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Int. J. Mol. Sci., Volume 19, Issue 2 (February 2018)

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Cover Story (view full-size image) The IL-23/IL-17 axis mediates the initiation and persistence of inflammation during Psoriasis and [...] Read more.
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Editorial

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Open AccessEditorial Molecular Research on Emerging Viruses: Evolution, Diagnostics, Pathogenesis, and Therapeutics
Int. J. Mol. Sci. 2018, 19(2), 398; doi:10.3390/ijms19020398
Received: 22 December 2017 / Revised: 17 January 2018 / Accepted: 26 January 2018 / Published: 30 January 2018
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Abstract
Viruses are increasingly recognized as emerging infectious disease agents in both humans and animals.[...] Full article
Open AccessEditorial Special Protein Molecules Computational Identification
Int. J. Mol. Sci. 2018, 19(2), 536; doi:10.3390/ijms19020536
Received: 16 January 2018 / Revised: 2 February 2018 / Accepted: 10 February 2018 / Published: 10 February 2018
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Abstract
Computational identification of special protein molecules is a key issue in understanding protein function. It can guide molecular experiments and help to save costs. I assessed 18 papers published in the special issue of Int. J. Mol. Sci., and also discussed the
[...] Read more.
Computational identification of special protein molecules is a key issue in understanding protein function. It can guide molecular experiments and help to save costs. I assessed 18 papers published in the special issue of Int. J. Mol. Sci., and also discussed the related works. The computational methods employed in this special issue focused on machine learning, network analysis, and molecular docking. New methods and new topics were also proposed. There were in addition several wet experiments, with proven results showing promise. I hope our special issue will help in protein molecules identification researches. Full article
(This article belongs to the Special Issue Special Protein Molecules Computational Identification)
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Research

Jump to: Editorial, Review, Other

Open AccessArticle Crosstalk of PmCBFs and PmDAMs Based on the Changes of Phytohormones under Seasonal Cold Stress in the Stem of Prunus mume
Int. J. Mol. Sci. 2018, 19(2), 15; doi:10.3390/ijms19020015
Received: 2 December 2017 / Revised: 8 January 2018 / Accepted: 9 January 2018 / Published: 23 January 2018
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Abstract
Plants facing the seasonal variations always need a growth restraining mechanism when temperatures turn down. C-repeat binding factor (CBF) genes work essentially in the cold perception. Despite lots of researches on CBFs, the multiple crosstalk is still interesting on their
[...] Read more.
Plants facing the seasonal variations always need a growth restraining mechanism when temperatures turn down. C-repeat binding factor (CBF) genes work essentially in the cold perception. Despite lots of researches on CBFs, the multiple crosstalk is still interesting on their interaction with hormones and dormancy-associated MADS (DAM) genes in the growth and dormancy control. Therefore, this study highlights roles of PmCBFs in cold-induced dormancy from different orgens. And a sense-response relationship between PmCBFs and PmDAMs is exhibited in this process, jointly regulated by six PmCBFs and PmDAM4–6. Meantime, GA3 and ABA showed negative and positive correlation with PmCBFs expression levels, respectively. We also find a high correlation between IAA and PmDAM1–3. Finally, we display the interaction mode of PmCBFs and PmDAMs, especially PmCBF1-PmDAM1. These results can disclose another view of molecular mechanism in plant growth between cold-response pathway and dormancy regulation together with genes and hormones. Full article
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Open AccessArticle Lasiodiplodia theobromae as a Producer of Biotechnologically Relevant Enzymes
Int. J. Mol. Sci. 2018, 19(2), 29; doi:10.3390/ijms19020029
Received: 19 December 2017 / Revised: 11 January 2018 / Accepted: 17 January 2018 / Published: 23 January 2018
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Abstract
Phytopathogenic fungi are known to produce several types of enzymes usually involved in plant cell wall degradation and pathogenesis. The increasing of global temperature may induce fungi, such as Lasiodiplodia theobromae (L. theobromae), to alter its behavior. Nonetheless, there is only
[...] Read more.
Phytopathogenic fungi are known to produce several types of enzymes usually involved in plant cell wall degradation and pathogenesis. The increasing of global temperature may induce fungi, such as Lasiodiplodia theobromae (L. theobromae), to alter its behavior. Nonetheless, there is only limited information regarding the effect of temperature on L. theobromae production of enzymes. The need for new, thermostable enzymes, that are biotechnologically relevant, led us to investigate the effect of temperature on the production of several extracellular enzymatic activities by different L. theobromae strains. Fungi were grown at 25 °C, 30 °C and 37 °C and the enzymatic activities were detected by plate assays, quantified by spectrophotometric methods and characterized by zymography. The thermostability (25–80 °C) of the enzymes produced was also tested. Strains CAA019, CBS339.90, LA-SOL3, LA-SV1 and LA-MA-1 produced amylases, gelatinases, caseinases, cellulases, lipases, laccases, xylanases, pectinases and pectin liases. Temperature modulated the expression of the enzymes, and this effect was more visible when fungi were grown at 37 °C than at lower temperatures. Contrary to proteolytic and endoglucanolytic activities, whose highest activities were detected when fungi were grown at 30 °C, lipolytic activity was not detected at this growth temperature. Profiles of proteases and endoglucanases of fungi grown at different temperatures were characterized by zymography. Enzymes were shown to be more thermostable when fungi were grown at 30 °C. Proteases were active up to 50 °C and endoglucanases up to 70 °C. Lipases were the least stable, with activities detected up to 45 °C. The enzymatic profiles detected for L. theobromae strains tested showed to be temperature and strain-dependent, making this species a good target for biotechnological applications. Full article
(This article belongs to the Special Issue Microbial Enzymes)
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Open AccessArticle Peretinoin, an Acyclic Retinoid, Inhibits Hepatitis B Virus Replication by Suppressing Sphingosine Metabolic Pathway In Vitro
Int. J. Mol. Sci. 2018, 19(2), 108; doi:10.3390/ijms19020108
Received: 11 December 2017 / Revised: 10 January 2018 / Accepted: 17 January 2018 / Published: 23 January 2018
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Abstract
Hepatocellular carcinoma (HCC) frequently develops from hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. We previously reported that peretinoin, an acyclic retinoid, inhibits HCV replication. This study aimed to examine the influence of peretinoin on the HBV lifecycle. HBV-DNA and covalently
[...] Read more.
Hepatocellular carcinoma (HCC) frequently develops from hepatitis C virus (HCV) and hepatitis B virus (HBV) infection. We previously reported that peretinoin, an acyclic retinoid, inhibits HCV replication. This study aimed to examine the influence of peretinoin on the HBV lifecycle. HBV-DNA and covalently closed circular DNA (cccDNA) were evaluated by a qPCR method in HepG2.2.15 cells. Peretinoin significantly reduced the levels of intracellular HBV-DNA, nuclear cccDNA, and HBV transcript at a concentration that did not induce cytotoxicity. Conversely, other retinoids, such as 9-cis, 13-cis retinoic acid (RA), and all-trans-retinoic acid (ATRA), had no effect or rather increased HBV replication. Mechanistically, although peretinoin increased the expression of HBV-related transcription factors, as observed for other retinoids, peretinoin enhanced the binding of histone deacetylase 1 (HDAC1) to cccDNA in the nucleus and negatively regulated HBV transcription. Moreover, peretinoin significantly inhibited the expression of SPHK1, a potential inhibitor of HDAC activity, and might be involved in hepatic inflammation, fibrosis, and HCC. SPHK1 overexpression in cells cancelled the inhibition of HBV replication induced by peretinoin. This indicates that peretinoin activates HDAC1 and thereby suppresses HBV replication by inhibiting the sphingosine metabolic pathway. Therefore, peretinoin may be a novel therapeutic agent for HBV replication and chemoprevention against HCC. Full article
(This article belongs to the Special Issue Inflammation and Cancer 2018)
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Open AccessArticle GC-MS Metabolomics to Evaluate the Composition of Plant Cuticular Waxes for Four Triticum aestivum Cultivars
Int. J. Mol. Sci. 2018, 19(2), 249; doi:10.3390/ijms19020249
Received: 1 December 2017 / Revised: 12 January 2018 / Accepted: 17 January 2018 / Published: 23 January 2018
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Abstract
Wheat (Triticum aestivum L.) is an important food crop, and biotic and abiotic stresses significantly impact grain yield. Wheat leaf and stem surface waxes are associated with traits of biological importance, including stress resistance. Past studies have characterized the composition of wheat
[...] Read more.
Wheat (Triticum aestivum L.) is an important food crop, and biotic and abiotic stresses significantly impact grain yield. Wheat leaf and stem surface waxes are associated with traits of biological importance, including stress resistance. Past studies have characterized the composition of wheat cuticular waxes, however protocols can be relatively low-throughput and narrow in the range of metabolites detected. Here, gas chromatography-mass spectrometry (GC-MS) metabolomics methods were utilized to provide a comprehensive characterization of the chemical composition of cuticular waxes in wheat leaves and stems. Further, waxes from four wheat cultivars were assayed to evaluate the potential for GC-MS metabolomics to describe wax composition attributed to differences in wheat genotype. A total of 263 putative compounds were detected and included 58 wax compounds that can be classified (e.g., alkanes and fatty acids). Many of the detected wax metabolites have known associations to important biological functions. Principal component analysis and ANOVA were used to evaluate metabolite distribution, which was attributed to both tissue type (leaf, stem) and cultivar differences. Leaves contained more primary alcohols than stems such as 6-methylheptacosan-1-ol and octacosan-1-ol. The metabolite data were validated using scanning electron microscopy of epicuticular wax crystals which detected wax tubules and platelets. Conan was the only cultivar to display alcohol-associated platelet-shaped crystals on its abaxial leaf surface. Taken together, application of GC-MS metabolomics enabled the characterization of cuticular wax content in wheat tissues and provided relative quantitative comparisons among sample types, thus contributing to the understanding of wax composition associated with important phenotypic traits in a major crop. Full article
(This article belongs to the Special Issue Metabolomics in the Plant Sciences 2017)
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Open AccessArticle Solid Lipid Nanoparticles Carrying Temozolomide for Melanoma Treatment. Preliminary In Vitro and In Vivo Studies
Int. J. Mol. Sci. 2018, 19(2), 255; doi:10.3390/ijms19020255
Received: 20 December 2017 / Revised: 11 January 2018 / Accepted: 18 January 2018 / Published: 24 January 2018
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Abstract
Aim: To develop an innovative delivery system for temozolomide (TMZ) in solid lipid nanoparticles (SLN), which has been preliminarily investigated for the treatment of melanoma. Materials and Methods: SLN-TMZ was obtained through fatty acid coacervation. Its pharmacological effects were assessed and compared with
[...] Read more.
Aim: To develop an innovative delivery system for temozolomide (TMZ) in solid lipid nanoparticles (SLN), which has been preliminarily investigated for the treatment of melanoma. Materials and Methods: SLN-TMZ was obtained through fatty acid coacervation. Its pharmacological effects were assessed and compared with free TMZ in in vitro and in vivo models of melanoma and glioblastoma. Results: Compared to the standard free TMZ, SLN-TMZ exerted larger effects, when cell proliferation of melanoma cells, and neoangiogeneis were evaluated. SLN-TMZ also inhibited growth and vascularization of B16-F10 melanoma in C57/BL6 mice, without apparent toxic effects. Conclusion: SLN could be a promising strategy for the delivery of TMZ, allowing an increased stability of the drug and thereby its employment in the treatment of aggressive malignacies. Full article
(This article belongs to the Special Issue Nanotechnology in Drug Delivery)
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Open AccessArticle Role of Disulfide Bonds in Activity and Stability of Tigerinin-1R
Int. J. Mol. Sci. 2018, 19(2), 288; doi:10.3390/ijms19020288
Received: 12 December 2017 / Revised: 12 January 2018 / Accepted: 17 January 2018 / Published: 23 January 2018
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Abstract
Tigerinin-1R (Arg–Val–Cys–Ser–Ala–Ile–Pro–Leu–Pro–Ile–Cys–His–NH2), a cationic 12-mer peptide containing a disulfide bond extracted from frog skin secretions, lacks antibacterial activity, but has the ability to stimulate insulin release both in vitro and in vivo. To study the structure–function relationships of tigerinin-1R, we designed
[...] Read more.
Tigerinin-1R (Arg–Val–Cys–Ser–Ala–Ile–Pro–Leu–Pro–Ile–Cys–His–NH2), a cationic 12-mer peptide containing a disulfide bond extracted from frog skin secretions, lacks antibacterial activity, but has the ability to stimulate insulin release both in vitro and in vivo. To study the structure–function relationships of tigerinin-1R, we designed and synthesized five analogs, including tigerinin-cyclic, tigerinin-1R-L4, tigerinin-linear, [C3K]tigerinin-1R, and [C11K]tigerinin-1R. Tigerinin-1R promoted insulin secretion in a concentration-dependent manner in INS-1 cells without obvious cytotoxicity. At a concentration of 10−5 M, [C11K]tigerinin-1R exhibited the highest stimulation ability, suggesting that the positive charge at the C-terminus may contribute to the in vitro insulin-releasing activity of tigerinin-1R. Tigerinin-1R peptides stimulated insulin release in INS-1 cells through a universal mechanism that involves mobilization of intracellular calcium without disrupting the cell membrane. In vivo experiments showed that both tigerinin-1R and [C11K]tigerinin-1R improved glucose tolerance in overnight-fasted mice. Due to its structural stability, tigerinin-1R showed superior hypoglycemic activity to [C11K]tigerinin-1R, which suggested a critical role of the disulfide bonds. In addition, we also identified a protective effect of tigerinin-1R peptides in apoptosis induced by oxidative stress. These results further confirm the potential for the development of tigerinin-1R as an anti-diabetic therapeutic agent in clinical practice. Full article
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Open AccessArticle Selective Blockade of the Metabotropic Glutamate Receptor mGluR5 Protects Mouse Livers in In Vitro and Ex Vivo Models of Ischemia Reperfusion Injury
Int. J. Mol. Sci. 2018, 19(2), 314; doi:10.3390/ijms19020314
Received: 20 November 2017 / Revised: 18 January 2018 / Accepted: 22 January 2018 / Published: 23 January 2018
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Abstract
2-Methyl-6-(phenylethynyl)pyridine (MPEP), a negative allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, protects hepatocytes from ischemic injury. In astrocytes and microglia, MPEP depletes ATP. These findings seem to be self-contradictory, since ATP depletion is a fundamental stressor in ischemia. This study attempted
[...] Read more.
2-Methyl-6-(phenylethynyl)pyridine (MPEP), a negative allosteric modulator of the metabotropic glutamate receptor (mGluR) 5, protects hepatocytes from ischemic injury. In astrocytes and microglia, MPEP depletes ATP. These findings seem to be self-contradictory, since ATP depletion is a fundamental stressor in ischemia. This study attempted to reconstruct the mechanism of MPEP-mediated ATP depletion and the consequences of ATP depletion on protection against ischemic injury. We compared the effects of MPEP and other mGluR5 negative modulators on ATP concentration when measured in rat hepatocytes and acellular solutions. We also evaluated the effects of mGluR5 blockade on viability in rat hepatocytes exposed to hypoxia. Furthermore, we studied the effects of MPEP treatment on mouse livers subjected to cold ischemia and warm ischemia reperfusion. We found that MPEP and 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) deplete ATP in hepatocytes and acellular solutions, unlike fenobam. This finding suggests that mGluR5s may not be involved, contrary to previous reports. MPEP, as well as MTEP and fenobam, improved hypoxic hepatocyte viability, suggesting that protection against ischemic injury is independent of ATP depletion. Significantly, MPEP protected mouse livers in two different ex vivo models of ischemia reperfusion injury, suggesting its possible protective deployment in the treatment of hepatic inflammatory conditions. Full article
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Open AccessArticle Identification of Proteins Involved in Carbohydrate Metabolism and Energy Metabolism Pathways and Their Regulation of Cytoplasmic Male Sterility in Wheat
Int. J. Mol. Sci. 2018, 19(2), 324; doi:10.3390/ijms19020324
Received: 19 December 2017 / Revised: 13 January 2018 / Accepted: 21 January 2018 / Published: 23 January 2018
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Abstract
Cytoplasmic male sterility (CMS) where no functional pollen is produced has important roles in wheat breeding. The anther is a unique organ for male gametogenesis and its abnormal development can cause male sterility. However, the mechanisms and regulatory networks related to plant male
[...] Read more.
Cytoplasmic male sterility (CMS) where no functional pollen is produced has important roles in wheat breeding. The anther is a unique organ for male gametogenesis and its abnormal development can cause male sterility. However, the mechanisms and regulatory networks related to plant male sterility are poorly understood. In this study, we conducted comparative analyses using isobaric tags for relative and absolute quantification (iTRAQ) of the pollen proteins in a CMS line and its wheat maintainer. Differentially abundant proteins (DAPs) were analyzed based on Gene Ontology classifications, metabolic pathways and transcriptional regulation networks using Blast2GO. We identified 5570 proteins based on 23,277 peptides, which matched with 73,688 spectra, including proteins in key pathways such as glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and 6-phosphofructokinase 1 in the glycolysis pathway, isocitrate dehydrogenase and citrate synthase in the tricarboxylic acid cycle and nicotinamide adenine dinucleotide (NADH)-dehydrogenase and adenosine-triphosphate (ATP) synthases in the oxidative phosphorylation pathway. These proteins may comprise a network that regulates male sterility in wheat. Quantitative real time polymerase chain reaction (qRT-PCR) analysis, ATP assays and total sugar assays validated the iTRAQ results. These DAPs could be associated with abnormal pollen grain formation and male sterility. Our findings provide insights into the molecular mechanism related to male sterility in wheat. Full article
(This article belongs to the Section Molecular Botany)
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Open AccessArticle Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells
Int. J. Mol. Sci. 2018, 19(2), 327; doi:10.3390/ijms19020327
Received: 11 November 2017 / Revised: 10 January 2018 / Accepted: 16 January 2018 / Published: 23 January 2018
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Abstract
The maintenance of ordinal cell cycle phases is a critical biological process in cancer genesis, which is a crucial target for anti-cancer drugs. As an important natural isoquinoline alkaloid from Chinese herbal medicine, Berberine (BBR) has been reported to possess anti-cancer potentiality to
[...] Read more.
The maintenance of ordinal cell cycle phases is a critical biological process in cancer genesis, which is a crucial target for anti-cancer drugs. As an important natural isoquinoline alkaloid from Chinese herbal medicine, Berberine (BBR) has been reported to possess anti-cancer potentiality to induce cell cycle arrest in hepatocellular carcinoma cells (HCC). However, the underlying mechanism remains to be elucidated. In our present study, G0/G1 phase cell cycle arrest was observed in berberine-treated Huh-7 and HepG2 cells. Mechanically, we observed that BBR could deactivate the Akt pathway, which consequently suppressed the S-phase kinase-associated protein 2 (Skp2) expression and enhanced the expression and translocation of Forkhead box O3a (FoxO3a) into nucleus. The translocated FoxO3a on one hand could directly promote the transcription of cyclin-dependent kinase inhibitors (CDKIs) p21Cip1 and p27Kip1, on the other hand, it could repress Skp2 expression, both of which lead to up-regulation of p21Cip1 and p27Kip1, causing G0/G1 phase cell cycle arrest in HCC. In conclusion, BBR promotes the expression of CDKIs p21Cip1 and p27Kip1 via regulating the Akt/FoxO3a/Skp2 axis and further induces HCC G0/G1 phase cell cycle arrest. This research uncovered a new mechanism of an anti-cancer effect of BBR. Full article
(This article belongs to the Special Issue Cell Growth Regulation)
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Open AccessArticle Pharmacokinetic Study of Bioactive Flavonoids in the Traditional Japanese Medicine Keigairengyoto Exerting Antibacterial Effects against Staphylococcus aureus
Int. J. Mol. Sci. 2018, 19(2), 328; doi:10.3390/ijms19020328
Received: 15 December 2017 / Revised: 12 January 2018 / Accepted: 22 January 2018 / Published: 23 January 2018
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Abstract
Recent studies have demonstrated that flavonoid glucuronides can be deconjugated to the active form aglycone by β-glucuronidase-expressing macrophages. Keigairengyoto (KRT) is a flavonoid-rich traditional Japanese medicine reported to enhance bacterial clearance through immune modulation. Our aims are to examine the pharmacokinetics of KRT
[...] Read more.
