694, BterF2: ABW21695, BterF3: ABW21696, and BterF4: ABW21697 (red) [<a href="#B26-ijms-17-01170" class="html-bibr">26</a>]. Full article ">Figure 4
The Raman amide I band of the hand drawn B. terrestris fibre. Spectral deconvolution (a); the underlying black trace represents the raw data, the red trace is the sum of the component peaks (dark grey) and components that are not associated with protein conformation and present for fitting purposes only (light grey). Polarized Raman spectra (b). Full article ">Figure 5
The graph of the limiting values of ‹P4› as a function of ‹P2›. The (‹P2›, ‹P4›) couple determined for B. terrestris (∆), A. Illawarra (○) fibres are presented along with those of B. mori (+) and S. c. ricini (□) cocoon silks and N. edulis (◊) dragline silk [<a href="#B13-ijms-17-01170" class="html-bibr">13</a>,<a href="#B15-ijms-17-01170" class="html-bibr">15</a>]. Note that the values determined for B. mori cocoon and S. c. ricini cocoon overlap. The dashed grey line represents the values of ‹P4› when λ4 = 0. Full article ">Figure 6
The most probable orientation distribution determined for the hand drawn B. terrestris silk fibre (black trace). A. illawarra fibres (dashed black trace) [<a href="#B13-ijms-17-01170" class="html-bibr">13</a>] presented along with those of B. mori and S. c. ricini cocoon (dark grey trace) and N. edulis dragline (light grey trace) silks [<a href="#B16-ijms-17-01170" class="html-bibr">16</a>]. The 0° of the polar plot coincides with the fibre direction. Full article ">Figure 7
Graph of ‹P2› values obtained from the deconvolution of the polarized hand drawn B. terrestris silk spectra (a); Proposed structure of the B. terrestris silk fibres based on the Raman results (b). Full article ">

400 KiB

Open AccessReview

Immune Checkpoint Inhibitors: A New Opportunity in the Treatment of Ovarian Cancer?

by Gloria Mittica, Sofia Genta, Massimo Aglietta and Giorgio Valabrega

Int. J. Mol. Sci. 2016, 17(7), 1169; https://doi.org/10.3390/ijms17071169 - 20 Jul 2016

Cited by 52 | Viewed by 8321

Abstract

Epithelial ovarian cancer (EOC) is the leading cause of death for gynecological cancer. The standard treatment for advanced stage is the combination of optimal debulking surgery and platinum-based chemotherapy. Nevertheless, recurrence is frequent (around 70%) and prognosis is globally poor. New therapeutic agents [...] Read more.

Epithelial ovarian cancer (EOC) is the leading cause of death for gynecological cancer. The standard treatment for advanced stage is the combination of optimal debulking surgery and platinum-based chemotherapy. Nevertheless, recurrence is frequent (around 70%) and prognosis is globally poor. New therapeutic agents are needed to improve survival. Since EOC is strongly immunogenic, immune checkpoint inhibitors are under evaluation for their capacity to contrast the “turn off” signals expressed by the tumor to escape the immune system and usually responsible for self-tolerance maintenance. This article reviews the literature on anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), anti-PD-1, anti-PD-L1, and anti-PD-L2 antibodies in EOC and highlights their possible lines of development. Further studies are needed to better define the prognostic role of the immune checkpoint inhibitors, to identify predictors of response and the optimal clinical setting in EOC. Full article

(This article belongs to the Special Issue Cancer Immunology with a Focus on Understudied Cancers as Targets for Immunotherapy)

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959 KiB

Open AccessCommunication

Inflammaging and Frailty Status Do Not Result in an Increased Extracellular Vesicle Concentration in Circulation

by Ainhoa Alberro, Matías Sáenz-Cuesta, Maider Muñoz-Culla, Maider Mateo-Abad, Esperanza Gonzalez, Estefania Carrasco-Garcia, Marcos J. Araúzo-Bravo, Ander Matheu, Itziar Vergara and David Otaegui

Int. J. Mol. Sci. 2016, 17(7), 1168; https://doi.org/10.3390/ijms17071168 - 20 Jul 2016

Cited by 24 | Viewed by 5631

Abstract

In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in [...] Read more.