Recent studies have demonstrated that flavonoid glucuronides can be deconjugated to the active form aglycone by β-glucuronidase-expressing macrophages. Keigairengyoto (KRT) is a flavonoid-rich traditional Japanese medicine reported to enhance bacterial clearance through immune modulation. Our aims are to examine the pharmacokinetics of KRT flavonoids and to identify active flavonoids contributing to the adjuvant effects of KRT. KRT was evaluated at pharmacokinetic analysis to quantify absorbed flavonoids, and cutaneous infection assay induced in mice by inoculation of Staphylococcus aureus. Preventive or therapeutic KRT administration reduced the number of bacteria in the infection site as well as macroscopic and microscopic lesion scores with efficacies similar to antibiotics. Pharmacokinetic study revealed low plasma levels of flavonoid aglycones after KRT administration; however, plasma concentrations were enhanced markedly by β-glucuronidase treatment, with baicalein the most abundant (Cmax, 1.32 µg/mL). In random screening assays, flavonoids such as bacalein, genistein, and apigenin enhanced bacteria phagocytosis by macrophages. Glucuronide bacalin was converted to aglycone baicalein by incubation with living macrophages, macrophage lysate, or skin homogenate. Taken together, the adjuvant effect of KRT may be due to some blood-absorbed flavonoids which enhance macrophage functions in host defense. Flavonoid-rich KRT may be a beneficial treatment for infectious skin inflammation. Full article
(This article belongs to the Special Issue Bioactive Phenolics and Polyphenols 2018)
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Open AccessArticle Biotherapeutic Effect of Gingival Stem Cells Conditioned Medium in Bone Tissue Restoration
Int. J. Mol. Sci. 2018, 19(2), 329; doi:10.3390/ijms19020329
Received: 28 December 2017 / Revised: 18 January 2018 / Accepted: 19 January 2018 / Published: 23 January 2018
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Abstract
Bone tissue engineering is one of the main branches of regenerative medicine. In this field, the use of a scaffold, which supported bone development, in combination with mesenchymal stem cells (MSCs), has promised better outcomes for bone regeneration. In particular, human gingival mesenchymal
[...] Read more.
Bone tissue engineering is one of the main branches of regenerative medicine. In this field, the use of a scaffold, which supported bone development, in combination with mesenchymal stem cells (MSCs), has promised better outcomes for bone regeneration. In particular, human gingival mesenchymal stem cells (hGMSCs) may present advantages compared to other MSCs, including the easier isolation. However, MSCs’ secretome has attracted much attention for its potential use in tissue regeneration, such as conditioned medium (CM) that contains different soluble factors proved to be useful for the regenerative purposes. In this study, we evaluated the osteogenic capacity of a poly-(lactide) (3D-PLA) scaffold enriched with hGMSCs and hGMSCs derived CM and its ability to regenerate bone defects in rat calvarias. 3D-PLA alone, 3D-PLA + CM or 3D-PLA + hGMSCs with/without CM were implanted in Wistar male rats subjected to calvarial defects. We observed that 3D-PLA scaffold enriched with hGMSCs and CM showed a better osteogenic capacity, being able to repair the calvarial defect as revealed in vivo by morphological evaluation. Moreover, transcriptomic analysis in vitro revealed the upregulation of genes involved in ossification and regulation of ossification in the 3D-PLA + CM + hGMSCs group. All of these results indicate the great osteogenic ability of 3D-PLA + CM + hGMSCs supporting its use in bone regenerative medicine, in particular in the repair of cranial bone defects. Especially, hGMSCs derived CM played a key role in the induction of the osteogenic process and in bone regeneration. Full article
(This article belongs to the Special Issue Cell Colonization in Scaffolds)
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Open AccessArticle Involvement of Bradykinin B2 Receptor in Pathological Vascularization in Oxygen-Induced Retinopathy in Mice and Rabbit Cornea
Int. J. Mol. Sci. 2018, 19(2), 330; doi:10.3390/ijms19020330
Received: 7 December 2017 / Revised: 15 January 2018 / Accepted: 19 January 2018 / Published: 23 January 2018
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Abstract
The identification of components of the kallikrein–kinin system in the vitreous from patients with microvascular retinal diseases suggests that bradykinin (BK) signaling may contribute to pathogenesis of retinal vascular complications. BK receptor 2 (B2R) signaling has been implicated in both pro-inflammatory and pro-angiogenic
[...] Read more.
The identification of components of the kallikrein–kinin system in the vitreous from patients with microvascular retinal diseases suggests that bradykinin (BK) signaling may contribute to pathogenesis of retinal vascular complications. BK receptor 2 (B2R) signaling has been implicated in both pro-inflammatory and pro-angiogenic effects promoted by BK. Here, we investigated the role of BK/B2R signaling in the retinal neovascularization in the oxygen-induced retinopathy (OIR) model. Blockade of B2R signaling by the antagonist fasitibant delayed retinal vascularization in mouse pups, indicating that the retinal endothelium is a target of the BK/B2R system. In the rabbit cornea assay, a model of pathological neoangiogenesis, the B2 agonist kallidin induced vessel sprouting and promoted cornea opacity, a sign of edema and tissue inflammation. In agreement with these results, in the OIR model, a blockade of B2R signaling significantly reduced retinal neovascularization, as determined by the area of retinal tufts, and, in the retinal vessel, it also reduced vascular endothelial growth factor and fibroblast growth factor-2 expression. All together, these findings show that B2R blockade reduces retinal neovascularization and inhibits the expression of proangiogenic and pro-inflammatory cytokines, suggesting that targeting B2R signaling may be an effective strategy for treating ischemic retinopathy. Full article
(This article belongs to the Special Issue Retinal Diseases: Bridging Basic and Clinical Research)
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Open AccessArticle The Differentiation of Rat Oligodendroglial Cells Is Highly Influenced by the Oxygen Tension: In Vitro Model Mimicking Physiologically Normoxic Conditions
Int. J. Mol. Sci. 2018, 19(2), 331; doi:10.3390/ijms19020331
Received: 19 December 2017 / Revised: 15 January 2018 / Accepted: 18 January 2018 / Published: 24 January 2018
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Abstract
Oligodendrocyte progenitor cells (OPCs) constitute one of the main populations of dividing cells in the central nervous system (CNS). Physiologically, OPCs give rise to mature, myelinating oligodendrocytes and confer trophic support to their neighboring cells within the nervous tissue. OPCs are known to
[...] Read more.
Oligodendrocyte progenitor cells (OPCs) constitute one of the main populations of dividing cells in the central nervous system (CNS). Physiologically, OPCs give rise to mature, myelinating oligodendrocytes and confer trophic support to their neighboring cells within the nervous tissue. OPCs are known to be extremely sensitive to the influence of exogenous clues which might affect their crucial biological processes, like survival, proliferation, differentiation, and the ability to generate a myelin membrane. Alterations in their differentiation influencing their final potential for myelinogenesis are usually the leading cause of CNS dys- and demyelination, contributing to the development of leukodystrophic disorders. The evaluation of the mechanisms that cause oligodendrocytes to malfunction requires detailed studies based on designed in vitro models. Since OPCs readily respond to changes in local homeostasis, it is crucial to establish restricted culture conditions to eliminate the potential stimuli that might influence oligodendrocyte biology. Additionally, the in vitro settings should mimic the physiological conditions to enable the obtained results to be translated to future preclinical studies. Therefore, the aim of our study was to investigate OPC differentiation in physiological normoxia (5% O2) and a restricted in vitro microenvironment. To evaluate the impact of the combined microenvironmental clues derived from other components of the nervous tissue, which are also influenced by the local oxygen concentration, the process of generating OPCs was additionally analyzed in organotypic hippocampal slices. The obtained results show that OPC differentiation, although significantly slowed down, proceeded correctly through its typical stages in the physiologically relevant conditions created in vitro. The established settings were also conducive to efficient cell proliferation, exerting also a neuroprotective effect by promoting the proliferation of neurons. In conclusion, the performed studies show how oxygen tension influences OPC proliferation, differentiation, and their ability to express myelin components, and should be taken into consideration while planning preclinical studies, e.g., to examine neurotoxic compounds or to test neuroprotective strategies. Full article
(This article belongs to the collection Neuroprotective Strategies)
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Open AccessArticle Visualization and Quantitative 3D Analysis of Intraocular Melanoma and Its Vascularization in a Hamster Eye
Int. J. Mol. Sci. 2018, 19(2), 332; doi:10.3390/ijms19020332
Received: 31 December 2017 / Revised: 16 January 2018 / Accepted: 18 January 2018 / Published: 24 January 2018
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Abstract
A tumor vasculature network undergoes intense growth and rebuilding during tumor growth. Traditionally, vascular networks are histologically examined using parameters such as vessel density determined from two-dimensional slices of the tumor. Two-dimensional probing of a complicated three-dimensional (3D) structure only provides partial information.
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A tumor vasculature network undergoes intense growth and rebuilding during tumor growth. Traditionally, vascular networks are histologically examined using parameters such as vessel density determined from two-dimensional slices of the tumor. Two-dimensional probing of a complicated three-dimensional (3D) structure only provides partial information. Therefore, we propose the use of microcomputed tomography (micro-CT) imaging to analyze the evolution of a tumor vasculature in an experimental ocular tumor model. A Bomirski Hamster Melanoma was implanted in the anterior chamber of a hamster eye. Ultrasound (US) imaging of the same tumor was performed in vivo, and the vascular results obtained using the two methods were compared. Normal ocular tissues, a tumor, and a tumor vascular structure were revealed with high accuracy using micro-CT. The vessels that grew within the tumor were chaotic, leaky, and contained many convoluted micro-vessels and embolizations. They comprised 20–38% of the tumor mass. The blood flow in the larger functional vessels was in the range from 10 to 25 mm/s, as determined by in vivo Doppler US. The micro-CT imaging of the hamster eyeball enabled both qualitative and quantitative 3D analyses of the globe at a histological level. Although the presented images were obtained ex vivo, micro-CT noninvasive imaging is being developed intensively, and high-resolution in vivo imaging is feasible. Full article
(This article belongs to the Special Issue Animal Models of Melanoma)
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Open AccessArticle Unveiling a Selective Mechanism for the Inhibition of α-Synuclein Aggregation by β-Synuclein
Int. J. Mol. Sci. 2018, 19(2), 334; doi:10.3390/ijms19020334
Received: 8 December 2017 / Revised: 15 January 2018 / Accepted: 19 January 2018 / Published: 24 January 2018
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Abstract
α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson’s disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as
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α-Synuclein (αS) is an intrinsically disordered protein that is associated with Parkinson’s disease (PD) through its ability to self-assemble into oligomers and fibrils. Inhibition of this oligomerization cascade is an interesting approach to developing therapeutical strategies and β-synuclein (βS) has been described as a natural negative regulator of this process. However, the biological background and molecular mechanisms by which this inhibition occurs is unclear. Herein, we focused on assessing the effect of βS on the aggregation of five αS pathological mutants linked to early-onset PD (A30P, E46K, H50Q, G51D and A53T). By coupling single molecule fluorescence spectroscopy to a cell-free protein expression system, we validated the ability of βS to act as a chaperone of αS, effectively inhibiting its aggregation. Interestingly, we found that βS does so in a selective manner, i.e., is a more effective inhibitor for certain αS pathological mutants—A30P and G51D—as compared to E46K, H50Q and A53T. Moreover, two-color coincidence experiments proved that this discrepancy is due to a preferential incorporation of βS into smaller oligomers of αS. This was validated by showing that the chaperoning effect was lost when proteins were mixed after being expressed individually. This study highlights the potential of fluorescence spectroscopy to deconstruct αS aggregation cascade and its interplay with βS. Full article
(This article belongs to the collection Protein Folding)
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Open AccessArticle An in Vitro Study on the Effect of Combined Treatment with Photodynamic and Chemical Therapies on Candida albicans
Int. J. Mol. Sci. 2018, 19(2), 337; doi:10.3390/ijms19020337
Received: 13 December 2017 / Revised: 15 January 2018 / Accepted: 18 January 2018 / Published: 24 January 2018
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Abstract
Candida albicans is the most commonly encountered human fungal pathogen, and it is traditionally treated with antimicrobial chemical agents. The antimicrobial effect of these agents is largely weakened by drug resistance and biofilm-associated virulence. Enhancement of the antimicrobial activity of existing agents is
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Candida albicans is the most commonly encountered human fungal pathogen, and it is traditionally treated with antimicrobial chemical agents. The antimicrobial effect of these agents is largely weakened by drug resistance and biofilm-associated virulence. Enhancement of the antimicrobial activity of existing agents is needed for effective candidiasis treatment. Our aim was to develop a therapy that combined biofilm disruption with existing antimicrobial agents. Photodynamic therapy (PDT) utilizing curcumin and blue light was tested as an independent therapy and in combination with fluconazole treatment. Viability assays and morphology analysis were used to assess the effectiveness of C. albicans treatment. Results showed that fluconazole treatment decreased the viability of planktonic C. albicans, but the decrease was not as pronounced in adherent C. albicans because its biofilm form was markedly more resistant to the antimicrobiotic. PDT effectively eradicated C. albicans biofilms, and when combined with fluconazole, PDT significantly inhibited C. albicans to a greater extent. This study suggests that the addition of PDT to fluconazole to treat C. albicans infection enhances its effectiveness and can potentially be used clinically. Full article
(This article belongs to the Special Issue Laser Application in Life Sciences 2018)
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Open AccessArticle The Prevalence of Helicobacter pylori in Estonian Bariatric Surgery Patients
Int. J. Mol. Sci. 2018, 19(2), 338; doi:10.3390/ijms19020338
Received: 12 December 2017 / Revised: 9 January 2018 / Accepted: 18 January 2018 / Published: 24 January 2018
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Abstract
Helicobacter pylori (Hp) is one of the most important human pathogens that can cause duodenal and gastric ulcers, gastritis and stomach cancer. Hp infection is considered to be a cause of limiting access to bariatric surgery. The aim of this study
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Helicobacter pylori (Hp) is one of the most important human pathogens that can cause duodenal and gastric ulcers, gastritis and stomach cancer. Hp infection is considered to be a cause of limiting access to bariatric surgery. The aim of this study was to determine the prevalence of Hp in patients with obesity going into bariatric surgery and to reveal the relationship between Hp and clinical data. The study group was formed of 68 preoperative bariatric surgery patients (body mass index (BMI) 44.7 ± 4.8). Gastric biopsies (antrum and corpus) were used for histological and molecular (caqA and glmM genes) examinations. The PCR method revealed Hp infection in 64.7% of obese patients that is higher in comparison with histological analysis (55.9%). The prevalence of cagA and glmM genes in antrum mucosa was 45.6% and 47.0% while in the corpus it was 41.2% and 38.3%, respectively. The coincidence of both cagA and glmM virulence genes in the antrum and corpus mucosa was 33.8% and 22.1%, respectively. Either of the genes was found in 58.8% of antrum and 57.3% of corpus mucosa. Presence of caqA and glmM genes was in association with active and atrophic chronic gastritis. In conclusion, our study demonstrated that two thirds of morbidly obese patients undergoing bariatric surgery are infected with Hp and have a high prevalence of cagA and glmM virulence genes that points out the necessity for diagnostics and treatment of this infection before surgery. Full article
(This article belongs to the Special Issue Helicobacter pylori Research)
Open AccessArticle Elucidating the Role of CD84 and AHR in Modulation of LPS-Induced Cytokines Production by Cruciferous Vegetable-Derived Compounds Indole-3-Carbinol and 3,3′-Diindolylmethane
Int. J. Mol. Sci. 2018, 19(2), 339; doi:10.3390/ijms19020339
Received: 8 December 2017 / Revised: 19 January 2018 / Accepted: 22 January 2018 / Published: 24 January 2018
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Abstract
Modulation of the immune system by cancer protective food bioactives has preventive and therapeutic importance in prostate cancer, but the mechanisms remain largely unclear. The current study tests the hypothesis that the diet-derived cancer protective compounds, indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM), affect the
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Modulation of the immune system by cancer protective food bioactives has preventive and therapeutic importance in prostate cancer, but the mechanisms remain largely unclear. The current study tests the hypothesis that the diet-derived cancer protective compounds, indole-3-carbinol (I3C) and 3,3′-diindolylmethane (DIM), affect the tumor microenvironment by regulation of inflammatory responses in monocytes and macrophages. We also ask whether I3C and DIM act through the aryl hydrocarbon (AHR)-dependent pathway or the signaling lymphocyte activation molecule (SLAM) family protein CD84-mediated pathway. The effect of I3C and DIM was examined using the human THP-1 monocytic cell in its un-differentiated (monocyte) and differentiated (macrophage) state. We observed that I3C and DIM inhibited lipopolysaccharide (LPS) induction of IL-1β mRNA and protein in the monocyte form but not the macrophage form of THP-1. Interestingly, CD84 mRNA but not protein was inhibited by I3C and DIM. AHR siRNA knockdown experiments confirmed that the inhibitory effects of I3C and DIM on IL-1β as well as CD84 mRNA are regulated through AHR-mediated pathways. Additionally, the AHR ligand appeared to differentially regulate other LPS-induced cytokines expression. Hence, cross-talk between AHR and inflammation-mediated pathways, but not CD84-mediated pathways, in monocytes but not macrophages may contribute to the modulation of tumor environments by I3C and DIM in prostate cancer. Full article
(This article belongs to the Special Issue Natural Bioactives and Phytochemicals in Cancer Prevention)
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Open AccessArticle Erinacine A-Enriched Hericium erinaceus Mycelium Produces Antidepressant-Like Effects through Modulating BDNF/PI3K/Akt/GSK-3β Signaling in Mice
Int. J. Mol. Sci. 2018, 19(2), 341; doi:10.3390/ijms19020341
Received: 25 December 2017 / Revised: 21 January 2018 / Accepted: 22 January 2018 / Published: 24 January 2018
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Abstract
Antidepressant-like effects of ethanolic extract of Hericium erinaceus (HE) mycelium enriched in erinacine A on depressive mice challenged by repeated restraint stress (RS) were examined. HE at 100, 200 or 400 mg/kg body weight/day was orally given to mice for four weeks. After
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Antidepressant-like effects of ethanolic extract of Hericium erinaceus (HE) mycelium enriched in erinacine A on depressive mice challenged by repeated restraint stress (RS) were examined. HE at 100, 200 or 400 mg/kg body weight/day was orally given to mice for four weeks. After two weeks of HE administration, all mice except the control group went through with 14 days of RS protocol. Stressed mice exhibited various behavioral alterations, such as extending immobility time in the tail suspension test (TST) and forced swimming test (FST), and increasing the number of entries in open arm (POAE) and the time spent in the open arm (PTOA). Moreover, the levels of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) were decreased in the stressed mice, while the levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were increased. These changes were significantly inverted by the administration of HE, especially at the dose of 200 or 400 mg/kg body weight/day. Additionally, HE was shown to activate the BDNF/TrkB/PI3K/Akt/GSK-3β pathways and block the NF-κB signals in mice. Taken together, erinacine A-enriched HE mycelium could reverse the depressive-like behavior caused by RS and was accompanied by the modulation of monoamine neurotransmitters as well as pro-inflammatory cytokines, and regulation of BDNF pathways. Therefore, erinacine A-enriched HE mycelium could be an attractive agent for the treatment of depressive disorders. Full article
(This article belongs to the Section Bioactives and Nutraceuticals)
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Open AccessArticle Molecular Understanding of the Activation of CB1 and Blockade of TRPV1 Receptors: Implications for Novel Treatment Strategies in Osteoarthritis
Int. J. Mol. Sci. 2018, 19(2), 342; doi:10.3390/ijms19020342
Received: 14 December 2017 / Revised: 18 January 2018 / Accepted: 19 January 2018 / Published: 24 January 2018
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Abstract
Osteoarthritis (OA) is a joint disease in which cartilage degenerates as a result of mechanical and biochemical changes. The main OA symptom is chronic pain involving both peripheral and central mechanisms of nociceptive processing. Our previous studies have implicated the benefits of dual-
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Osteoarthritis (OA) is a joint disease in which cartilage degenerates as a result of mechanical and biochemical changes. The main OA symptom is chronic pain involving both peripheral and central mechanisms of nociceptive processing. Our previous studies have implicated the benefits of dual- over single-acting compounds interacting with the endocannabinoid system (ECS) in OA treatment. In the present study, we focused on the specific molecular alterations associated with pharmacological treatment. OA was induced in Wistar rats by intra-articular injection of 3 mg of monoiodoacetate (MIA). Single target compounds (URB597, an FAAH inhibitor, and SB366791, a TRPV1 antagonist) and a dual-acting compound OMDM198 (FAAH inhibitor/TRPV1 antagonist) were used in the present study. At day 21 post-MIA injection, rats were sacrificed 1 h after i.p. treatment, and changes in mRNA expression were evaluated in the lumbar spinal cord by RT-qPCR. Following MIA administration, we observed 2-4-fold increase in mRNA expression of targeted receptors (Cnr1, Cnr2, and Trpv1), endocannabinoid degradation enzymes (Faah, Ptgs2, and Alox12), and TRPV1 sensitizing kinases (Mapk3, Mapk14, Prkcg, and Prkaca). OMDM198 treatment reversed some of the MIA effects on the spinal cord towards intact levels (Alox12, Mapk14, and Prkcg). Apparent regulation of ECS and TRPV1 in response to pharmacological intervention is a strong justification for novel ECS-based multi-target drug treatment in OA. Full article
(This article belongs to the Special Issue Cannabinoid Signaling in Nervous System)
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Open AccessArticle Disrupting the Btk Pathway Suppresses COPD-Like Lung Alterations in Atherosclerosis Prone ApoE−/− Mice Following Regular Exposure to Cigarette Smoke
Int. J. Mol. Sci. 2018, 19(2), 343; doi:10.3390/ijms19020343
Received: 15 November 2017 / Revised: 19 January 2018 / Accepted: 21 January 2018 / Published: 24 January 2018
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Abstract
Chronic obstructive pulmonary disease (COPD) is associated with severe chronic inflammation that promotes irreversible tissue destruction. Moreover, the most broadly accepted cause of COPD is exposure to cigarette smoke. There is no effective cure and significantly, the mechanism behind the development and progression
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Chronic obstructive pulmonary disease (COPD) is associated with severe chronic inflammation that promotes irreversible tissue destruction. Moreover, the most broadly accepted cause of COPD is exposure to cigarette smoke. There is no effective cure and significantly, the mechanism behind the development and progression of this disease remains unknown. Our laboratory has demonstrated that Bruton’s tyrosine kinase (Btk) is a critical regulator of pro-inflammatory processes in the lungs and that Btk controls expression of matrix metalloproteinase-9 (MMP-9) in the alveolar compartment. For this study apolipoprotein E null (ApoE−/−) mice were exposed to SHS to facilitate study in a COPD/atherosclerosis comorbidity model. We applied two types of treatments, animals received either a pharmacological inhibitor of Btk or MMP-9 specific siRNA to minimize MMP-9 expression in endothelial cells or neutrophils. We have shown that these treatments had a protective effect in the lung. We have noted a decrease in alveolar changes related to SHS induced inflammation in treated animals. In summary, we are presenting a novel concept in the field of COPD, i.e., that Btk may be a new drug target for this disease. Moreover, cell specific targeting of MMP-9 may also benefit patients affected by this disease. Full article
(This article belongs to the Special Issue Lung Diseases: Chronic Respiratory Infections)
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Open AccessArticle Lycopene Attenuates Tulathromycin and Diclofenac Sodium-Induced Cardiotoxicity in Mice
Int. J. Mol. Sci. 2018, 19(2), 344; doi:10.3390/ijms19020344
Received: 11 December 2017 / Revised: 15 January 2018 / Accepted: 17 January 2018 / Published: 24 January 2018
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Abstract
Recent experiments showed a potential cardiotoxic effect of the macrolide antibiotic (tulathromycin). This study was performed to investigate whether diclofenac sodium (DFS) potentiates the cardiotoxicity of tulathromycin and increases the cardioprotective effects of lycopene against DFS and tulathromycin. Seven groups (eight per group)
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Recent experiments showed a potential cardiotoxic effect of the macrolide antibiotic (tulathromycin). This study was performed to investigate whether diclofenac sodium (DFS) potentiates the cardiotoxicity of tulathromycin and increases the cardioprotective effects of lycopene against DFS and tulathromycin. Seven groups (eight per group) of adult Swiss albino mice received saline (control), tulathromycin (a single subcutaneous dose of 28 mg/kg/bw on day 14), DFS (a single oral dose of 100 mg/kg/bw on day 14), tulathromycin plus DFS, or lycopene (oral, 10 mg/kg/bw daily for 15 d) combined with tulathromycin, DFS, or both. Compared to the control group, the administration of tulathromycin or DFS (individually or in combination) caused significantly elevated (p < 0.05) serum levels of Creatine kinase-myocardial B fraction (CK-MB), lactate dehydrogenase, and cardiac-specific troponin-T and tissue levels of nitric oxide and malondialdehyde that were accompanied by significantly decreased tissue reduced glutathione content and glutathione peroxidase, superoxide dismutase, and catalase antioxidant enzyme activity. Upon histopathological and immunohistochemical examination, the mean pathology scores and the percentages of caspase-3-, Bax-, and CK-positive regions were significantly higher in the tulathromycin- and/or DFS-treated groups than in control mice. For all these parameters, the pathological changes were more significant in the tulathromycin–DFS combination group than in mice treated with either drug individually. Interestingly, co-administration of lycopene with tulathromycin and/or DFS significantly ameliorated the changes described above. In conclusion, DFS could potentiate the cardiotoxic effects of tulathromycin, whereas lycopene can serve as a cardioprotective agent against DFS and tulathromycin. Full article
(This article belongs to the Special Issue The Mechanism of Action of Food Components in Disease Prevention 2017)
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Open AccessArticle Deciphering the Functional Composition of Fusogenic Liposomes
Int. J. Mol. Sci. 2018, 19(2), 346; doi:10.3390/ijms19020346
Received: 27 December 2017 / Revised: 15 January 2018 / Accepted: 19 January 2018 / Published: 24 January 2018
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Abstract
Cationic liposomes are frequently used as carrier particles for nucleic acid delivery. The most popular formulation is the equimolar mixture of two components, a cationic lipid and a neutral phosphoethanolamine. Its uptake pathway has been described as endocytosis. The presence of an aromatic
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Cationic liposomes are frequently used as carrier particles for nucleic acid delivery. The most popular formulation is the equimolar mixture of two components, a cationic lipid and a neutral phosphoethanolamine. Its uptake pathway has been described as endocytosis. The presence of an aromatic molecule as a third component strongly influences the cellular uptake process and results in complete membrane fusion instead of endocytosis. Here, we systematically varied all three components of this lipid mixture and determined how efficiently the resulting particles fused with the plasma membrane of living mammalian cells. Our results show that an aromatic molecule and a cationic lipid component with conical molecular shape are essential for efficient fusion induction. While a neutral lipid is not mandatory, it can be used to control fusion efficiency and, in the most extreme case, to revert the uptake mechanism back to endocytosis. Full article
(This article belongs to the Special Issue Membrane Fusion)
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Open AccessArticle Cytoprotective Mechanisms in Fatty Liver Preservation against Cold Ischemia Injury: A Comparison between IGL-1 and HTK
Int. J. Mol. Sci. 2018, 19(2), 348; doi:10.3390/ijms19020348
Received: 29 November 2017 / Revised: 16 January 2018 / Accepted: 19 January 2018 / Published: 24 January 2018
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Abstract
Institute Goeorges Lopez 1 (IGL-1) and Histidine-Tryptophan-Ketoglutarate (HTK) preservation solutions are regularly used in clinical for liver transplantation besides University of Wisconsin (UW) solution and Celsior. Several clinical trials and experimental works have been carried out comparing all the solutions, however the comparative
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Institute Goeorges Lopez 1 (IGL-1) and Histidine-Tryptophan-Ketoglutarate (HTK) preservation solutions are regularly used in clinical for liver transplantation besides University of Wisconsin (UW) solution and Celsior. Several clinical trials and experimental works have been carried out comparing all the solutions, however the comparative IGL-1 and HTK appraisals are poor; especially when they deal with the underlying protection mechanisms of the fatty liver graft during cold storage. Fatty livers from male obese Zücker rats were conserved for 24 h at 4 °C in IGL-1 or HTK preservation solutions. After organ recovery and rinsing of fatty liver grafts with Ringer Lactate solution, we measured the changes in mechanistic target of rapamycin (mTOR) signaling activation, liver autophagy markers (Beclin-1, Beclin-2, LC3B and ATG7) and apoptotic markers (caspase 3, caspase 9 and TUNEL). These determinations were correlated with the prevention of liver injury (aspartate and alanine aminostransferase (AST/ALT), histology) and mitochondrial damage (glutamate dehydrogenase (GLDH) and confocal microscopy findings). Liver grafts preserved in IGL-1 solution showed a marked reduction on p-TOR/mTOR ratio when compared to HTK. This was concomitant with significant increased cyto-protective autophagy and prevention of liver apoptosis, including inflammatory cytokines such as HMGB1. Together, our results revealed that IGL-1 preservation solution better protected fatty liver grafts against cold ischemia damage than HTK solution. IGL-1 protection was associated with a reduced liver damage, higher induced autophagy and decreased apoptosis. All these effects would contribute to limit the subsequent extension of reperfusion injury after graft revascularization in liver transplantation procedures. Full article
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Open AccessArticle Workers’ Exposure to Nano-Objects with Different Dimensionalities in R&D Laboratories: Measurement Strategy and Field Studies
Int. J. Mol. Sci. 2018, 19(2), 349; doi:10.3390/ijms19020349
Received: 7 December 2017 / Revised: 11 January 2018 / Accepted: 16 January 2018 / Published: 24 January 2018
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Abstract
With the increasing interest in the potential benefits of nanotechnologies, concern is still growing that they may present emerging risks for workers. Various strategies have been developed to assess the exposure to nano-objects and their agglomerates and aggregates (NOAA) in the workplace, integrating
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With the increasing interest in the potential benefits of nanotechnologies, concern is still growing that they may present emerging risks for workers. Various strategies have been developed to assess the exposure to nano-objects and their agglomerates and aggregates (NOAA) in the workplace, integrating different aerosol measurement instruments and taking into account multiple parameters that may influence NOAA toxicity. The present study proposes a multi-metric approach for measuring and sampling NOAA in the workplace, applied to three case studies in laboratories each dedicated to materials with different shapes and dimensionalities: graphene, nanowires, and nanoparticles. The study is part of a larger project with the aim of improving risk management tools in nanomaterials research laboratories. The harmonized methodology proposed by the Organization for Economic Cooperation and Development (OECD) has been applied, including information gathering about materials and processes, measurements with easy-to-use and hand-held real-time devices, air sampling with personal samplers, and off-line analysis using scanning electron microscopy. Significant values beyond which an emission can be attributed to the NOAA production process were identified by comparison of the particle number concentration (PNC) time series and the corresponding background levels in the three laboratories. We explored the relations between background PNC and microclimatic parameters. Morphological and elemental analysis of sampled filters was done to identify possible emission sources of NOAA during the production processes: rare particles, spherical, with average diameter similar to the produced NOAA were identified in the nanoparticles laboratory, so further investigation is recommended to confirm the potential for worker exposure. In conclusion, the information obtained should provide a valuable basis for improving risk management strategies in the laboratory at work. Full article
(This article belongs to the Special Issue Nanotoxicology and Nanosafety)
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Open AccessArticle Comparative Digital Gene Expression Analysis of Tissue-Cultured Plantlets of Highly Resistant and Susceptible Banana Cultivars in Response to Fusarium oxysporum
Int. J. Mol. Sci. 2018, 19(2), 350; doi:10.3390/ijms19020350
Received: 8 December 2017 / Revised: 15 January 2018 / Accepted: 16 January 2018 / Published: 24 January 2018
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Abstract
Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive soil-borne diseases. In this study, young tissue-cultured plantlets of banana (Musa spp. AAA) cultivars differing in Foc susceptibility were used to reveal their
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Banana Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc) is one of the most destructive soil-borne diseases. In this study, young tissue-cultured plantlets of banana (Musa spp. AAA) cultivars differing in Foc susceptibility were used to reveal their differential responses to this pathogen using digital gene expression (DGE). Data were evaluated by various bioinformatic tools (Venn diagrams, gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses) and immunofluorescence labelling method to support the identification of gene candidates determining the resistance of banana against Foc. Interestingly, we have identified MaWRKY50 as an important gene involved in both constitutive and induced resistance. We also identified new genes involved in the resistance of banana to Foc, including several other transcription factors (TFs), pathogenesis-related (PR) genes and some genes related to the plant cell wall biosynthesis or degradation (e.g., pectinesterases, β-glucosidases, xyloglucan endotransglucosylase/hydrolase and endoglucanase). The resistant banana cultivar shows activation of PR-3 and PR-4 genes as well as formation of different constitutive cell barriers to restrict spreading of the pathogen. These data suggest new mechanisms of banana resistance to Foc. Full article
(This article belongs to the Section Molecular Botany)
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Open AccessArticle Tauroursodeoxycholic Acid Protects against the Effects of P-Cresol-Induced Reactive Oxygen Species via the Expression of Cellular Prion Protein
Int. J. Mol. Sci. 2018, 19(2), 352; doi:10.3390/ijms19020352
Received: 29 November 2017 / Revised: 16 January 2018 / Accepted: 22 January 2018 / Published: 25 January 2018
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Abstract
Mesenchymal stem cells (MSCs) could be a promising solution in the treatment of various diseases including chronic kidney disease (CKD). However, endoplasmic reticulum (ER) stress induced by ischemia in the area of application limits the integration and survival of MSCs in patients. In
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Mesenchymal stem cells (MSCs) could be a promising solution in the treatment of various diseases including chronic kidney disease (CKD). However, endoplasmic reticulum (ER) stress induced by ischemia in the area of application limits the integration and survival of MSCs in patients. In our study, we generated ER stress-induced conditions in MSCs using P-cresol. As P-cresol is a toxic compound accumulated in the body of CKD patients and induces apoptosis and inflammation through reactive oxygen species (ROS), we observed ER stress-induced MSC apoptosis activated by oxidative stress, which in turn resulted from ROS generation. To overcome stress-induced apoptosis, we investigated the protective effects of tauroursodeoxycholic acid (TUDCA), a bile acid, on ER stress in MSCs. In ER stress, TUDCA treatment of MSCs reduced ER stress-associated protein activation, including GRP78, PERK, eIF2α, ATF4, IRE1α, and CHOP. Next, to explore the protective mechanism adopted by TUDCA, TUDCA-mediated cellular prion protein (PrPC) activation was assessed. We confirmed that PrPC expression significantly increased ROS, which was eliminated by superoxide dismutase and catalase in MSCs. These findings suggest that TUDCA protects from inflammation and apoptosis in ER stress via PrPC expression. Our study demonstrates that TUDCA protects MSCs against inflammation and apoptosis in ER stress by PrPC expression in response to P-cresol exposure. Full article
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Open AccessArticle Mitochondrial BK Channel Openers CGS7181 and CGS7184 Exhibit Cytotoxic Properties
Int. J. Mol. Sci. 2018, 19(2), 353; doi:10.3390/ijms19020353
Received: 16 November 2017 / Revised: 15 January 2018 / Accepted: 22 January 2018 / Published: 25 January 2018
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Abstract
Potassium channel openers (KCOs) have been shown to play a role in cytoprotection through the activation of mitochondrial potassium channels. Recently, in several reports, a number of data has been described as off-target actions for KCOs. In the present study, we investigated the
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Potassium channel openers (KCOs) have been shown to play a role in cytoprotection through the activation of mitochondrial potassium channels. Recently, in several reports, a number of data has been described as off-target actions for KCOs. In the present study, we investigated the effects of BKCa channel openers CGS7181, CGS7184, NS1619, and NS004 in neuronal cells. For the purpose of this research, we used a rat brain, the mouse hippocampal HT22 cells, and the human astrocytoma U-87 MG cell line. We showed that CGS7184 activated the mitochondrial BKCa (mitoBKCa) channel in single-channel recordings performed on astrocytoma mitoplasts. Moreover, when applied to the rat brain homogenate or isolated rat brain mitochondria, CGS7184 increased the oxygen consumption rate, and can thus be considered a potentially cytoprotective agent. However, experiments on intact neuronal HT22 cells revealed that both CGS7181 and CGS7184 induced HT22 cell death in a concentration- and time-dependent manner. By contrast, we did not observe cell death when NS1619 or NS004 was applied. CGS7184 toxicity was not abolished by BKCa channel inhibitors, suggesting that the observed effects were independent of a BKCa-type channel activity. CGS7184 treatment resulted in an increase of cytoplasmic Ca2+ concentration that likely involved efflux from internal calcium stores and the activation of calpains (calcium-dependent proteases). The cytotoxic effect of the channel opener was partially reversed by a calpain inhibitor. Our data show that KCOs under study not only activate mitoBKCa channels from brain tissue, but also induce cell death when used in cellular models. Full article
(This article belongs to the Special Issue Ion Transporters and Channels in Physiology and Pathophysiology)
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Open AccessArticle The Effects of Varying Degree of MWCNT Carboxylation on Bioactivity in Various In Vivo and In Vitro Exposure Models
Int. J. Mol. Sci. 2018, 19(2), 354; doi:10.3390/ijms19020354
Received: 13 December 2017 / Revised: 16 January 2018 / Accepted: 23 January 2018 / Published: 25 January 2018
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Abstract
Functionalization has been shown to alter toxicity of multi-walled carbon nanotube (MWCNT) in several studies. This study varied the degree of functionalization (viz., amount of MWCNT surface carboxylation) to define the relationship between the extent of carboxylation and effects in a variety of
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Functionalization has been shown to alter toxicity of multi-walled carbon nanotube (MWCNT) in several studies. This study varied the degree of functionalization (viz., amount of MWCNT surface carboxylation) to define the relationship between the extent of carboxylation and effects in a variety of in vitro cell models and short-term ex vivo/in vivo particle exposures. Studies with vitamin D3 plus phorbol ester transformed THP-1 macrophages demonstrated that functionalization, regardless of amount, corresponded with profoundly decreased NLRP3 inflammasome activation. However, all MWCNT variants were slightly toxic in this model. Alternatively, studies with A549 epithelial cells showed some varied effects. For example, IL-33 and TNF-α release were related to varying amounts of functionalization. For in vivo particle exposures, autophagy of alveolar macrophages, measured using green fluorescent protein (GFP)- fused-LC3 transgenic mice, increased for all MWCNT tested three days after exposure, but, by Day 7, autophagy was clearly dependent on the amount of carboxylation. The instilled source MWCNT continued to produce cellular injury in alveolar macrophages over seven days. In contrast, the more functionalized MWCNT initially showed similar effects, but reduced over time. Dark-field imaging showed the more functionalized MWCNTs were distributed more uniformly throughout the lung and not isolated to macrophages. Taken together, the results indicated that in vitro and in vivo bioactivity of MWCNT decreased with increased carboxylation. Functionalization by carboxylation eliminated the bioactive potential of the MWCNT in the exposure models tested. The observation that maximally functionalized MWCNT distribute more freely throughout the lung with the absence of cellular damage, and extended deposition, may establish a practical use for these particles as a safer alternative for unmodified MWCNT. Full article
(This article belongs to the Special Issue Macrophages in Inflammation)
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Open AccessArticle The Anti-Stress Effect of Mentha arvensis in Immobilized Rats
Int. J. Mol. Sci. 2018, 19(2), 355; doi:10.3390/ijms19020355
Received: 12 December 2017 / Revised: 16 January 2018 / Accepted: 22 January 2018 / Published: 25 January 2018
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Abstract
Stress can lead to inflammation, accelerated aging, and some chronic diseases condition. Mentha arvensis (MA) is a traditional medicine having antioxidant and anti-inflammatory activities. The present study investigated the anti-stress role of MA and fermented MA (FMA) extract in immobilized rats. We studied
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Stress can lead to inflammation, accelerated aging, and some chronic diseases condition. Mentha arvensis (MA) is a traditional medicine having antioxidant and anti-inflammatory activities. The present study investigated the anti-stress role of MA and fermented MA (FMA) extract in immobilized rats. We studied the lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and rats were immobilized for 2 h per day for 14 days using a restraining cage. MA (100 mg/kg) and FMA (100 mg/kg) were orally administered to rats 1 h prior to immobilization. Using high-performance liquid chromatography (HPLC) analysis, we determined the rosmarinic acid content of MA and FMA. The generation of malondialdehyde (MDA) and nitric oxide (NO) in RAW 246.7 cells were suppressed by both MA and FMA. In rats, MA and FMA notably improved the body weight, daily food intake, and duodenum histology. MDA and NO level were gradually decreased by MA and FMA treatment. MA and FMA significantly controlled the stress-related hormones by decreasing corticosterone and β-endorphin and increasing serotonin level. Moreover, protein expression levels of mitogen activated protein kinases (MAPK) and cyclooxygenase-2 (COX-2) were markedly downregulated by MA and FMA. Taken together, MA and FMA could ameliorate immobilized-stress by reducing oxidative stress, regulating stress-related hormones, and MAPK/COX-2 signaling pathways in rats. Particularly, FMA has shown greater anti-stress activities than MA. Full article
(This article belongs to the Section Bioactives and Nutraceuticals)
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Open AccessArticle Genotyping by Sequencing Highlights a Polygenic Resistance to Ralstonia pseudosolanacearum in Eggplant (Solanum melongena L.)