In the last decades extracellular vesicles (EVs) have emerged as key players for intercellular communication. In the case of inflammation, several studies have reported that EV levels are increased in circulation during inflammatory episodes. Based on this, we investigated whether aging results in elevated EV number, as a basal proinflammatory status termed “inflammaging” has been described in aged individuals. Moreover, we also hypothesized that frailty and dependence conditions of the elderly could affect EV concentration in plasma. Results showed that inflammaging, frailty or dependence status do not result in EV increase, at least in the total number of EVs in circulation. These results open a new perspective for investigating the role of EVs in human aging and in the inflammaging process. Full article

(This article belongs to the Special Issue Focus on Extracellular Vesicles)

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Full article ">Figure 1
An elevated concentration of interleukin-6 (IL-6) is observed in aged subjects. IL-6 levels were measured by ELISA and concentration values above 4.7 pg/mL were considered detectable. (A) Elderly individuals have a higher concentration of IL-6 than adults (*** p < 0.001); and (B) there is a high variability among Robust, Frail and Non-autonomous elderly, but an increasing concentration with dependency can be observed. Each dot shows the concentration of a subject and the line represents the average value of the group. Full article ">Figure 2
Nanoparticle tracking analysis (NTA) of extracellular vesicles (EVs). Particle size and EV concentration were measured by NTA. (A) Size distribution of EVs. Each line represents one sample. Despite the particle concentration difference, all samples have a similar size distribution—they are enriched in small EVs (50–300 nm); (B) EV concentration of different age ranges were compared and samples from elder people (79–92 years) do not show an increased EV number; and (C) among elder individuals, the frailty status does also not alter EV concentration. In figures (B) and (C) each dot shows the EV concentration of a subject and the line represents the average value of the group. Full article ">Figure 2 Cont.
Nanoparticle tracking analysis (NTA) of extracellular vesicles (EVs). Particle size and EV concentration were measured by NTA. (A) Size distribution of EVs. Each line represents one sample. Despite the particle concentration difference, all samples have a similar size distribution—they are enriched in small EVs (50–300 nm); (B) EV concentration of different age ranges were compared and samples from elder people (79–92 years) do not show an increased EV number; and (C) among elder individuals, the frailty status does also not alter EV concentration. In figures (B) and (C) each dot shows the EV concentration of a subject and the line represents the average value of the group. Full article ">

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Open AccessReview

Clinical Metabolomics: The New Metabolic Window for Inborn Errors of Metabolism Investigations in the Post-Genomic Era

by Abdellah Tebani, Lenaig Abily-Donval, Carlos Afonso, Stéphane Marret and Soumeya Bekri

Int. J. Mol. Sci. 2016, 17(7), 1167; https://doi.org/10.3390/ijms17071167 - 20 Jul 2016

Cited by 95 | Viewed by 12606

Abstract

Inborn errors of metabolism (IEM) represent a group of about 500 rare genetic diseases with an overall estimated incidence of 1/2500. The diversity of metabolic pathways involved explains the difficulties in establishing their diagnosis. However, early diagnosis is usually mandatory for successful treatment. [...] Read more.

Inborn errors of metabolism (IEM) represent a group of about 500 rare genetic diseases with an overall estimated incidence of 1/2500. The diversity of metabolic pathways involved explains the difficulties in establishing their diagnosis. However, early diagnosis is usually mandatory for successful treatment. Given the considerable clinical overlap between some inborn errors, biochemical and molecular tests are crucial in making a diagnosis. Conventional biological diagnosis procedures are based on a time-consuming series of sequential and segmented biochemical tests. The rise of “omic” technologies offers holistic views of the basic molecules that build a biological system at different levels. Metabolomics is the most recent “omic” technology based on biochemical characterization of metabolites and their changes related to genetic and environmental factors. This review addresses the principles underlying metabolomics technologies that allow them to comprehensively assess an individual biochemical profile and their reported applications for IEM investigations in the precision medicine era. Full article

(This article belongs to the Special Issue Metabolomic Technologies in Medicine)

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9951 KiB

Open AccessArticle

Leptin Receptor Metabolism Disorder in Primary Chondrocytes from Adolescent Idiopathic Scoliosis Girls

by Yun-Jia Wang, Hong-Gui Yu, Zhen-Hai Zhou, Qiang Guo, Long-Jie Wang and Hong-Qi Zhang

Int. J. Mol. Sci. 2016, 17(7), 1160; https://doi.org/10.3390/ijms17071160 - 20 Jul 2016

Cited by 30 | Viewed by 5345

Abstract

To investigate the underlying mechanisms of low metabolic activity of primary chondrocytes obtained from girls with adolescent idiopathic scoliosis (AIS); AIS is a spine-deforming disease that often occurs in girls. AIS is associated with a lower bone mass than that of healthy individuals [...] Read more.