Int. J. Mol. Sci. 2018, 19(2), 357; doi:10.3390/ijms19020357
Received: 25 December 2017 / Revised: 19 January 2018 / Accepted: 22 January 2018 / Published: 25 January 2018
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Abstract
Eggplant cultivation is limited by numerous diseases, including the devastating bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC). Within the RSSC, Ralstonia pseudosolanacearum (including phylotypes I and III) causes severe damage to all solanaceous crops, including eggplant. Therefore, the creation
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Eggplant cultivation is limited by numerous diseases, including the devastating bacterial wilt (BW) caused by the Ralstonia solanacearum species complex (RSSC). Within the RSSC, Ralstonia pseudosolanacearum (including phylotypes I and III) causes severe damage to all solanaceous crops, including eggplant. Therefore, the creation of cultivars resistant to R. pseudosolanacearum strains is a major goal for breeders. An intraspecific eggplant population, segregating for resistance, was created from the cross between the susceptible MM738 and the resistant EG203 lines. The population of 123 doubled haploid lines was challenged with two strains belonging to phylotypes I (PSS4) and III (R3598), which both bypass the published EBWR9 BW-resistance quantitative trait locus (QTL). Ten and three QTLs of resistance to PSS4 and to R3598, respectively, were detected and mapped. All were strongly influenced by environmental conditions. The most stable QTLs were found on chromosomes 3 and 6. Given their estimated physical position, these newly detected QTLs are putatively syntenic with BW-resistance QTLs in tomato. In particular, the QTLs’ position on chromosome 6 overlaps with that of the major broad-spectrum tomato resistance QTL Bwr-6. The present study is a first step towards understanding the complex polygenic system, which underlies the high level of BW resistance of the EG203 line. Full article
(This article belongs to the Special Issue Plant Defense Genes Against Biotic Stresses)
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Open AccessArticle Aging Donor-Derived Human Mesenchymal Stem Cells Exhibit Reduced Reactive Oxygen Species Loads and Increased Differentiation Potential Following Serial Expansion on a PEG-PCL Copolymer Substrate
Int. J. Mol. Sci. 2018, 19(2), 359; doi:10.3390/ijms19020359
Received: 9 January 2018 / Revised: 22 January 2018 / Accepted: 23 January 2018 / Published: 25 January 2018
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Abstract
Human mesenchymal stem cells (hMSCs) have been widely studied for therapeutic development in tissue engineering and regenerative medicine. They can be harvested from human donors via tissue biopsies, such as bone marrow aspiration, and cultured to reach clinically relevant cell numbers. However, an
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Human mesenchymal stem cells (hMSCs) have been widely studied for therapeutic development in tissue engineering and regenerative medicine. They can be harvested from human donors via tissue biopsies, such as bone marrow aspiration, and cultured to reach clinically relevant cell numbers. However, an unmet issue lies in the fact that the hMSC donors for regenerative therapies are more likely to be of advanced age. Their stem cells are not as potent compared to those of young donors, and continue to lose healthy, stemness-related activities when the hMSCs are serially passaged in tissue culture plates. Here, we have developed a cheap, scalable, and effective copolymer film to culture hMSCs obtained from aged human donors over several passages without loss of reactive oxygen species (ROS) handling or differentiation capacity. Assays of cell morphology, reactive oxygen species load, and differentiation potential demonstrate the effectiveness of copolymer culture on reduction in senescence-related activities of aging donor-derived hMSCs that could hinder the therapeutic potential of autologous stem cell therapies. Full article
(This article belongs to the Special Issue Novel Biomaterials for Tissue Engineering 2018)
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Open AccessArticle A Pilot Study of Stress System Activation in Children Enrolled in a Targeted Prevention Program: Implications for Personalization
Int. J. Mol. Sci. 2018, 19(2), 361; doi:10.3390/ijms19020361
Received: 10 January 2018 / Revised: 17 January 2018 / Accepted: 17 January 2018 / Published: 25 January 2018
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Abstract
Empirically validated interventions addressing childhood psychological problems are now readily available, but success likely depends in part on accurately identifying which children will benefit from which intervention. This pilot study examined the stress activation and response system, first as a way to differentiate
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Empirically validated interventions addressing childhood psychological problems are now readily available, but success likely depends in part on accurately identifying which children will benefit from which intervention. This pilot study examined the stress activation and response system, first as a way to differentiate high versus low-risk children, and second to explore indicators of the stress system associated with favorable intervention response. Method. Participants (N = 43, 58% male) were school-aged children who qualified for inclusion in the Early Risers “Skills for Success” Prevention Program based on their elevated levels of aggressive and/or socially withdrawn behavior and a normally developing comparison group. Compared to the normally developing group, children who were participants in the intervention exhibited a more blunted cortisol response to the stress paradigm. However, for the children in the intervention group, elevated cortisol levels at the start of the stress paradigm were concurrently associated with internalizing problems and predictive of improvement in internalizing problems over time. These findings provide preliminary evidence that hypothalamic pituitary adrenal (HPA) axis biological variables may be helpful tools for identifying children who would benefit from intervention and personalizing interventions. Full article
Open AccessArticle Acidic Chitinase-Chitin Complex Is Dissociated in a Competitive Manner by Acetic Acid: Purification of Natural Enzyme for Supplementation Purposes
Int. J. Mol. Sci. 2018, 19(2), 362; doi:10.3390/ijms19020362
Received: 19 November 2017 / Revised: 10 January 2018 / Accepted: 22 January 2018 / Published: 25 January 2018
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Abstract
Acidic chitinase (Chia) has been implicated in asthma, allergic inflammations, and food processing. We have purified Chia enzymes with striking acid stability and protease resistance from chicken and pig stomach tissues using a chitin column and 8 M urea (urea-Chia). Here, we report
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Acidic chitinase (Chia) has been implicated in asthma, allergic inflammations, and food processing. We have purified Chia enzymes with striking acid stability and protease resistance from chicken and pig stomach tissues using a chitin column and 8 M urea (urea-Chia). Here, we report that acetic acid is a suitable agent for native Chia purification from the stomach tissues using a chitin column (acetic acid-Chia). Chia protein can be eluted from a chitin column using 0.1 M acetic acid (pH 2.8), but not by using Gly-HCl (pH 2.5) or sodium acetate (pH 4.0 or 5.5). The melting temperatures of Chia are not affected substantially in the elution buffers, as assessed by differential scanning fluorimetry. Interestingly, acetic acid appears to be more effective for Chia-chitin dissociation than do other organic acids with similar structures. We propose a novel concept of this dissociation based on competitive interaction between chitin and acetic acid rather than on acid denaturation. Acetic acid-Chia also showed similar chitinolytic activity to urea-Chia, indicating that Chia is extremely stable against acid, proteases, and denaturing agents. Both acetic acid- and urea-Chia seem to have good potential for supplementation or compensatory purposes in agriculture or even biomedicine. Full article
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Open AccessArticle Ethanol Extract of Oldenlandia diffusa Herba Attenuates Scopolamine-Induced Cognitive Impairments in Mice via Activation of BDNF, P-CREB and Inhibition of Acetylcholinesterase
Int. J. Mol. Sci. 2018, 19(2), 363; doi:10.3390/ijms19020363
Received: 19 December 2017 / Revised: 16 January 2018 / Accepted: 25 January 2018 / Published: 25 January 2018
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Abstract
Though Oldenlandia diffusa Herba (ODH) has been known to exhibit anti-cancer and anti-inflammatory effects, its anti-amnestic effect has never been reported so far. The aim of this present study was to elucidate the anti-amnestic effect of ODH. ODH pretreatment significantly reduced escape latency
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Though Oldenlandia diffusa Herba (ODH) has been known to exhibit anti-cancer and anti-inflammatory effects, its anti-amnestic effect has never been reported so far. The aim of this present study was to elucidate the anti-amnestic effect of ODH. ODH pretreatment significantly reduced escape latency of scopolamine treated Institute of Cancer Research (ICR) mice compared to untreated control groups in a Morris water maze test. Similarly, the passive avoidance test showed that ODH treatment recovered the scopolamine induced amnesia in the ICR mouse model. Concentration of Ach in brains of ODH treated mice was increased compared to that of scopolamine treated mice. In addition, activity of acetylcholinesterase (AChE) was notably decreased by ODH. The protein expression of brain-derived neurotrophic factor (BDNF) and phospho-cAMP response element-binding protein (p-CREB) (Ser133) was increased in ODH pretreated group compared to control group. Consistently, immunohistochemistry (IHC) revealed the elevated expression of brain-derived neurotrophic factor (BDNF) and p-CREB in brains of ODH treated mice compared to the control group. Overall, these findings suggest that ODH has anti-amnestic potential via activation of BDNF and p-CREB and inhibition of AChE in mice with scopolamine induced amnesia. Full article
(This article belongs to the Special Issue Traditional Medicine – Unraveling Its Molecular Mechanism)
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Open AccessArticle Inadequate Dietary Phosphorus Levels Cause Skeletal Anomalies and Alter Osteocalcin Gene Expression in Zebrafish
Int. J. Mol. Sci. 2018, 19(2), 364; doi:10.3390/ijms19020364
Received: 14 September 2017 / Revised: 16 January 2018 / Accepted: 17 January 2018 / Published: 25 January 2018
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Abstract
Phosphorus (P) is an essential mineral for the development and maintenance of the vertebrate skeletal system. Modulation of P levels is believed to influence metabolism and the physiological responses of gene expression. In this study, we investigated the influence of dietary P on
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Phosphorus (P) is an essential mineral for the development and maintenance of the vertebrate skeletal system. Modulation of P levels is believed to influence metabolism and the physiological responses of gene expression. In this study, we investigated the influence of dietary P on skeletal deformities and osteocalcin gene expression in zebrafish (Danio rerio), and sought to determine appropriate levels in a diet. We analyzed a total of 450 zebrafish within 31 days of hatching. Animals were distributed in a completely randomized experimental design that consisted of five replications. After an eight-week experiment, fish were diaphanized to evaluate cranial and spinal bone deformities. Increases in dietary phosphorus were inversely proportional to the occurrence of partial spine fusions, the absence of spine fusions, absence of parallelism between spines, intervertebral spacing, vertebral compression, scoliosis, lordosis, ankylosis, fin caudal insertion, and craniofacial deformities. Additionally, osteocalcin expression was inversely correlated to P levels, suggesting a physiological recovery response for bone mineralization deficiency. Our data showed that dietary P concentration was a critical factor in the occurrence of zebrafish skeletal abnormalities. We concluded that 1.55% P in the diet significantly reduces the appearance of skeletal deformities and favors adequate bone mineralization through the adjustment of osteocalcin expression. Full article
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Open AccessArticle Size-Dependent Affinity of Glycine and Its Short Oligomers to Pyrite Surface: A Model for Prebiotic Accumulation of Amino Acid Oligomers on a Mineral Surface
Int. J. Mol. Sci. 2018, 19(2), 365; doi:10.3390/ijms19020365
Received: 30 November 2017 / Revised: 23 December 2017 / Accepted: 23 December 2017 / Published: 25 January 2018
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Abstract
The interaction strength of progressively longer oligomers of glycine, (Gly), di-Gly, tri-Gly, and penta-Gly, with a natural pyrite surface was directly measured using the force mode of an atomic force microscope (AFM). In recent years, selective activation of abiotically formed amino acids on
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The interaction strength of progressively longer oligomers of glycine, (Gly), di-Gly, tri-Gly, and penta-Gly, with a natural pyrite surface was directly measured using the force mode of an atomic force microscope (AFM). In recent years, selective activation of abiotically formed amino acids on mineral surfaces, especially that of pyrite, has been proposed as an important step in many origins of life scenarios. To investigate such notions, we used AFM-based force measurements to probe possible non-covalent interactions between pyrite and amino acids, starting from the simplest amino acid, Gly. Although Gly itself interacted with the pyrite surface only weakly, progressively larger unbinding forces and binding frequencies were obtained using oligomers from di-Gly to penta-Gly. In addition to an expected increase of the configurational entropy and size-dependent van der Waals force, the increasing number of polar peptide bonds, among others, may be responsible for this observation. The effect of chain length was also investigated by performing similar experiments using l-lysine vs. poly-l-lysine (PLL), and l-glutamic acid vs. poly-l-glutamic acid. The results suggest that longer oligomers/polymers of amino acids can be preferentially adsorbed on pyrite surfaces. Full article
(This article belongs to the Special Issue Atomic Force Microscopy for Biological Applications)
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Open AccessArticle Mechanisms of Cardiovascular Protection Associated with Intermittent Hypobaric Hypoxia Exposure in a Rat Model: Role of Oxidative Stress
Int. J. Mol. Sci. 2018, 19(2), 366; doi:10.3390/ijms19020366
Received: 2 November 2017 / Revised: 8 January 2018 / Accepted: 9 January 2018 / Published: 26 January 2018
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Abstract
More than 140 million people live and works (in a chronic or intermittent form) above 2500 m worldwide and 35 million live in the Andean Mountains. Furthermore, in Chile, it is estimated that 55,000 persons work in high altitude shifts, where stays at
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More than 140 million people live and works (in a chronic or intermittent form) above 2500 m worldwide and 35 million live in the Andean Mountains. Furthermore, in Chile, it is estimated that 55,000 persons work in high altitude shifts, where stays at lowlands and interspersed with working stays at highlands. Acute exposure to high altitude has been shown to induce oxidative stress in healthy human lowlanders, due to an increase in free radical formation and a decrease in antioxidant capacity. However, in animal models, intermittent hypoxia (IH) induce preconditioning, like responses and cardioprotection. Here, we aimed to describe in a rat model the responses on cardiac and vascular function to 4 cycles of intermittent hypobaric hypoxia (IHH). Twelve adult Wistar rats were randomly divided into two equal groups, a four-cycle of IHH, and a normobaric hypoxic control. Intermittent hypoxia was induced in a hypobaric chamber in four continuous cycles (1 cycle = 4 days hypoxia + 4 days normoxia), reaching a barometric pressure equivalent to 4600 m of altitude (428 Torr). At the end of the first and fourth cycle, cardiac structural, and functional variables were determined by echocardiography. Thereafter, ex vivo vascular function and biomechanical properties were determined in femoral arteries by wire myography. We further measured cardiac oxidative stress biomarkers (4-Hydroxy-nonenal, HNE; nytrotirosine, NT), reactive oxygen species (ROS) sources (NADPH and mitochondrial), and antioxidant enzymes activity (catalase, CAT; glutathione peroxidase, GPx, and superoxide dismutase, SOD). Our results show a higher ejection and shortening fraction of the left ventricle function by the end of the 4th cycle. Further, femoral vessels showed an improvement of vasodilator capacity and diminished stiffening. Cardiac tissue presented a higher expression of antioxidant enzymes and mitochondrial ROS formation in IHH, as compared with normobaric hypoxic controls. IHH exposure determines a preconditioning effect on the heart and femoral artery, both at structural and functional levels, associated with the induction of antioxidant defence mechanisms. However, mitochondrial ROS generation was increased in cardiac tissue. These findings suggest that initial states of IHH are beneficial for cardiovascular function and protection. Full article
(This article belongs to the Special Issue Adaptation to Chronic Hypoxia: The Last Word Has Not yet Been Said)
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Open AccessArticle Skeleton-Controlled pDNA Delivery of Renewable Steroid-Based Cationic Lipids, the Endocytosis Pathway Analysis and Intracellular Localization
Int. J. Mol. Sci. 2018, 19(2), 369; doi:10.3390/ijms19020369
Received: 18 December 2017 / Revised: 11 January 2018 / Accepted: 18 January 2018 / Published: 26 January 2018
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Abstract
Using renewable and biocompatible natural-based resources to construct functional biomaterials has attracted great attention in recent years. In this work, we successfully prepared a series of steroid-based cationic lipids by integrating various steroid skeletons/hydrophobes with (l-)-arginine headgroups via facile and efficient
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Using renewable and biocompatible natural-based resources to construct functional biomaterials has attracted great attention in recent years. In this work, we successfully prepared a series of steroid-based cationic lipids by integrating various steroid skeletons/hydrophobes with (l-)-arginine headgroups via facile and efficient synthetic approach. The plasmid DNA (pDNA) binding affinity of the steroid-based cationic lipids, average particle sizes, surface potentials, morphologies and stability of the steroid-based cationic lipids/pDNA lipoplexes were disclosed to depend largely on the steroid skeletons. Cellular evaluation results revealed that cytotoxicity and gene transfection efficiency of the steroid-based cationic lipids in H1299 and HeLa cells strongly relied on the steroid hydrophobes. Interestingly, the steroid lipids/pDNA lipoplexes inclined to enter H1299 cells mainly through caveolae and lipid-raft mediated endocytosis pathways, and an intracellular trafficking route of “lipid-raft-mediated endocytosis→lysosome→cell nucleic localization” was accordingly proposed. The study provided possible approach for developing high-performance steroid-based lipid gene carriers, in which the cytotoxicity, gene transfection capability, endocytosis pathways, and intracellular trafficking/localization manners could be tuned/controlled by introducing proper steroid skeletons/hydrophobes. Noteworthy, among the lipids, Cho-Arg showed remarkably high gene transfection efficacy, even under high serum concentration (50% fetal bovine serum), making it an efficient gene transfection agent for practical application. Full article
(This article belongs to the Special Issue Nucleic Acid Nanotechnology)
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Open AccessArticle Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds
Int. J. Mol. Sci. 2018, 19(2), 370; doi:10.3390/ijms19020370
Received: 29 December 2017 / Revised: 20 January 2018 / Accepted: 22 January 2018 / Published: 26 January 2018
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Abstract
In this study, we first used gelatin/chondroitin-6-sulfate/hyaluronan/chitosan highly elastic cryogels, which showed total recovery from large strains during repeated compression cycles, as 3D scaffolds to study the effects of cyclic dynamic compressive loading on chondrocyte gene expression and extracellular matrix (ECM) production. Dynamic
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In this study, we first used gelatin/chondroitin-6-sulfate/hyaluronan/chitosan highly elastic cryogels, which showed total recovery from large strains during repeated compression cycles, as 3D scaffolds to study the effects of cyclic dynamic compressive loading on chondrocyte gene expression and extracellular matrix (ECM) production. Dynamic culture of porcine chondrocytes was studied at 1 Hz, 10% to 40% strain and 1 to 9 h/day stimulation duration, in a mechanical-driven multi-chamber bioreactor for 14 days. From the experimental results, we could identify the optimum dynamic culture condition (20% and 3 h/day) to enhance the chondrocytic phenotype of chondrocytes from the expression of marker (Col I, Col II, Col X, TNF-α, TGF-β1 and IGF-1) genes by quantitative real-time polymerase chain reactions (qRT-PCR) and production of ECM (GAGs and Col II) by biochemical analysis and immunofluorescence staining. With up-regulated growth factor (TGF-β1 and IGF-1) genes, co-culture of chondrocytes with porcine adipose-derived stem cells (ASCs) was employed to facilitate chondrogenic differentiation of ASCs during dynamic culture in cryogel scaffolds. By replacing half of the chondrocytes with ASCs during co-culture, we could obtain similar production of ECM (GAGs and Col II) and expression of Col II, but reduced expression of Col I, Col X and TNF-α. Subcutaneous implantation of cells/scaffold constructs in nude mice after mono-culture (chondrocytes or ASCs) or co-culture (chondrocytes + ASCs) and subject to static or dynamic culture condition in vitro for 14 days was tested for tissue-engineering applications. The constructs were retrieved 8 weeks post-implantation for histological analysis by Alcian blue, Safranin O and Col II immunohistochemical staining. The most abundant ectopic cartilage tissue was found for the chondrocytes and chondrocytes + ASCs groups using dynamic culture, which showed similar neo-cartilage formation capability with half of the chondrocytes replaced by ASCs for co-culture. This combined co-culture/dynamic culture strategy is expected to cut down the amount of donor chondrocytes needed for cartilage-tissue engineering. Full article
(This article belongs to the Section Biomaterial Sciences)
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Open AccessArticle Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense
Int. J. Mol. Sci. 2018, 19(2), 371; doi:10.3390/ijms19020371
Received: 20 December 2017 / Revised: 15 January 2018 / Accepted: 24 January 2018 / Published: 26 January 2018
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Abstract
Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To
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Expression cloning from cDNA is an important technique for acquiring genes encoding novel fluorescent proteins. However, the probability of in-frame cDNA insertion following the first start codon of the vector is normally only 1/3, which is a cause of low cloning efficiency. To overcome this issue, we developed a new expression plasmid vector, pRSET-TriEX, in which transcriptional slippage was induced by introducing a DNA sequence of (dT)14 next to the first start codon of pRSET. The effectiveness of frame-insensitive cloning was validated by inserting the gene encoding eGFP with all three possible frames to the vector. After transformation with one of these plasmids, E. coli cells expressed eGFP with no significant difference in the expression level. The pRSET-TriEX vector was then used for expression cloning of a novel fluorescent protein from Scolionema suvaense. We screened 3658 E. coli colonies transformed with pRSET-TriEX containing Scolionema suvaense cDNA, and found one colony expressing a novel green fluorescent protein, ScSuFP. The highest score in protein sequence similarity was 42% with the chain c of multi-domain green fluorescent protein like protein “ember” from Anthoathecata sp. Variations in the N- and/or C-terminal sequence of ScSuFP compared to other fluorescent proteins indicate that the expression cloning, rather than the sequence similarity-based methods, was crucial for acquiring the gene encoding ScSuFP. The absorption maximum was at 498 nm, with an extinction efficiency of 1.17 × 105 M−1·cm−1. The emission maximum was at 511 nm and the fluorescence quantum yield was determined to be 0.6. Pseudo-native gel electrophoresis showed that the protein forms obligatory homodimers. Full article
(This article belongs to the Special Issue Fluorescent Proteins)
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Open AccessArticle Poly-N-Acetyllactosamine Neo-Glycoproteins as Nanomolar Ligands of Human Galectin-3: Binding Kinetics and Modeling
Int. J. Mol. Sci. 2018, 19(2), 372; doi:10.3390/ijms19020372
Received: 15 January 2018 / Revised: 22 January 2018 / Accepted: 23 January 2018 / Published: 26 January 2018
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Abstract
Galectin-3 (Gal-3) is recognized as a prognostic marker in several cancer types. Its involvement in tumor development and proliferation makes this lectin a promising target for early cancer diagnosis and anti-cancer therapies. Gal-3 recognizes poly-N-acetyllactosamine (LacNAc)-based carbohydrate motifs of glycoproteins and
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Galectin-3 (Gal-3) is recognized as a prognostic marker in several cancer types. Its involvement in tumor development and proliferation makes this lectin a promising target for early cancer diagnosis and anti-cancer therapies. Gal-3 recognizes poly-N-acetyllactosamine (LacNAc)-based carbohydrate motifs of glycoproteins and glycolipids with a high specificity for internal LacNAc epitopes. This study analyzes the mode and kinetics of binding of Gal-3 to a series of multivalent neo-glycoproteins presenting complex poly-LacNAc-based oligosaccharide ligands on a scaffold of bovine serum albumin. These neo-glycoproteins rank among the strongest Gal-3 ligands reported, with Kd reaching sub-nanomolar values as determined by surface plasmon resonance. Significant differences in the binding kinetics were observed within the ligand series, showing the tetrasaccharide capped with N,N′-diacetyllactosamine (LacdiNAc) as the strongest ligand of Gal-3 in this study. A molecular model of the Gal-3 carbohydrate recognition domain with docked oligosaccharide ligands is presented that shows the relations in the binding site at the molecular level. The neo-glycoproteins presented herein may be applied for selective recognition of Gal-3 both on the cell surface and in blood serum. Full article
(This article belongs to the Special Issue Galectins in Cancer and Translational Medicine)
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Open AccessArticle Transcriptome Analysis of Kiwifruit in Response to Pseudomonas syringae pv. actinidiae Infection
Int. J. Mol. Sci. 2018, 19(2), 373; doi:10.3390/ijms19020373
Received: 21 November 2017 / Revised: 23 January 2018 / Accepted: 23 January 2018 / Published: 26 January 2018
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Abstract
Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has brought about a severe threat to the kiwifruit industry worldwide since its first outbreak in 2008. Studies on other pathovars of P. syringae are revealing the pathogenesis of these pathogens, but little
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Kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa) has brought about a severe threat to the kiwifruit industry worldwide since its first outbreak in 2008. Studies on other pathovars of P. syringae are revealing the pathogenesis of these pathogens, but little about the mechanism of kiwifruit bacterial canker is known. In order to explore the species-specific interaction between Psa and kiwifruit, we analyzed the transcriptomic profile of kiwifruit infected by Psa. After 48 h, 8255 differentially expressed genes were identified, including those involved in metabolic process, secondary metabolites metabolism and plant response to stress. Genes related to biosynthesis of terpens were obviously regulated, indicating terpens may play roles in suppressing the growth of Psa. We identified 283 differentially expressed resistant genes, of which most U-box domain containing genes were obviously up regulated. Expression of genes involved in plant immunity was detected and some key genes showed differential expression. Our results suggest that Psa induced defense response of kiwifruit, including PAMP (pathogen/microbe-associated molecular patterns)-triggered immunity, effector-triggered immunity and hypersensitive response. Metabolic process was adjusted to adapt to these responses and production of secondary metabolites may be altered to suppress the growth of Psa. Full article
(This article belongs to the Special Issue Plant Microbe Interaction 2017)
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Open AccessArticle Characterization and Oral Delivery of Proinsulin-Transferrin Fusion Protein Expressed Using ExpressTec
Int. J. Mol. Sci. 2018, 19(2), 378; doi:10.3390/ijms19020378
Received: 26 November 2017 / Revised: 23 January 2018 / Accepted: 24 January 2018 / Published: 26 January 2018
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Abstract
Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific
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Proinsulin-transferrin fusion protein (ProINS-Tf) has been designed and successfully expressed from the mammalian HEK293 cells (HEK-ProINS-Tf). It was found that HEK-ProINS-Tf could be converted into an activated form in the liver. Furthermore, HEK-ProINS-Tf was demonstrated as an extra-long acting insulin analogue with liver-specific insulin action in streptozotocin (STZ)-induced type 1 diabetic mice. However, due to the low production yield from transfected HEK293 cells, there are other interesting features, including the oral bioavailability, which have not been fully explored and characterized. To improve the protein production yield, an alternative protein expression system, ExpressTec using transgenic rice (Oryza sativa L.), was used. The intact and active rice-derived ProINS-Tf (ExpressTec-ProINS-Tf) was successfully expressed from the transgenic rice expression system. Our results suggested that, although the insulin-like bioactivity of ExpressTec-ProINS-Tf was slightly lower in vitro, its potency of in vivo blood glucose control was considerably stronger than that of HEK-ProINS-Tf. The oral delivery studies in type 1 diabetic mice demonstrated a prolonged control of blood glucose to near-normal levels after oral administration of ExpressTec-ProINS-Tf. Results in this report suggest that ExpressTec-ProINS-Tf is a promising insulin analog with advantages including low cost, prolonged and liver targeting effects, and most importantly, oral bioactivity. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle Corylin Suppresses Hepatocellular Carcinoma Progression via the Inhibition of Epithelial-Mesenchymal Transition, Mediated by Long Noncoding RNA GAS5
Int. J. Mol. Sci. 2018, 19(2), 380; doi:10.3390/ijms19020380
Received: 30 December 2017 / Revised: 13 January 2018 / Accepted: 18 January 2018 / Published: 27 January 2018
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Abstract
Corylin is a flavonoid extracted from the nuts of Psoralea corylifolia L. (Fabaceae), which is a widely used anti-inflammatory and anticancer herb in China. Recent studies revealed antioxidant, anti-inflammatory, and bone differentiation–promoting effects of corylin. However, there are no studies examining the anticancer
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Corylin is a flavonoid extracted from the nuts of Psoralea corylifolia L. (Fabaceae), which is a widely used anti-inflammatory and anticancer herb in China. Recent studies revealed antioxidant, anti-inflammatory, and bone differentiation–promoting effects of corylin. However, there are no studies examining the anticancer activity of corylin. In this study, we used cells and animal models to examine the antitumor effects of corylin on hepatocellular carcinoma (HCC) and then studied its downstream regulatory mechanisms. The results showed that corylin significantly inhibited the proliferation, migration, and invasiveness of HCC cells and suppressed epithelial–mesenchymal transition. We found that the anti-HCC mechanism of corylin’s action lies in the upregulation of tumor suppressor long noncoding RNA growth arrest-specific transcript 5 (GAS5) and the activation of its downstream anticancer pathways. In animal experiments, we also found that corylin can significantly inhibit tumor growth without significant physiological toxicity. The above results suggest that corylin has anti-HCC effects and good potential as a clinical treatment. Full article
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Open AccessArticle Resistance to the Antiproliferative In Vitro Effect of PI3K-Akt-mTOR Inhibition in Primary Human Acute Myeloid Leukemia Cells Is Associated with Altered Cell Metabolism
Int. J. Mol. Sci. 2018, 19(2), 382; doi:10.3390/ijms19020382
Received: 6 December 2017 / Revised: 17 January 2018 / Accepted: 23 January 2018 / Published: 27 January 2018
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Abstract
Constitutive signaling through the phosphatidylinositol-3-kinase-Akt-mechanistic target of rapamycin (PI3K-Akt-mTOR) pathway is present in acute myeloid leukemia (AML) cells. However, AML is a heterogeneous disease, and we therefore investigated possible associations between cellular metabolism and sensitivity to PI3K-Akt-mTOR pathway inhibitors. We performed non-targeted metabolite
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Constitutive signaling through the phosphatidylinositol-3-kinase-Akt-mechanistic target of rapamycin (PI3K-Akt-mTOR) pathway is present in acute myeloid leukemia (AML) cells. However, AML is a heterogeneous disease, and we therefore investigated possible associations between cellular metabolism and sensitivity to PI3K-Akt-mTOR pathway inhibitors. We performed non-targeted metabolite profiling to compare the metabolome differences of primary human AML cells derived from patients susceptible or resistant to the in vitro antiproliferative effects of mTOR and PI3K inhibitors. In addition, the phosphorylation status of 18 proteins involved in PI3K-Akt-mTOR signaling and the effect of the cyclooxygenase inhibitor indomethacin on their phosphorylation status was investigated by flow cytometry. Strong antiproliferative effects by inhibitors were observed only for a subset of patients. We compared the metabolite profiles for responders and non-responders towards PI3K-mTOR inhibitors, and 627 metabolites could be detected. Of these metabolites, 128 were annotated and 15 of the annotated metabolites differed significantly between responders and non-responders, including metabolites involved in energy, amino acid, and lipid metabolism. To conclude, leukemia cells that are susceptible or resistant to PI3K-Akt-mTOR inhibitors differ in energy, amino acid, and arachidonic acid metabolism, and modulation of arachidonic acid metabolism alters the activation of mTOR and its downstream mediators. Full article
(This article belongs to the Special Issue mTOR in Human Diseases)
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Open AccessArticle VEGF Triggers the Activation of Cofilin and the Arp2/3 Complex within the Growth Cone
Int. J. Mol. Sci. 2018, 19(2), 384; doi:10.3390/ijms19020384
Received: 22 December 2017 / Revised: 16 January 2018 / Accepted: 24 January 2018 / Published: 27 January 2018
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Abstract
A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor
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A crucial neuronal structure for the development and regeneration of neuronal networks is the axonal growth cone. Affected by different guidance cues, it grows in a predetermined direction to reach its final destination. One of those cues is the vascular endothelial growth factor (VEGF), which was identified as a positive effector for growth cone movement. These positive effects are mainly mediated by a reorganization of the actin network. This study shows that VEGF triggers a tight colocalization of cofilin and the Arp2/3 complex to the actin cytoskeleton within chicken dorsal root ganglia (DRG). Live cell imaging after microinjection of GFP (green fluorescent protein)-cofilin and RFP (red fluorescent protein)-LifeAct revealed that both labeled proteins rapidly redistributed within growth cones, and showed a congruent distribution pattern after VEGF supplementation. Disruption of signaling upstream of cofilin via blocking LIM-kinase (LIMK) activity resulted in growth cones displaying regressive growth behavior. Microinjection of GFP-p16b (a subunit of the Arp2/3 complex) and RFP-LifeAct revealed that both proteins redistributed into lamellipodia of the growth cone within minutes after VEGF stimulation. Disruption of the signaling to the Arp2/3 complex in the presence of VEGF by inhibition of N-WASP (neuronal Wiskott–Aldrich–Scott protein) caused retraction of growth cones. Hence, cofilin and the Arp2/3 complex appear to be downstream effector proteins of VEGF signaling to the actin cytoskeleton of DRG growth cones. Our data suggest that VEGF simultaneously affects different pathways for signaling to the actin cytoskeleton, since activation of cofilin occurs via inhibition of LIMK, whereas activation of Arp2/3 is achieved by stimulation of N-WASP. Full article
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Open AccessArticle A Simple Method to Reduce both Lactic Acid and Ammonium Production in Industrial Animal Cell Culture
Int. J. Mol. Sci. 2018, 19(2), 385; doi:10.3390/ijms19020385
Received: 23 December 2017 / Revised: 16 January 2018 / Accepted: 23 January 2018 / Published: 28 January 2018
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Abstract
Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to
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Fed-batch animal cell culture is the most common method for commercial production of recombinant proteins. However, higher cell densities in these platforms are still limited due to factors such as excessive ammonium production, lactic acid production, nutrient limitation, and/or hyperosmotic stress related to nutrient feeds and base additions to control pH. To partly overcome these factors, we investigated a simple method to reduce both ammonium and lactic acid production—termed Lactate Supplementation and Adaptation (LSA) technology—through the use of CHO cells adapted to a lactate-supplemented medium. Using this simple method, we achieved a reduction of nearly 100% in lactic acid production with a simultaneous 50% reduction in ammonium production in batch shaker flasks cultures. In subsequent fed-batch bioreactor cultures, lactic acid production and base addition were both reduced eight-fold. Viable cell densities of 35 million cells per mL and integral viable cell days of 273 million cell-days per mL were achieved, both among the highest currently reported for a fed-batch animal cell culture. Investigating the benefits of LSA technology in animal cell culture is worthy of further consideration and may lead to process conditions more favorable for advanced industrial applications. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle Structural Studies of Predicted Ligand Binding Sites and Molecular Docking Analysis of Slc2a4 as a Therapeutic Target for the Treatment of Cancer
Int. J. Mol. Sci. 2018, 19(2), 386; doi:10.3390/ijms19020386
Received: 4 November 2017 / Revised: 27 November 2017 / Accepted: 28 November 2017 / Published: 28 January 2018
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Abstract
Presently, many studies have focused on exploring in silico approaches in the identification and development of alternative therapy for the treatment and management of cancer. Solute carrier family-2-member-4-gene (Slc2a4) which encodes glucose transporter 4 protein (GLUT4), has been identified as a
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Presently, many studies have focused on exploring in silico approaches in the identification and development of alternative therapy for the treatment and management of cancer. Solute carrier family-2-member-4-gene (Slc2a4) which encodes glucose transporter 4 protein (GLUT4), has been identified as a promising therapeutic target for cancer. Though Slc2a4 is known to play a major regulatory role in the pathophysiology of type 2 diabetes, emerging evidence suggests that successful pharmacological inhibition of this protein may lead to the development of a novel drug candidate for the treatment of cancer. In this study, Slc2a4 protein sequence was retrieved and analysed using in silico approaches, and we identified seven putative antimicrobial peptides (AMPs; RAB1-RAB7) as anti-cancer. The structures of the protein and AMPs were modelled using I-TASSER server, and the overall quality of the Slc2a4 model was validated using PROCHECK. Subsequently, the probable motifs and active site of the protein were forecasted. Also, the molecular interaction between the AMPs and Slc2a4 was ascertained using PatchDock. The result revealed that, all the AMPs are good Slc2a4 inhibitors with RAB1 having the highest binding affinity of 12,392 and binding energy of −39.13 kcal/mol. Hence, this study reveals that all the generated AMPs can serve as therapeutic drug in treating cancer by inhibiting Slc2a4 which is responsible for the production of energy for cancer cells during angiogenesis. This is the first report on AMPs as inhibitors of Slc2a4 for the treatment of cancer. Full article
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Open AccessArticle Multifunctional Tannic Acid/Silver Nanoparticle-Based Mucoadhesive Hydrogel for Improved Local Treatment of HSV Infection: In Vitro and In Vivo Studies
Int. J. Mol. Sci. 2018, 19(2), 387; doi:10.3390/ijms19020387
Received: 11 December 2017 / Revised: 16 January 2018 / Accepted: 23 January 2018 / Published: 28 January 2018
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Abstract
Mucoadhesive gelling systems with tannic acid modified silver nanoparticles were developed for effective treatment of herpes virus infections. To increase nanoparticle residence time after local application, semi solid formulations designed from generally regarded as safe (GRAS) excipients were investigated for their rheological and