To investigate the underlying mechanisms of low metabolic activity of primary chondrocytes obtained from girls with adolescent idiopathic scoliosis (AIS); AIS is a spine-deforming disease that often occurs in girls. AIS is associated with a lower bone mass than that of healthy individuals and osteopenia. Leptin was shown to play an important role in bone growth. It can also regulate the function of chondrocytes. Changes in leptin and Ob-R levels in AIS patients have been reported in several studies. The underlying mechanisms between the dysfunction of peripheral leptin signaling and abnormal chondrocytes remain unclear; The following parameters were evaluated in AIS patients and the control groups: total serum leptin levels; Ob-R expression in the plasma membrane of primary chondrocytes; JAK2 and STAT3 phosphorylation status. Then, we inhibited the lysosome and proteasome and knocked down clathrin heavy chain (CHC) expression in primary chondrocytes isolated from girls with AIS and evaluated Ob-R expression. We investigated the effects of leptin combined with a lysosome inhibitor or CHC knockdown in primary chondrocytes obtained from AIS patients; Compared with the controls, AIS patients showed similar total serum leptin levels, reduced JAK2 and STAT3 phosphorylation, and decreased cartilage matrix synthesis in the facet joint. Lower metabolic activity and lower membrane expression of Ob-R were observed in primary chondrocytes from the AIS group than in the controls. Lysosome inhibition increased the total Ob-R content but had no effect on the membrane expression of Ob-R or leptin’s effects on AIS primary chondrocytes. CHC knockdown upregulated the membrane Ob-R levels and enhanced leptin’s effects on AIS primary chondrocytes; The underlying mechanism of chondrocytes that are hyposensitive to leptin in some girls with AIS is low plasma membrane Ob-R expression that results from an imbalance between the rate of receptor endocytosis and the insertion of newly synthesized receptors into the membrane. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms of Bone Metastasis)

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759 KiB

Open AccessReview

Angiotensin A/Alamandine/MrgD Axis: Another Clue to Understanding Cardiovascular Pathophysiology

by Jaroslav Hrenak, Ludovit Paulis and Fedor Simko

Int. J. Mol. Sci. 2016, 17(7), 1098; https://doi.org/10.3390/ijms17071098 - 20 Jul 2016

Cited by 80 | Viewed by 9870

Abstract

The renin-angiotensin system (RAS) plays a crucial role in cardiovascular regulations and its modulation is a challenging target for the vast majority of cardioprotective strategies. However, many biological effects of these drugs cannot be explained by the known mode of action. Our comprehension [...] Read more.

The renin-angiotensin system (RAS) plays a crucial role in cardiovascular regulations and its modulation is a challenging target for the vast majority of cardioprotective strategies. However, many biological effects of these drugs cannot be explained by the known mode of action. Our comprehension of the RAS is thus far from complete. The RAS represents an ingenious system of “checks and balances”. It incorporates vasoconstrictive, pro-proliferative, and pro-inflammatory compounds on one hand and molecules with opposing action on the other hand. The list of these molecules is still not definitive because new biological properties can be achieved by minor alteration of the molecular structure. The angiotensin A/alamandine-MrgD cascade associates the deleterious and protective branches of the RAS. Its identification provided a novel clue to the understanding of the RAS. Angiotensin A (Ang A) is positioned at the “crossroad” in this system since it either elicits direct vasoconstrictive and pro-proliferative actions or it is further metabolized to alamandine, triggering opposing effects. Alamandine, the central molecule of this cascade, can be generated both from the “deleterious” Ang A as well as from the “protective” angiotensin 1–7. This pathway modulates peripheral and central blood pressure regulation and cardiovascular remodeling. Further research will elucidate its interactions in cardiovascular pathophysiology and its possible therapeutic implications. Full article