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Mucoadhesive gelling systems with tannic acid modified silver nanoparticles were developed for effective treatment of herpes virus infections. To increase nanoparticle residence time after local application, semi solid formulations designed from generally regarded as safe (GRAS) excipients were investigated for their rheological and mechanical properties followed with ex vivo mucoadhesive behavior to the porcine vaginal mucosa. Particular effort was made to evaluate the activity of nanoparticle-based hydrogels toward herpes simplex virus (HSV) type 1 and 2 infection in vitro in immortal human keratinocyte cell line and in vivo using murine model of HSV-2 genital infection. The effect of infectivity was determined by real time quantitative polymerase chain reaction, plaque assay, inactivation, attachment, penetration and cell-to-cell assessments. All analyzed nanoparticle-based hydrogels exhibited pseudoplastic and thixotropic properties. Viscosity and mechanical measurements of hydrogels were found to correlate with the mucoadhesive properties. The results confirmed the ability of nanoparticle-based hydrogels to affect viral attachment, impede penetration and cell-to-cell transmission, although profound differences in the activity evoked by tested preparations toward HSV-1 and HSV-2 were noted. In addition, these findings demonstrated the in vivo potential of tannic acid modified silver nanoparticle-based hydrogels for vaginal treatment of HSV-2 genital infection. Full article
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Open AccessArticle Comparison of Human Dermal Fibroblasts and HaCat Cells Cultured in Medium with or without Serum via a Generic Tissue Engineering Research Platform
Int. J. Mol. Sci. 2018, 19(2), 388; doi:10.3390/ijms19020388
Received: 11 December 2017 / Revised: 25 January 2018 / Accepted: 25 January 2018 / Published: 28 January 2018
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Abstract
A generic research platform with 2-dimensional (2D) cell culture technology, a 3-dimensional (3D) in vitro tissue model, and a scaled-down cell culture and imaging system in between, was utilized to address the problematic issues associated with the use of serum in skin tissue
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A generic research platform with 2-dimensional (2D) cell culture technology, a 3-dimensional (3D) in vitro tissue model, and a scaled-down cell culture and imaging system in between, was utilized to address the problematic issues associated with the use of serum in skin tissue engineering. Human dermal fibroblasts (HDFs) and immortalized keratinocytes (HaCat cells) mono- or co-cultured in serum or serum-free medium were compared and analyzed via the platform. It was demonstrated that serum depletion had significant influence on the attachment of HaCat cells onto tissue culture plastic (TCP), porous substrates and cellulosic scaffolds, which was further enhanced by the pre-seeded HDFs. The complex structures formed by the HDFs colonized within the porous substrates and scaffolds not only prevented the seeded HaCat cells from filtering through the open pores, but also acted as cellular substrates for HaCat cells to attach onto. When mono-cultured on TCP, both HDFs and HaCat cells were less proliferative in medium without serum than with serum. However, both cell types were successfully co-cultured in 2D using serum-free medium if the initial cell seeding density was higher than 80,000 cells/cm2 (with 1:1 ratio). Based on the results from 2D cultures, co-culture of both cell types on modular substrates with small open pores (125 μm) and cellulosic scaffolds with open pores of varying sizes (50–300 µm) were then conducted successfully in serum-free medium. This study demonstrated that the generic research platform had great potential for in-depth understanding of HDFs and HaCat cells cultivated in serum-free medium, which could inform the processes for manufacturing skin cells or tissues for clinical applications. Full article
(This article belongs to the Special Issue Cell Colonization in Scaffolds)
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Open AccessArticle S1P Signalling Differentially Affects Migration of Peritoneal B Cell Populations In Vitro and Influences the Production of Intestinal IgA In Vivo
Int. J. Mol. Sci. 2018, 19(2), 391; doi:10.3390/ijms19020391
Received: 5 December 2017 / Revised: 15 January 2018 / Accepted: 24 January 2018 / Published: 29 January 2018
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Abstract
Introduction: Sphingosine-1-phosphate (S1P) regulates the migration of follicular B cells (B2 cells) and directs the positioning of Marginal zone B cells (MZ B cells) within the spleen. The function of S1P signalling in the third B cell lineage, B1 B cells, mainly present
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Introduction: Sphingosine-1-phosphate (S1P) regulates the migration of follicular B cells (B2 cells) and directs the positioning of Marginal zone B cells (MZ B cells) within the spleen. The function of S1P signalling in the third B cell lineage, B1 B cells, mainly present in the pleural and peritoneal cavity, has not yet been determined. Methods: S1P receptor expression was analysed in peritoneal B cells by real-time polymerase chain reaction (qPCR). The chemotactic response to S1P was studied in vitro. The role of S1P signalling was further explored in a s1p4−/− mouse strain. Results: Peritoneal B cells expressed considerable amounts of the S1P receptors 1 and 4 (S1P1 and S1P4, respectively). S1P1 showed differential expression between the distinct peritoneal B cell lineages. While B2 cells showed no chemotactic response to S1P, B1 B cells showed a migration response to S1P. s1p4−/− mice displayed significant alterations in the composition of peritoneal B cell populations, as well as a significant reduction of mucosal immunoglobulin A (IgA) in the gut. Discussion: S1P signalling influences peritoneal B1 B cell migration. S1P4 deficiency alters the composition of peritoneal B cell populations and reduces secretory IgA levels. These findings suggest that S1P signalling may be a target to modulate B cell function in inflammatory intestinal pathologies. Full article
(This article belongs to the Special Issue Sphingolipids: Signals and Disease)
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Open AccessArticle Anti-Cancerous Effect of Inonotus taiwanensis Polysaccharide Extract on Human Acute Monocytic Leukemia Cells through ROS-Independent Intrinsic Mitochondrial Pathway
Int. J. Mol. Sci. 2018, 19(2), 393; doi:10.3390/ijms19020393
Received: 13 November 2017 / Revised: 3 January 2018 / Accepted: 19 January 2018 / Published: 29 January 2018
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Abstract
Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set
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Acute leukemia is one of the commonly diagnosed neoplasms and causes human death. However, the treatment for acute leukemia is not yet satisfactory. Studies have shown that mushroom-derived polysaccharides display low toxicity and have been used clinically for cancer therapy. Therefore, we set out to evaluate the anti-cancerous efficacy of a water-soluble polysaccharide extract from Inonotus taiwanensis (WSPIS) on human acute monocytic leukemia THP-1 and U937 cell lines in vitro. Under our experimental conditions, WSPIS elicited dose-dependent growth retardation and induced apoptotic cell death. Further analysis showed that WSPIS-induced apoptosis was associated with a mitochondrial apoptotic pathway, such as the disruption of mitochondrial membrane potential (MMP), followed by the activation of caspase-9, caspase-3, and PARP (poly(ADP-ribose) polymerase) cleavage. However, a broad caspase inhibitor, Z-VAD.fmk, could not prevent WSPIS-induced apoptosis. These data imply that mechanism(s) other than caspase might be involved. Thus, the involvement of endonuclease G (endoG), a mediator arbitrating caspase-independent oligonucleosomal DNA fragmentation, was examined. Western blotting demonstrated that WSPIS could elicit nuclear translocation of endoG. MMP disruption after WSPIS treatment was accompanied by intracellular reactive oxygen species (ROS) generation. However, pretreatment with N-acetyl-l-cysteine (NAC) could not attenuate WSPIS-induced apoptosis. In addition, our data also show that WSPIS could inhibit autophagy. Activation of autophagy by rapamycin decreased WSPIS-induced apoptosis and cell death. Taken together, our findings suggest that cell cycle arrest, endonuclease G-mediated apoptosis, and autophagy inhibition contribute to the anti-cancerous effect of WSPIS on human acute monocytic leukemia cells. Full article
(This article belongs to the Section Bioactives and Nutraceuticals)
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Open AccessArticle Comparative Transcriptome Analyses Uncover Key Candidate Genes Mediating Flight Capacity in Bactrocera dorsalis (Hendel) and Bactrocera correcta (Bezzi) (Diptera: Tephritidae)
Int. J. Mol. Sci. 2018, 19(2), 396; doi:10.3390/ijms19020396
Received: 5 December 2017 / Revised: 8 January 2018 / Accepted: 22 January 2018 / Published: 30 January 2018
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Abstract
Flight capacity is important for invasive pests during entry, establishment and spreading. Both Bactrocera dorsalis Hendel and Bactrocera correcta Bezzi are invasive fruit flies but their flight capacities differ. Here, a tethered flight mill test demonstrated that B. dorsalis exhibits a greater flight
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Flight capacity is important for invasive pests during entry, establishment and spreading. Both Bactrocera dorsalis Hendel and Bactrocera correcta Bezzi are invasive fruit flies but their flight capacities differ. Here, a tethered flight mill test demonstrated that B. dorsalis exhibits a greater flight capacity than B. correcta. RNA-Seq was used to determine the transcriptomic differences associated with the flight capacity of two Bactrocera species. Transcriptome data showed that 6392 unigenes were differentially expressed between the two species in the larval stage, whereas in the adult stage, 4104 differentially expressed genes (DEGs) were identified in females, and 3445 DEGs were observed in males. The flight capacity appeared to be correlated with changes in the transcriptional levels of genes involved in wing formation, flight muscle structure, energy metabolism, and hormonal control. Using RNA interference (RNAi) to verify the function of one DEG, the epidermal growth factor receptor (EGFR), we confirmed the role of this gene in regulating wing development, and thereby flight capacity, in both species. This work reveals the flight mechanism of fruit flies and provides insight into fundamental transcriptomics for further studies on the flight performance of insects. Full article
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Open AccessArticle Sex Hormone Receptors in Benign and Malignant Salivary Gland Tumors: Prognostic and Predictive Role
Int. J. Mol. Sci. 2018, 19(2), 399; doi:10.3390/ijms19020399
Received: 21 November 2017 / Revised: 3 January 2018 / Accepted: 19 January 2018 / Published: 30 January 2018
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Abstract
The role of sex hormone receptors in human cancer development and progression has been well documented in numerous studies, as has the success of sex hormone antagonists in the biological therapy of many human tumors. In salivary gland tumors (SGTs), little and conflicting
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The role of sex hormone receptors in human cancer development and progression has been well documented in numerous studies, as has the success of sex hormone antagonists in the biological therapy of many human tumors. In salivary gland tumors (SGTs), little and conflicting information about the role of the estrogen receptor alpha (ERα), progesterone receptor (PgR) and androgen receptor (AR) has been described and in most cases the use of sex hormone antagonists is not contemplated in clinical practice. In this study, we analyzed a panel of sex hormone receptors that have not been widely investigated in SGTs—ERα, PgR, AR, but also ERβ and GPR30—to define their expression pattern and their prognostic and predictive value in a case series of 69 benign and malignant SGTs. We showed the aberrant expression of AR in mucoepidermoid and oncocytic carcinoma, a strong relation between cytoplasmic ERβ expression and tumor grade, and a strong correlation between nuclear GPR30 expression and disease-free survival (DFS) of SGT patients. Full article
(This article belongs to the Special Issue Sex Hormone Receptor Signals in Human Malignancies)
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Open AccessArticle Discovery of Cryoprotective Activity in Human Genome-Derived Intrinsically Disordered Proteins
Int. J. Mol. Sci. 2018, 19(2), 401; doi:10.3390/ijms19020401
Received: 20 December 2017 / Revised: 17 January 2018 / Accepted: 22 January 2018 / Published: 30 January 2018
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Abstract
Intrinsically disordered proteins (IDPs) are an emerging phenomenon. They may have a high degree of flexibility in their polypeptide chains, which lack a stable 3D structure. Although several biological functions of IDPs have been proposed, their general function is not known. The only
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Intrinsically disordered proteins (IDPs) are an emerging phenomenon. They may have a high degree of flexibility in their polypeptide chains, which lack a stable 3D structure. Although several biological functions of IDPs have been proposed, their general function is not known. The only finding related to their function is the genetically conserved YSK2 motif present in plant dehydrins. These proteins were shown to be IDPs with the YSK2 motif serving as a core region for the dehydrins’ cryoprotective activity. Here we examined the cryoprotective activity of randomly selected IDPs toward the model enzyme lactate dehydrogenase (LDH). All five IDPs that were examined were in the range of 35–45 amino acid residues in length and were equally potent at a concentration of 50 μg/mL, whereas folded proteins, the PSD-95/Dlg/ZO-1 (PDZ) domain, and lysozymes had no potency. We further examined their cryoprotective activity toward glutathione S-transferase as an example of the other enzyme, and toward enhanced green fluorescent protein as a non-enzyme protein example. We further examined the lyophilization protective activity of the peptides toward LDH, which revealed that some IDPs showed a higher activity than that of bovine serum albumin (BSA). Based on these observations, we propose that cryoprotection is a general feature of IDPs. Our findings may become a clue to various industrial applications of IDPs in the future. Full article
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Open AccessArticle Differential Glycosylation and Modulation of Camel and Human HSP Isoforms in Response to Thermal and Hypoxic Stresses
Int. J. Mol. Sci. 2018, 19(2), 402; doi:10.3390/ijms19020402
Received: 8 December 2017 / Revised: 18 January 2018 / Accepted: 26 January 2018 / Published: 30 January 2018
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Abstract
Increased expression of heat shock proteins (HSPs) following heat stress or other stress conditions is a common physiological response in almost all living organisms. Modification of cytosolic proteins including HSPs by O-GlcNAc has been shown to enhance their capabilities for counteracting lethal
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Increased expression of heat shock proteins (HSPs) following heat stress or other stress conditions is a common physiological response in almost all living organisms. Modification of cytosolic proteins including HSPs by O-GlcNAc has been shown to enhance their capabilities for counteracting lethal levels of cellular stress. Since HSPs are key players in stress resistance and protein homeostasis, we aimed to analyze their forms at the cellular and molecular level using camel and human HSPs as models for efficient and moderate thermotolerant mammals, respectively. In this study, we cloned the cDNA encoding two inducible HSP members, HSPA6 and CRYAB from both camel (Camelus dromedarius) and human in a Myc-tagged mammalian expression vector. Expression of these chaperones in COS-1 cells revealed protein bands of approximately 25-kDa for both camel and human CRYAB and 70-kDa for camel HSPA6 and its human homologue. While localization and trafficking of the camel and human HSPs revealed similar cytosolic localization, we could demonstrate altered glycan structure between camel and human HSPA6. Interestingly, the glycoform of camel HSPA6 was rapidly formed and stabilized under normal and stress culture conditions whereas human HSPA6 reacted differently under similar thermal and hypoxic stress conditions. Our data suggest that efficient glycosylation of camel HSPA6 is among the mechanisms that provide camelids with a superior capability for alleviating stressful environmental circumstances. Full article
(This article belongs to the collection Protein Folding)
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Open AccessCommunication Nanobody Based Dual Specific CARs
Int. J. Mol. Sci. 2018, 19(2), 403; doi:10.3390/ijms19020403
Received: 27 December 2017 / Revised: 22 January 2018 / Accepted: 24 January 2018 / Published: 30 January 2018
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Abstract
Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due
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Recent clinical trials have shown that adoptive chimeric antigen receptor (CAR) T cell therapy is a very potent and possibly curative option in the treatment of B cell leukemias and lymphomas. However, targeting a single antigen may not be sufficient, and relapse due to the emergence of antigen negative leukemic cells may occur. A potential strategy to counter the outgrowth of antigen escape variants is to broaden the specificity of the CAR by incorporation of multiple antigen recognition domains in tandem. As a proof of concept, we here describe a bispecific CAR in which the single chain variable fragment (scFv) is replaced by a tandem of two single-antibody domains or nanobodies (nanoCAR). High membrane nanoCAR expression levels are observed in retrovirally transduced T cells. NanoCARs specific for CD20 and HER2 induce T cell activation, cytokine production and tumor lysis upon incubation with transgenic Jurkat cells expressing either antigen or both antigens simultaneously. The use of nanobody technology allows for the production of compact CARs with dual specificity and predefined affinity. Full article
(This article belongs to the Special Issue Chimeric Antigen Receptor (CAR) T Cell Therapy)
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Open AccessArticle Different Achilles Tendon Pathologies Show Distinct Histological and Molecular Characteristics
Int. J. Mol. Sci. 2018, 19(2), 404; doi:10.3390/ijms19020404
Received: 10 January 2018 / Revised: 25 January 2018 / Accepted: 26 January 2018 / Published: 30 January 2018
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Abstract
Reasons for the development of chronic tendon pathologies are still under debate and more basic knowledge is needed about the different diseases. The aim of the present study was therefore to characterize different acute and chronic Achilles tendon disorders. Achilles tendon samples from
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Reasons for the development of chronic tendon pathologies are still under debate and more basic knowledge is needed about the different diseases. The aim of the present study was therefore to characterize different acute and chronic Achilles tendon disorders. Achilles tendon samples from patients with chronic tendinopathy (n = 7), chronic ruptures (n = 6), acute ruptures (n = 13), and intact tendons (n = 4) were analyzed. The histological score investigating pathological changes was significantly increased in tendinopathy and chronic ruptures compared to acute ruptures. Inflammatory infiltration was detected by immunohistochemistry in all tendon pathology groups, but was significantly lower in tendinopathy compared to chronic ruptures. Quantitative real-time PCR (qRT-PCR) analysis revealed significantly altered expression of genes related to collagens and matrix modeling/remodeling (matrix metalloproteinases, tissue inhibitors of metalloproteinases) in tendinopathy and chronic ruptures compared to intact tendons and/or acute ruptures. In all three tendon pathology groups markers of inflammation (interleukin (IL) , tumor necrosis factor α, IL6, IL10, IL33, soluble ST2, transforming growth factor β1, cyclooxygenase 2), inflammatory cells (cluster of differentaition (CD) 3, CD68, CD80, CD206), fat metabolism (fatty acid binding protein 4, peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein α, adiponectin), and innervation (protein gene product 9.5, growth associated protein 43, macrophage migration inhibitory factor) were detectable, but only in acute ruptures significantly regulated compared to intact tendons. The study gives an insight into structural and molecular changes of pathological processes in tendons and might be used to identify targets for future therapy of tendon pathologies. Full article
(This article belongs to the Special Issue Biological Basis of Musculoskeletal Regeneration)
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Open AccessArticle Inhibition of GLO1 in Glioblastoma Multiforme Increases DNA-AGEs, Stimulates RAGE Expression, and Inhibits Brain Tumor Growth in Orthotopic Mouse Models
Int. J. Mol. Sci. 2018, 19(2), 406; doi:10.3390/ijms19020406
Received: 23 December 2017 / Revised: 22 January 2018 / Accepted: 26 January 2018 / Published: 30 January 2018
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Abstract
Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as
[...] Read more.
Cancers that exhibit the Warburg effect may elevate expression of glyoxylase 1 (GLO1) to detoxify the toxic glycolytic byproduct methylglyoxal (MG) and inhibit the formation of pro-apoptotic advanced glycation endproducts (AGEs). Inhibition of GLO1 in cancers that up-regulate glycolysis has been proposed as a therapeutic targeting strategy, but this approach has not been evaluated for glioblastoma multiforme (GBM), the most aggressive and difficult to treat malignancy of the brain. Elevated GLO1 expression in GBM was established in patient tumors and cell lines using bioinformatics tools and biochemical approaches. GLO1 inhibition in GBM cell lines and in an orthotopic xenograft GBM mouse model was examined using both small molecule and short hairpin RNA (shRNA) approaches. Inhibition of GLO1 with S-(p-bromobenzyl) glutathione dicyclopentyl ester (p-BrBzGSH(Cp)2) increased levels of the DNA-AGE N2-1-(carboxyethyl)-2′-deoxyguanosine (CEdG), a surrogate biomarker for nuclear MG exposure; substantially elevated expression of the immunoglobulin-like receptor for AGEs (RAGE); and induced apoptosis in GBM cell lines. Targeting GLO1 with shRNA similarly increased CEdG levels and RAGE expression, and was cytotoxic to glioma cells. Mice bearing orthotopic GBM xenografts treated systemically with p-BrBzGSH(Cp)2 exhibited tumor regression without significant off-target effects suggesting that GLO1 inhibition may have value in the therapeutic management of these drug-resistant tumors. Full article
(This article belongs to the Special Issue Glyoxalase System in Health and Disease 2017)
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Open AccessArticle The Design and Development of Potent Small Molecules as Anticancer Agents Targeting EGFR TK and Tubulin Polymerization
Int. J. Mol. Sci. 2018, 19(2), 408; doi:10.3390/ijms19020408
Received: 31 December 2017 / Revised: 22 January 2018 / Accepted: 23 January 2018 / Published: 30 January 2018
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Abstract
Some novel anthranilate diamides derivatives 4ae, 6ac and 9ad were designed and synthesized to be evaluated for their in vitro anticancer activity. Structures of all newly synthesized compounds were confirmed by infra-red (IR), high-resolution mass (HR-MS)
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Some novel anthranilate diamides derivatives 4ae, 6ac and 9ad were designed and synthesized to be evaluated for their in vitro anticancer activity. Structures of all newly synthesized compounds were confirmed by infra-red (IR), high-resolution mass (HR-MS) spectra, 1H nuclear magnetic resonance (NMR) and 13C nuclear magnetic resonance (NMR) analyses. Cytotoxic screening was performed according to (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium (MTT) assay method using erlotinib as a reference drug against two different types of breast cancer cells. The molecular docking study was performed for representative compounds against two targets, epidermal growth factor receptor (EGFR) and tubulin in colchicine binding site to assess their binding affinities in order to rationalize their anticancer activity in a qualitative way. The data obtained from the molecular modeling was correlated with that obtained from the biological screening. These data showed considerable anticancer activity for these newly synthesized compounds. Biological data for most of the anthranilate diamide showed excellent activity with nanomolar or sub nanomolar half maximal inhibitory concentration (IC50) values against tumor cells. EGFR tyrosine kinase (TK) inhibition assay, tubulin inhibition assay and apoptosis analysis were performed for selected compounds to get more details about their mechanism of action. Extensive structure activity relationship (SAR) analyses were also carried out. Full article
(This article belongs to the Section Molecular Biophysics)
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Open AccessArticle Antioxidative and Anti-Melanogenic Activities of Bamboo Stems (Phyllostachys nigra variety henosis) via PKA/CREB-Mediated MITF Downregulation in B16F10 Melanoma Cells
Int. J. Mol. Sci. 2018, 19(2), 409; doi:10.3390/ijms19020409
Received: 10 January 2018 / Revised: 24 January 2018 / Accepted: 26 January 2018 / Published: 30 January 2018
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Abstract
Phyllostachys nigra var. henosis, a domestic bamboo species, has been attracting much attention; its bioactive compounds (especially in the leaf) show antioxidant, anti-inflammatory, and anti-obesity activities. Little information is available on the antioxidative and anti-melanogenetic activities of the bioactive compounds in bamboo stems.
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Phyllostachys nigra var. henosis, a domestic bamboo species, has been attracting much attention; its bioactive compounds (especially in the leaf) show antioxidant, anti-inflammatory, and anti-obesity activities. Little information is available on the antioxidative and anti-melanogenetic activities of the bioactive compounds in bamboo stems. The anti-melanogenic and antioxidative activities of the EtOAc fraction (PN3) of a P. nigra stem extract were investigated in a cell-free system and in B16F10 melanoma cells. PN3 consisted of a mixture of flavonoids, such as catechin, chlorogenic acid, caffeic acid, and p-coumaric acid. The antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)), and hydroxyl radical scavenging) was evaluated, as well as the inhibition of reactive oxygen species (ROS) produced by the Fenton reaction. PN3 showed in vitro tyrosinase inhibition activity with the half maximal inbihitory concentration (IC50) values of 240 μg/mL, and in vivo cytotoxic concentration ranges > 100 μg/mL. The protein expression levels and mRNA transcription levels of TYR, TRP-1, and MITF were decreased in a dose-dependent manner by the treatment with PN3. PN3 interfered with the phosphorylation of intracellular protein kinase A (PKA)/cAMP response element-binding protein (CREB), demonstrating potent anti-melanogenic effects. PN3 could inhibit PKA/CREB and the subsequent degradation of microphthalmia-associated transcription factor (MITF), resulting in the suppression of melanogenic enzymes and melanin production, probably because of the presence of flavonoid compounds. These properties make it a candidate as an additive to whitening cosmetics. Full article
(This article belongs to the Special Issue Melanins and Melanogenesis: From Nature to Applications)
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Open AccessArticle Comparison of Compressive Stress-Relaxation Behavior in Osteoarthritic (ICRS Graded) Human Articular Cartilage
Int. J. Mol. Sci. 2018, 19(2), 413; doi:10.3390/ijms19020413
Received: 5 November 2017 / Revised: 13 December 2017 / Accepted: 25 January 2018 / Published: 31 January 2018
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Abstract
Osteoarthritis (OA) is a common joint disorder found mostly in elderly people. The role of mechanical behavior in the progression of OA is complex and remains unclear. The stress-relaxation behavior of human articular cartilage in clinically defined osteoarthritic stages may have importance in
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Osteoarthritis (OA) is a common joint disorder found mostly in elderly people. The role of mechanical behavior in the progression of OA is complex and remains unclear. The stress-relaxation behavior of human articular cartilage in clinically defined osteoarthritic stages may have importance in diagnosis and prognosis of OA. In this study we investigated differences in the biomechanical responses among human cartilage of ICRS grades I, II and III using polymer dynamics theory. We collected 24 explants of human articular cartilage (eight each of ICRS grade I, II and III) and acquired stress-relaxation data applying a continuous load on the articular surface of each cartilage explant for 1180 s. We observed a significant decrease in Young’s modulus, stress-relaxation time, and stretching exponent in advanced stages of OA (ICRS grade III). The stretch exponential model speculated that significant loss in hyaluronic acid polymer might be the reason for the loss of proteoglycan in advanced OA. This work encourages further biomechanical modelling of osteoarthritic cartilage utilizing these data as input parameters to enhance the fidelity of computational models aimed at revealing how mechanical behaviors play a role in pathogenesis of OA. Full article
(This article belongs to the Section Molecular Biophysics)
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Open AccessArticle Changes in the Distribution of Cocaine- and Amphetamine-Regulated Transcript-Containing Neural Structures in the Human Colon Affected by the Neoplastic Process
Int. J. Mol. Sci. 2018, 19(2), 414; doi:10.3390/ijms19020414
Received: 5 January 2018 / Revised: 25 January 2018 / Accepted: 26 January 2018 / Published: 31 January 2018
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Abstract
The present study analysed changes in the distribution pattern of cocaine- and amphetamine-regulated transcript (CART) in the enteric nervous system (ENS) of the human colon challenged by adenocarcinoma invasion, using the double-labelling immunofluorescence technique. In control specimens, CART immunoreactivity was found in neurons
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The present study analysed changes in the distribution pattern of cocaine- and amphetamine-regulated transcript (CART) in the enteric nervous system (ENS) of the human colon challenged by adenocarcinoma invasion, using the double-labelling immunofluorescence technique. In control specimens, CART immunoreactivity was found in neurons of all studied plexuses, representing 30.1 ± 4.1%, 12.9 ± 5.2%, and 4.1 ± 1.3% of all neurons forming the myenteric plexus (MP), outer submucous plexus (OSP), and inner submucous plexus (ISP), respectively. Tumour growth into the colon wall caused an increase in the relative frequency of CART-like immunoreactive (CART-LI) neurons in enteric plexuses located in the vicinity of the infiltrating neoplasm (to 36.1 ± 6.7%, 32.7 ± 7.3% and 12.1 ± 3.8% of all neurons in MP, OSP and ISP, respectively). The density of CART-LI nerves within particular layers of the intestinal wall did not differ between control and adenocarcinoma-affected areas of the human colon. This is the first detailed description of the CART distribution pattern within the ENS during the adenocarcinoma invasion of the human colon wall. The obtained results suggest that CART probably acts as a neuroprotective factor and may be involved in neuronal plasticity evoked by the progression of a neoplastic process. Full article
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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Open AccessArticle A Pathological Study of Acute Pulmonary Toxicity Induced by Inhaled Kanto Loam Powder
Int. J. Mol. Sci. 2018, 19(2), 416; doi:10.3390/ijms19020416
Received: 15 January 2018 / Revised: 23 January 2018 / Accepted: 30 January 2018 / Published: 31 January 2018
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Abstract
The frequency and volume of Asian sand dust (ASD) (Kosa) are increasing in Japan, and it has been reported that ASD may cause adverse respiratory effects. The pulmonary toxicity of ASD has been previously analyzed in mice exposed to ASD particles by intratracheal
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The frequency and volume of Asian sand dust (ASD) (Kosa) are increasing in Japan, and it has been reported that ASD may cause adverse respiratory effects. The pulmonary toxicity of ASD has been previously analyzed in mice exposed to ASD particles by intratracheal instillation. To study the pulmonary toxicity induced by inhalation of ASD, ICR mice were exposed by inhalation to 50 or 200 mg/m3 Kanto loam powder, which resembles ASD in elemental composition and particle size, for 6 h a day over 1, 3, 6, 9, or 15 consecutive days. Histological examination revealed that Kanto loam powder induced acute inflammation in the whole lung at all the time points examined. The lesions were characterized by infiltration of neutrophils and macrophages. The intensity of the inflammatory changes in the lung and number of neutrophils in both histological lesions and bronchoalveolar lavage fluid (BALF) appeared to increase over time. Immunohistochemical staining showed interleukin (IL)-6- and tumor necrosis factor (TNF)-α-positive macrophages and a decrease in laminin positivity in the inflammatory lesions of the lung tissues. Electron microscopy revealed vacuolar degeneration in the alveolar epithelial cells close to the Kanto loam particles. The nitric oxide level in the BALF increased over time. These results suggest that inhaled Kanto loam powder may induce diffuse and acute pulmonary inflammation, which is associated with increased expression of inflammatory cytokines and oxidative stress. Full article
(This article belongs to the Section Molecular Toxicology)
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Open AccessArticle Synthesis of a Cleaved Form of Osteopontin by THP-1 Cells and Its Alteration by Phorbol 12-Myristate 13-Acetate and BCG Infection
Int. J. Mol. Sci. 2018, 19(2), 418; doi:10.3390/ijms19020418
Received: 6 January 2018 / Revised: 26 January 2018 / Accepted: 26 January 2018 / Published: 31 January 2018
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Abstract
The protease-cleaved osteopontin (OPN) was proposed to enhance the migration of memory T cells to granulomas in tuberculosis. Various forms of OPN were identified in human monocytic THP-1 cells stimulated by phorbol 12-myristate 13-acetate (PMA). Antibodies O-17, 10A16 and 34E3, which recognize N-terminus,
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The protease-cleaved osteopontin (OPN) was proposed to enhance the migration of memory T cells to granulomas in tuberculosis. Various forms of OPN were identified in human monocytic THP-1 cells stimulated by phorbol 12-myristate 13-acetate (PMA). Antibodies O-17, 10A16 and 34E3, which recognize N-terminus, the C-half, and thrombin-cleaved site of OPN, respectively, all detected distinct bands on Western blots following PMA stimulation. Bands corresponding to 18 and 30 kD were detected by antibodies 34E3 and 10A16, indicating that OPN cleavage occurred by endogenous proteases in the PMA-stimulated THP-1 cells. In immune-fluorescence (IF) assay, 34E3 positive signals were detected in intracellular space of non-infected and bacillus Calmette-Guérin (BCG)-infected cells; however, 10A16 positive signals were confirmed in extracellular area in PMA-stimulated cells followed by BCG infection. Small amounts of full-length (FL) and thrombin-cleaved (Tr) OPN were detected by ELISA in the supernatants of non-PMA-stimulated cells, and increased levels of all forms, including undefined (Ud) OPN, in PMA-stimulated cells. ELISA showed a decrease in OPN synthesis during BCG infection. To our knowledge, this is the first report of OPN cleavage in THP-1 macrophages after PMA stimulation, and of enhanced cleavage induced by BCG infection. Full article
(This article belongs to the Special Issue Molecular Mechanism of Infectious Disease)
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Open AccessArticle HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes
Int. J. Mol. Sci. 2018, 19(2), 419; doi:10.3390/ijms19020419
Received: 14 December 2017 / Revised: 23 January 2018 / Accepted: 29 January 2018 / Published: 31 January 2018
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Abstract
SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can
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SERCA2a is the Ca2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency. Full article
(This article belongs to the Special Issue Calcium Signaling in Human Health and Diseases)
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Open AccessArticle Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies
Int. J. Mol. Sci. 2018, 19(2), 421; doi:10.3390/ijms19020421
Received: 2 December 2017 / Revised: 9 January 2018 / Accepted: 27 January 2018 / Published: 31 January 2018
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Abstract
N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment
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N-glycosylation profoundly affects the biological stability and function of therapeutic proteins, which explains the recent interest in glycoengineering technologies as methods to develop biobetter therapeutics. In current manufacturing processes, N-glycosylation is host-specific and remains difficult to control in a production environment that changes with scale and production batches leading to glycosylation heterogeneity and inconsistency. On the other hand, in vitro chemoenzymatic glycan remodeling has been successful in producing homogeneous pre-defined protein glycoforms, but needs to be combined with a cost-effective and scalable production method. An efficient chemoenzymatic glycan remodeling technology using a plant expression system that combines in vivo deglycosylation with an in vitro chemoenzymatic glycosylation is described. Using the monoclonal antibody rituximab as a model therapeutic protein, a uniform Gal2GlcNAc2Man3GlcNAc2 (A2G2) glycoform without α-1,6-fucose, plant-specific α-1,3-fucose or β-1,2-xylose residues was produced. When compared with the innovator product Rituxan®, the plant-made remodeled afucosylated antibody showed similar binding affinity to the CD20 antigen but significantly enhanced cell cytotoxicity in vitro. Using a scalable plant expression system and reducing the in vitro deglycosylation burden creates the potential to eliminate glycan heterogeneity and provide affordable customization of therapeutics’ glycosylation for maximal and targeted biological activity. This feature can reduce cost and provide an affordable platform to manufacture biobetter antibodies. Full article
(This article belongs to the Special Issue Recombinant Proteins)
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Open AccessArticle Cigarette Smoking Promotes Infection of Cervical Cells by High-Risk Human Papillomaviruses, but not Subsequent E7 Oncoprotein Expression
Int. J. Mol. Sci. 2018, 19(2), 422; doi:10.3390/ijms19020422
Received: 5 January 2018 / Revised: 28 January 2018 / Accepted: 29 January 2018 / Published: 31 January 2018
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Abstract
Persistent cervical infection with high-risk human papillomaviruses (hrHPVs) is a necessary, but not sufficient, condition for the development of cervical cancer. Therefore, there are other co-factors facilitating the hrHPV carcinogenic process, one of which is smoking. To assess the effect of smoking on
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Persistent cervical infection with high-risk human papillomaviruses (hrHPVs) is a necessary, but not sufficient, condition for the development of cervical cancer. Therefore, there are other co-factors facilitating the hrHPV carcinogenic process, one of which is smoking. To assess the effect of smoking on high-risk (hr) HPV DNA positivity and on the expression of HPV E7 oncoprotein, as a surrogate of persistent hrHPV infection, we used data from women recruited for the PIPAVIR project, which examined the role of E7 protein detection in cervical cancer screening. Women were tested for hrHPV DNA, using Multiplex Genotyping (MPG), and E7 protein, using a novel sandwich ELISA method, and gave information on their smoking habits. Among 1473 women, hrHPV prevalence was 19.1%. The odds ratio (OR) for hrHPV positivity of smokers compared to non-smokers was 1.785 (95% confidence intervals (CI): 1.365–2.332, p < 0.001). The ORs for E7 positivity, concerning hrHPV positive women, ranged from 0.720 to 1.360 depending on the E7 detection assay used, but this was not statistically significant. Smoking increases the probability of hrHPV infection, and smoking intensity is positively associated to this increase. Smoking is not related to an increased probability of E7 protein positivity for hrHPV positive women. Full article
(This article belongs to the Special Issue Human Polyomaviruses and Papillomaviruses)
Open AccessArticle Enhanced Thermostability of Glucose Oxidase through Computer-Aided Molecular Design
Int. J. Mol. Sci. 2018, 19(2), 425; doi:10.3390/ijms19020425
Received: 5 November 2017 / Revised: 6 December 2017 / Accepted: 26 January 2018 / Published: 31 January 2018
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Abstract
Glucose oxidase (GOD, EC.1.1.3.4) specifically catalyzes the reaction of β-d-glucose to gluconic acid and hydrogen peroxide in the presence of oxygen, which has become widely used in the food industry, gluconic acid production and the feed industry. However, the poor thermostability
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Glucose oxidase (GOD, EC.1.1.3.4) specifically catalyzes the reaction of β-d-glucose to gluconic acid and hydrogen peroxide in the presence of oxygen, which has become widely used in the food industry, gluconic acid production and the feed industry. However, the poor thermostability of the current commercial GOD is a key limiting factor preventing its widespread application. In the present study, amino acids closely related to the thermostability of glucose oxidase from Penicillium notatum were predicted with a computer-aided molecular simulation analysis, and mutant libraries were established following a saturation mutagenesis strategy. Two mutants with significantly improved thermostabilities, S100A and D408W, were subsequently obtained. Their protein denaturing temperatures were enhanced by about 4.4 °C and 1.2 °C, respectively, compared with the wild-type enzyme. Treated at 55 °C for 3 h, the residual activities of the mutants were greater than 72%, while that of the wild-type enzyme was only 20%. The half-lives of S100A and D408W were 5.13- and 4.41-fold greater, respectively, than that of the wild-type enzyme at the same temperature. This work provides novel and efficient approaches for enhancing the thermostability of GOD by reducing the protein free unfolding energy or increasing the interaction of amino acids with the coenzyme. Full article
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Open AccessArticle Preventive Effects of Resveratrol on Endocannabinoid System and Synaptic Protein Modifications in Rat Cerebral Cortex Challenged by Bilateral Common Carotid Artery Occlusion and Reperfusion
Int. J. Mol. Sci. 2018, 19(2), 426; doi:10.3390/ijms19020426
Received: 27 December 2017 / Revised: 18 January 2018 / Accepted: 19 January 2018 / Published: 31 January 2018
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Abstract
This study aims to evaluate the putative roles of a single acute dose of resveratrol (RVT) in preventing cerebral oxidative stress induced by bilateral common carotid artery occlusion, followed by reperfusion (BCCAO/R) and to investigate RVT’s ability to preserve the neuronal structural integrity.