(This article belongs to the Special Issue Molecular Research on Hypertension)

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1642 KiB

Open AccessArticle

Tissue-Specific Effects of Vitamin E Supplementation

by Eugene Jansen, Dale Viezeliene, Piet Beekhof, Eric Gremmer and Leonid Ivanov

Int. J. Mol. Sci. 2016, 17(7), 1166; https://doi.org/10.3390/ijms17071166 - 19 Jul 2016

Cited by 10 | Viewed by 5796

Abstract

A multivitamin and mineral supplementation study of 6 weeks was conducted with male and female mice. The control group received a standard dose of vitamins and minerals of 1× the Recommended Daily Intake (RDI), whereas a second group received 3× RDI. A third [...] Read more.

A multivitamin and mineral supplementation study of 6 weeks was conducted with male and female mice. The control group received a standard dose of vitamins and minerals of 1× the Recommended Daily Intake (RDI), whereas a second group received 3× RDI. A third group received a high dose of vitamin E (25× RDI), close to the upper limit of toxicity (UL), but still recommended and considered to be harmless and beneficial. The high dose of vitamin E caused a number of beneficial, but also adverse effects. Different biomarkers of tissue toxicity, oxidative stress related processes and inflammation were determined. These biomarkers did not change in plasma and erythrocytes to a large extent. In the liver of male mice, some beneficial effects were observed by a lower concentration of several biomarkers of inflammation. However, in the kidney of male mice, a number of biomarkers increased substantially with the higher dose of vitamin E, indicating tissue toxicity and an increased level of inflammation. Since this dose of vitamin E, which is lower than the UL, cause some adverse effects, even after a short exposure period, further studies are required to reconsider the UL for vitamin E. Full article

(This article belongs to the Special Issue Tocopherols and Tocotrienols: Metabolism and Properties)

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816 KiB

Open AccessReview

Senescence in Human Mesenchymal Stem Cells: Functional Changes and Implications in Stem Cell-Based Therapy

by Valentina Turinetto, Emanuela Vitale and Claudia Giachino

Int. J. Mol. Sci. 2016, 17(7), 1164; https://doi.org/10.3390/ijms17071164 - 19 Jul 2016

Cited by 371 | Viewed by 20086

Abstract

Regenerative medicine is extensively interested in developing cell therapies using mesenchymal stem cells (MSCs), with applications to several aging-associated diseases. For successful therapies, a substantial number of cells are needed, requiring extensive ex vivo cell expansion. However, MSC proliferation is limited and it [...] Read more.

Regenerative medicine is extensively interested in developing cell therapies using mesenchymal stem cells (MSCs), with applications to several aging-associated diseases. For successful therapies, a substantial number of cells are needed, requiring extensive ex vivo cell expansion. However, MSC proliferation is limited and it is quite likely that long-term culture evokes continuous changes in MSCs. Therefore, a substantial proportion of cells may undergo senescence. In the present review, we will first present the phenotypic characterization of senescent human MSCs (hMSCs) and their possible consequent functional alterations. The accumulation of oxidative stress and dysregulation of key differentiation regulatory factors determine decreased differentiation potential of senescent hMSCs. Senescent hMSCs also show a marked impairment in their migratory and homing ability. Finally, many factors present in the secretome of senescent hMSCs are able to exacerbate the inflammatory response at a systemic level, decreasing the immune modulation activity of hMSCs and promoting either proliferation or migration of cancer cells. Considering the deleterious effects that these changes could evoke, it would appear of primary importance to monitor the occurrence of senescent phenotype in clinically expanded hMSCs and to evaluate possible ways to prevent in vitro MSC senescence. An updated critical presentation of the possible strategies for in vitro senescence monitoring and prevention constitutes the second part of this review. Understanding the mechanisms that drive toward hMSC growth arrest and evaluating how to counteract these for preserving a functional stem cell pool is of fundamental importance for the development of efficient cell-based therapeutic approaches. Full article

(This article belongs to the Special Issue DNA Damage and Repair in Degenerative Diseases 2016)

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