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This study aims to evaluate the putative roles of a single acute dose of resveratrol (RVT) in preventing cerebral oxidative stress induced by bilateral common carotid artery occlusion, followed by reperfusion (BCCAO/R) and to investigate RVT’s ability to preserve the neuronal structural integrity. Frontal and temporal-occipital cortices were examined in two groups of adult Wistar rats, sham-operated and submitted to BCCAO/R. In both groups, 6 h before surgery, half the rats were gavage-fed with a single dose of RVT (40 mg/per rat in 300 µL of sunflower oil as the vehicle), while the second half received the vehicle alone. In the frontal cortex, RVT pre-treatment prevented the BCCAO/R-induced increase of lipoperoxides, augmented concentrations of palmitoylethanolamide and docosahexaenoic acid, increased relative levels of the cannabinoid receptors type 1 (CB1) and 2 (CB2), and peroxisome-proliferator-activated-receptor (PPAR)-α proteins. Increased expression of CB1/CB2 receptors mirrored that of synaptophysin and post-synaptic density-95 protein. No BCCAO/R-induced changes occurred in the temporal-occipital cortex. Collectively, our results demonstrate that, in the frontal cortex, RVT pre-treatment prevents the BCCAO/R-induced oxidative stress and modulates the endocannabinoid and PPAR-α systems. The increased expression of synaptic structural proteins further suggests the possible efficacy of RVT as a dietary supplement to preserve the nervous tissue metabolism and control the physiological response to the hypoperfusion/reperfusion challenge. Full article
(This article belongs to the Special Issue The Beneficial Effects of Plant Oil on Human Health)
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Open AccessArticle Amorphous, Smart, and Bioinspired Polyphosphate Nano/Microparticles: A Biomaterial for Regeneration and Repair of Osteo-Articular Impairments In-Situ
Int. J. Mol. Sci. 2018, 19(2), 427; doi:10.3390/ijms19020427
Received: 23 November 2017 / Revised: 27 January 2018 / Accepted: 29 January 2018 / Published: 31 January 2018
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Abstract
Using femur explants from mice as an in vitro model, we investigated the effect of the physiological polymer, inorganic polyphosphate (polyP), on differentiation of the cells of the bone marrow in their natural microenvironment into the osteogenic and chondrogenic lineages. In the form
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Using femur explants from mice as an in vitro model, we investigated the effect of the physiological polymer, inorganic polyphosphate (polyP), on differentiation of the cells of the bone marrow in their natural microenvironment into the osteogenic and chondrogenic lineages. In the form of amorphous Ca-polyP nano/microparticles, polyP retains its function to act as both an intra- and extracellular metabolic fuel and a stimulus eliciting morphogenetic signals. The method for synthesis of the nano/microparticles with the polyanionic polyP also allowed the fabrication of hybrid particles with the bisphosphonate zoledronic acid, a drug used in therapy of bone metastases in cancer patients. The results revealed that the amorphous Ca-polyP particles promote the growth/viability of mesenchymal stem cells, as well as the osteogenic and chondrogenic differentiation of the bone marrow cells in rat femur explants, as revealed by an upregulation of the expression of the transcription factors SOX9 (differentiation towards osteoblasts) and RUNX2 (chondrocyte differentiation). In parallel to this bone anabolic effect, incubation of the femur explants with these particles significantly reduced the expression of the gene encoding the osteoclast bone-catabolic enzyme, cathepsin-K, while the expression of the tartrate-resistant acid phosphatase remained unaffected. The gene expression data were supported by the finding of an increased mineralization of the cells in the femur explants in response to the Ca-polyP particles. Finally, we show that the hybrid particles of polyP complexed with zoledronic acid exhibit both the cytotoxic effect of the bisphosphonate and the morphogenetic and mineralization inducing activity of polyP. Our results suggest that the Ca-polyP nano/microparticles are not only a promising scaffold material for repairing long bone osteo-articular damages but can also be applied, as a hybrid with zoledronic acid, as a drug delivery system for treatment of bone metastases. The polyP particles are highlighted as genuine, smart, bioinspired nano/micro biomaterials. Full article
(This article belongs to the collection Bioactive Nanoparticles)
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Open AccessArticle Protic Ionic Liquids for Lignin Extraction—A Lignin Characterization Study
Int. J. Mol. Sci. 2018, 19(2), 428; doi:10.3390/ijms19020428
Received: 23 October 2017 / Revised: 20 November 2017 / Accepted: 27 November 2017 / Published: 31 January 2018
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Abstract
Protic ionic liquids (PILs) have been established as effective solvents for the selective extraction and recovery of lignin from lignocellulosic biomass. In this study, we utilize extensive analytical techniques to characterize the PIL-extracted lignins to (1) expand on the physical/chemical structure, and to
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Protic ionic liquids (PILs) have been established as effective solvents for the selective extraction and recovery of lignin from lignocellulosic biomass. In this study, we utilize extensive analytical techniques to characterize the PIL-extracted lignins to (1) expand on the physical/chemical structure, and to (2) develop a better understanding of the mechanism behind the lignin dissolution process. The PIL-lignins were characterized using elemental and FT-IR analyses, alongside molecular weight distribution and chemical modeling via MM2. For the more ionic pyrrolidinium acetate ([Pyrr][Ac]), there is an increase in the fragmentation of lignin, resulting in lignin with a smaller average molecular weight and a more uniform dispersity. This lends better understanding to previous findings indicating that higher ionicity in a PIL leads to increased lignin extraction. Full article
(This article belongs to the Special Issue The Lignin Challenge: Exploring Innovative Applications)
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Open AccessArticle A Phenotyping Method of Giant Cells from Root-Knot Nematode Feeding Sites by Confocal Microscopy Highlights a Role for CHITINASE-LIKE 1 in Arabidopsis
Int. J. Mol. Sci. 2018, 19(2), 429; doi:10.3390/ijms19020429
Received: 21 December 2017 / Revised: 19 January 2018 / Accepted: 26 January 2018 / Published: 1 February 2018
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Abstract
Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls
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Most effective nematicides for the control of root-knot nematodes are banned, which demands a better understanding of the plant-nematode interaction. Understanding how gene expression in the nematode-feeding sites relates to morphological features may assist a better characterization of the interaction. However, nematode-induced galls resulting from cell-proliferation and hypertrophy hinders such observation, which would require tissue sectioning or clearing. We demonstrate that a method based on the green auto-fluorescence produced by glutaraldehyde and the tissue-clearing properties of benzyl-alcohol/benzyl-benzoate preserves the structure of the nematode-feeding sites and the plant-nematode interface with unprecedented resolution quality. This allowed us to obtain detailed measurements of the giant cells’ area in an Arabidopsis line overexpressing CHITINASE-LIKE-1 (CTL1) from optical sections by confocal microscopy, assigning a role for CTL1 and adding essential data to the scarce information of the role of gene repression in giant cells. Furthermore, subcellular structures and features of the nematodes body and tissues from thick organs formed after different biotic interactions, i.e., galls, syncytia, and nodules, were clearly distinguished without embedding or sectioning in different plant species (Arabidopsis, cucumber or Medicago). The combination of this method with molecular studies will be valuable for a better understanding of the plant-biotic interactions. Full article
(This article belongs to the Special Issue Plant Defense Genes Against Biotic Stresses)
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Open AccessArticle Analysis of Transcriptional Responses of the Inflorescence Meristems in Jatropha curcas Following Gibberellin Treatment
Int. J. Mol. Sci. 2018, 19(2), 432; doi:10.3390/ijms19020432
Received: 27 December 2017 / Revised: 16 January 2018 / Accepted: 25 January 2018 / Published: 1 February 2018
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Abstract
Jatropha curcas L. seeds an oilseed plant with great potential for biodiesel production. However, low seed yield, which was limited by its lower female flowers, was a major drawback for its utilization. Our previous study found that the flower number and female-to-male ratio
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Jatropha curcas L. seeds an oilseed plant with great potential for biodiesel production. However, low seed yield, which was limited by its lower female flowers, was a major drawback for its utilization. Our previous study found that the flower number and female-to-male ratio were increased by gibberellin treatment. Here, we compared the transcriptomic profiles of inflorescence meristem at different time points after gibberellic acid A3 (GA3) treatment. The present study showed that 951 differentially expressed genes were obtained in response to gibberellin treatment, compared with control samples. The 6-h time point was an important phase in the response to exogenous gibberellin. Furthermore, the plant endogenous gibberellin, auxin, ethylene, abscisic acid, and brassinolide-signaling transduction pathways were repressed, whereas the genes associated with cytokinin and jasmonic acid signaling were upregulated for 24-h time point following GA3 treatment. In addition, the floral meristem determinacy genes (JcLFY, JcSOC1) and floral organ identity genes (JcAP3, JcPI, JcSEP1-3) were significantly upregulated, but their negative regulator (JcSVP) was downregulated after GA3 treatment. Moreover, the effects of phytohormone, which was induced by exogenous plant growth regulator, mainly acted on the female floral differentiation process. To the best of our knowledge, this data is the first comprehensive analysis of the underlying transcriptional response mechanism of floral differentiation following GA3 treatment in J. curcas, which helps in engineering high-yielding varieties of Jatropha. Full article
(This article belongs to the Section Molecular Botany)
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Open AccessArticle Variation on Molecular Structure, Crystallinity, and Optical Properties of Dentin Due to Nd:YAG Laser and Fluoride Aimed at Tooth Erosion Prevention
Int. J. Mol. Sci. 2018, 19(2), 433; doi:10.3390/ijms19020433
Received: 29 December 2017 / Revised: 25 January 2018 / Accepted: 26 January 2018 / Published: 1 February 2018
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Abstract
This in vitro study evaluated the compositional, crystalline, and morphological effects promoted by Nd:YAG laser on root dentin, and verified the effects of laser and topical acidulated phosphate fluoride application (APF-gel) on dentin erosion. 180 bovine dentin slabs were randomized into 4 groups
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This in vitro study evaluated the compositional, crystalline, and morphological effects promoted by Nd:YAG laser on root dentin, and verified the effects of laser and topical acidulated phosphate fluoride application (APF-gel) on dentin erosion. 180 bovine dentin slabs were randomized into 4 groups (n = 45): G1–untreated, G2–APF-gel (1.23% F, 4 min), G3–Nd:YAG (1064 nm, 84.9 J/cm2, 10 Hz), and G4–APF-gel application followed by Nd:YAG laser irradiation. The compositional, crystalline, and morphological effects promoted by treatments were investigated on five samples of each experimental group. The other samples were submitted to a 5-day, 10-day, or 15-day erosive and abrasive demineralization and remineralization cycling in order to create erosion lesions. The area and depth of lesions, as well as the optical attenuation coefficient, were assessed, and all data were statistically analysed (p < 0.05). Nd:YAG laser promoted the reduction of carbonate, the formation of tetracalcium phosphate, as well as the melting and recrystallization of the dentin surface. Laser significantly decreased the area and depth of erosion lesions and altered the optical attenuation coefficient when compared to untreated and APF-gel groups, but the association of APF-gel and laser did not promote an additional effect. Nd:YAG laser irradiation can be a promissory treatment to prevent dentin erosion and the abrasion process. Full article
(This article belongs to the Special Issue Laser Application in Life Sciences 2017)
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Open AccessArticle Photodynamic Inactivation Potentiates the Susceptibility of Antifungal Agents against the Planktonic and Biofilm Cells of Candida albicans
Int. J. Mol. Sci. 2018, 19(2), 434; doi:10.3390/ijms19020434
Received: 20 December 2017 / Revised: 23 January 2018 / Accepted: 29 January 2018 / Published: 1 February 2018
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Abstract
Photodynamic inactivation (PDI) has been shown to be a potential treatment modality against Candida infection. However, limited light penetration might leave some cells alive and undergoing regrowth. In this study, we explored the possibility of combining PDI and antifungal agents to enhance the
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Photodynamic inactivation (PDI) has been shown to be a potential treatment modality against Candida infection. However, limited light penetration might leave some cells alive and undergoing regrowth. In this study, we explored the possibility of combining PDI and antifungal agents to enhance the therapeutic efficacy of Candida albicans and drug-resistant clinical isolates. We found that planktonic cells that had survived toluidine blue O (TBO)-mediated PDI were significantly susceptible to fluconazole within the first 2 h post PDI. Following PDI, the killing efficacy of antifungal agents relates to the PDI dose in wild-type and drug-resistant clinical isolates. However, only a 3-log reduction was found in the biofilm cells, suggesting limited therapeutic efficacy under the combined treatment of PDI and azole antifungal drugs. Using confocal microscopic analysis, we showed that TBO-mediated PDI could partially remove the extracellular polymeric substance (EPS) of biofilm. Finally, we showed that a combination of PDI with caspofungin could result in the complete killing of biofilms compared to those treated with caspofungin or PDI alone. These results clearly indicate that the combination of PDI and antifungal agents could be a promising treatment against C. albicans infections. Full article
(This article belongs to the Special Issue Laser Application in Life Sciences 2017)
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Open AccessArticle Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index
Int. J. Mol. Sci. 2018, 19(2), 435; doi:10.3390/ijms19020435
Received: 13 December 2017 / Revised: 26 January 2018 / Accepted: 30 January 2018 / Published: 1 February 2018
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Abstract
Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss
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Articular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Similar musculoskeletal disorders also affect horses and OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-β1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine. Full article
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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Open AccessArticle Adsorption Behavior of Surfactant on Lignite Surface: A Comparative Experimental and Molecular Dynamics Simulation Study
Int. J. Mol. Sci. 2018, 19(2), 437; doi:10.3390/ijms19020437
Received: 9 November 2017 / Revised: 17 January 2018 / Accepted: 30 January 2018 / Published: 1 February 2018
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Abstract
Experimental and computational simulation methods are used to investigate the adsorption behavior of the surfactant nonylphenol ethoxylate (NPEO10), which contains 10 ethylene oxide groups, on the lignite surface. The adsorption of NPEO10 on lignite follow a Langmuir-type isotherm. The thermodynamic parameters of the
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Experimental and computational simulation methods are used to investigate the adsorption behavior of the surfactant nonylphenol ethoxylate (NPEO10), which contains 10 ethylene oxide groups, on the lignite surface. The adsorption of NPEO10 on lignite follow a Langmuir-type isotherm. The thermodynamic parameters of the adsorption process show that the whole process is spontaneous. X-ray photoelectron spectroscopic (XPS) analysis indicates that a significant fraction of the oxygen-containing functional groups on the lignitic surface were covered by NPEO10. Molecular dynamics (MD) simulations show that the NPEO10 molecules were found to adsorb at the water-coal interface. Moreover, polar interactions are the main effect in the adsorption process. The density distributions of coal, NPEO10, and water molecules along the Z axis show that the remaining hydrophobic portions of the surfactant extend into the solution, creating a more hydrophobic coal surface that repels water molecules. The negative interaction energy calculated from the density profiles of the head and tail groups along the three spatial directions between the surfactant and the lignitic surface suggest that the adsorption process is spontaneous. The self-diffusion coefficients show that the presence of NPEO10 causes higher water mobility by improving the hydrophobicity of lignite. Full article
(This article belongs to the Section Molecular Biophysics)
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Open AccessArticle Rings and Bricks: Expression of Cohesin Components is Dynamic during Development and Adult Life
Int. J. Mol. Sci. 2018, 19(2), 438; doi:10.3390/ijms19020438
Received: 11 January 2018 / Revised: 26 January 2018 / Accepted: 28 January 2018 / Published: 1 February 2018
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Abstract
Cohesin complex components exert fundamental roles in animal cells, both canonical in cell cycle and non-canonical in gene expression regulation. Germline mutations in genes coding for cohesins result in developmental disorders named cohesinopaties, of which Cornelia de Lange syndrome (CdLS) is the best-known
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Cohesin complex components exert fundamental roles in animal cells, both canonical in cell cycle and non-canonical in gene expression regulation. Germline mutations in genes coding for cohesins result in developmental disorders named cohesinopaties, of which Cornelia de Lange syndrome (CdLS) is the best-known entity. However, a basic description of mammalian expression pattern of cohesins in a physiologic condition is still needed. Hence, we report a detailed analysis of expression in murine and human tissues of cohesin genes defective in CdLS. Using both quantitative and qualitative methods in fetal and adult tissues, cohesin genes were found to be ubiquitously and differentially expressed in human tissues. In particular, abundant expression was observed in hematopoietic and central nervous system organs. Findings of the present study indicate tissues which should be particularly sensitive to mutations, germline and/or somatic, in cohesin genes. Hence, this expression analysis in physiological conditions may represent a first core reference for cohesinopathies. Full article
(This article belongs to the Special Issue Rare Diseases: Molecular Mechanisms and Therapeutic Strategies)
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Open AccessArticle QTL Mapping of Fiber Quality and Yield-Related Traits in an Intra-Specific Upland Cotton Using Genotype by Sequencing (GBS)
Int. J. Mol. Sci. 2018, 19(2), 441; doi:10.3390/ijms19020441
Received: 20 December 2017 / Revised: 12 January 2018 / Accepted: 28 January 2018 / Published: 1 February 2018
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Abstract
Fiber quality and yield improvement are crucial for cotton domestication and breeding. With the transformation in spinning techniques and multiplicity needs, the development of cotton fiber quality and yield is of great importance. A genetic map of 5178 Single Nucleotide Polymorphism (SNP) markers
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Fiber quality and yield improvement are crucial for cotton domestication and breeding. With the transformation in spinning techniques and multiplicity needs, the development of cotton fiber quality and yield is of great importance. A genetic map of 5178 Single Nucleotide Polymorphism (SNP) markers were generated using 277 F2:3 population, from an intra-specific cross between two upland cotton accessions, CCRI35 a high fiber quality as female and Nan Dan Ba Di Da Hua (NH), with good yield properties as male parent. The map spanned 4768.098 cM with an average distance of 0.92 cM. A total of 110 Quantitative Traits Loci (QTLs) were identified for 11 traits, but only 30 QTLs were consistent in at least two environments. The highest percentage of phenotypic variance explained by a single QTL was 15.45%. Two major cluster regions were found, cluster 1 (chromosome17-D03) and cluster 2 (chromosome26-D12). Five candidate genes were identified in the two QTL cluster regions. Based on GO functional annotation, all the genes were highly correlated with fiber development, with functions such as protein kinase and phosphorylation. The five genes were associated with various fiber traits as follows: Gh_D03G0889 linked to qFM-D03_cb, Gh_D12G0093, Gh_D12G0410, Gh_D12G0435 associated with qFS-D12_cb and Gh_D12G0969 linked to qFY-D12_cb. Further structural annotation and fine mapping is needed to determine the specific role played by the five identified genes in fiber quality and yield related pathway. Full article
(This article belongs to the Section Molecular Botany)
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Open AccessArticle Ablation of the Right Cardiac Vagus Nerve Reduces Acetylcholine Content without Changing the Inflammatory Response during Endotoxemia
Int. J. Mol. Sci. 2018, 19(2), 442; doi:10.3390/ijms19020442
Received: 12 December 2017 / Revised: 23 January 2018 / Accepted: 26 January 2018 / Published: 1 February 2018
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Abstract
Acetylcholine is the main transmitter of the parasympathetic vagus nerve. According to the cholinergic anti-inflammatory pathway (CAP) concept, acetylcholine has been shown to be important for signal transmission within the immune system and also for a variety of other functions throughout the organism.
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Acetylcholine is the main transmitter of the parasympathetic vagus nerve. According to the cholinergic anti-inflammatory pathway (CAP) concept, acetylcholine has been shown to be important for signal transmission within the immune system and also for a variety of other functions throughout the organism. The spleen is thought to play an important role in regulating the CAP. In contrast, the existence of a “non-neuronal cardiac cholinergic system” that influences cardiac innervation during inflammation has been hypothesized, with recent publications introducing the heart instead of the spleen as a possible interface between the immune and nervous systems. To prove this hypothesis, we investigated whether selectively disrupting vagal stimulation of the right ventricle plays an important role in rat CAP regulation during endotoxemia. We performed a selective resection of the right cardiac branch of the Nervus vagus (VGX) with a corresponding sham resection in vehicle-injected and endotoxemic rats. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg body weight, intravenously) and observed for 4 h. Intraoperative blood gas analysis was performed, and hemodynamic parameters were assessed using a left ventricular pressure-volume catheter. Rat hearts and blood were collected, and the expression and concentration of proinflammatory cytokines using quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were measured, respectively. Four hours after injection, LPS induced a marked deterioration in rat blood gas parameters such as pH value, potassium, base excess, glucose, and lactate. The mean arterial blood pressure and the end-diastolic volume had decreased significantly. Further, significant increases in blood cholinesterases and in proinflammatory (IL-1β, IL-6, TNF-α) cytokine concentration and gene expression were obtained. Right cardiac vagus nerve resection (VGX) led to a marked decrease in heart acetylcholine concentration and an increase in cardiac acetylcholinesterase activity. Without LPS, VGX changed rat hemodynamic parameters, including heart frequency, cardiac output, and end-diastolic volume. In contrast, VGX during endotoxemia did not significantly change the concentration and expression of proinflammatory cytokines in the heart. In conclusion we demonstrate that right cardiac vagal innervation regulates cardiac acetylcholine content but neither improves nor worsens systemic inflammation. Full article
(This article belongs to the Section Molecular Toxicology)
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