Open AccessReview
The Role of the Estrogen Pathway in the Tumor Microenvironment
Int. J. Mol. Sci. 2018, 19(2), 611; doi:10.3390/ijms19020611 (registering DOI) -
Abstract
Estrogen receptors are broadly expressed in many cell types involved in the innate and adaptive immune responses, and differentially regulate the production of cytokines. While both genomic and non-genomic tumor cell promoting mechanisms of estrogen signaling are well characterized in multiple carcinomas including
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Estrogen receptors are broadly expressed in many cell types involved in the innate and adaptive immune responses, and differentially regulate the production of cytokines. While both genomic and non-genomic tumor cell promoting mechanisms of estrogen signaling are well characterized in multiple carcinomas including breast, ovarian, and lung, recent investigations have identified a potential immune regulatory role of estrogens in the tumor microenvironment. Tumor immune tolerance is a well-established mediator of oncogenesis, with increasing evidence indicating the importance of the immune response in tumor progression. Immune-based therapies such as antibodies that block checkpoint signals have emerged as exciting therapeutic approaches for cancer treatment, offering durable remissions and prolonged survival. However, only a subset of patients demonstrate clinical response to these agents, prompting efforts to elucidate additional immunosuppressive mechanisms within the tumor microenvironment. Evidence drawn from multiple cancer types, including carcinomas traditionally classified as non-immunogenic, implicate estrogen as a potential mediator of immunosuppression through modulation of protumor responses independent of direct activity on tumor cells. Herein, we review the interplay between estrogen and the tumor microenvironment and the clinical implications of endocrine therapy as a novel treatment strategy within immuno-oncology. Full article
Open AccessArticle
Mathematical Modelling of Nitric Oxide/Cyclic GMP/Cyclic AMP Signalling in Platelets
Int. J. Mol. Sci. 2018, 19(2), 612; doi:10.3390/ijms19020612 (registering DOI) -
Abstract
Platelet activation contributes to normal haemostasis but also to pathologic conditions like stroke and cardiac infarction. Signalling by cGMP and cAMP inhibit platelet activation and are therefore attractive targets for thrombosis prevention. However, extensive cross-talk between the cGMP and cAMP signalling pathways in
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Platelet activation contributes to normal haemostasis but also to pathologic conditions like stroke and cardiac infarction. Signalling by cGMP and cAMP inhibit platelet activation and are therefore attractive targets for thrombosis prevention. However, extensive cross-talk between the cGMP and cAMP signalling pathways in multiple tissues complicates the selective targeting of their activities. We have used mathematical modelling based on experimental data from the literature to quantify the steady state behaviour of nitric oxide (NO)/cGMP/cAMP signalling in platelets. The analysis provides an assessment of NO-induced cGMP synthesis and PKG activation as well as cGMP-mediated cAMP and PKA activation though modulation of phosphodiesterase (PDE2 and 3) activities. Both one- and two-compartment models of platelet cyclic nucleotide signalling are presented. The models provide new insight for understanding how NO signalling to cGMP and indirectly cAMP, can inhibit platelet shape-change, the initial step of platelet activation. Only the two-compartment models could account for the experimental observation that NO-mediated PKA activation can occur when the bulk platelet cAMP level is unchanged. The models revealed also a potential for hierarchical interplay between the different platelet phosphodiesterases. Specifically, the models predict, unexpectedly, a strong effect of pharmacological inhibitors of cGMP-specific PDE5 on the cGMP/cAMP cross-talk. This may explain the successful use of weak PDE5-inhibitors, such as dipyridamole, in anti-platelet therapy. In conclusion, increased NO signalling or PDE5 inhibition are attractive ways of increasing cGMP-cAMP cross-talk selectively in platelets. Full article
Open AccessArticle
Exploring the History of Chloroplast Capture in Arabis Using Whole Chloroplast Genome Sequencing
Int. J. Mol. Sci. 2018, 19(2), 602; doi:10.3390/ijms19020602 (registering DOI) -
Abstract
Chloroplast capture occurs when the chloroplast of one plant species is introgressed into another plant species. The phylogenies of nuclear and chloroplast markers from East Asian Arabis species are incongruent, which indicates hybrid origin and shows chloroplast capture. In the present study, the
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Chloroplast capture occurs when the chloroplast of one plant species is introgressed into another plant species. The phylogenies of nuclear and chloroplast markers from East Asian Arabis species are incongruent, which indicates hybrid origin and shows chloroplast capture. In the present study, the complete chloroplast genomes of A. hirsuta, A. nipponica, and A. flagellosa were sequenced in order to analyze their divergence and their relationships. The chloroplast genomes of A. nipponica and A. flagellosa were similar, which indicates chloroplast replacement. If hybridization causing chloroplast capture occurred once, divergence between recipient species would be lower than between donor species. However, the chloroplast genomes of species with possible hybrid origins, A. nipponica and A. stelleri, differ at similar levels to possible maternal donor species A. flagellosa, which suggests that multiple hybridization events have occurred in their respective histories. The mitochondrial genomes exhibited similar patterns, while A. nipponica and A. flagellosa were more similar to each other than to A. hirsuta. This suggests that the two organellar genomes were co-transferred during the hybridization history of the East Asian Arabis species. Full article
Open AccessArticle
Overexpression of the Vitronectin V10 Subunit in Patients with Nonalcoholic Steatohepatitis: Implications for Noninvasive Diagnosis of NASH
Int. J. Mol. Sci. 2018, 19(2), 603; doi:10.3390/ijms19020603 (registering DOI) -
Abstract
Nonalcoholic steatohepatitis (NASH) is the critical stage of nonalcoholic fatty liver disease (NAFLD). The persistence of necroinflammatory lesions and fibrogenesis in NASH is the leading cause of liver cirrhosis and, ultimately, hepatocellular carcinoma. To date, the histological examination of liver biopsies, albeit invasive,
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Nonalcoholic steatohepatitis (NASH) is the critical stage of nonalcoholic fatty liver disease (NAFLD). The persistence of necroinflammatory lesions and fibrogenesis in NASH is the leading cause of liver cirrhosis and, ultimately, hepatocellular carcinoma. To date, the histological examination of liver biopsies, albeit invasive, remains the means to distinguish NASH from simple steatosis (NAFL). Therefore, a noninvasive diagnosis by serum biomarkers is eagerly needed. Here, by a proteomic approach, we analysed the soluble low-molecular-weight protein fragments flushed out from the liver tissue of NAFL and NASH patients. On the basis of the assumption that steatohepatitis leads to the remodelling of the liver extracellular matrix (ECM), NASH-specific fragments were in silico analysed for their involvement in the ECM molecular composition. The 10 kDa C-terminal fragment of the ECM protein vitronectin (VTN) was then selected as a promising circulating biomarker in discriminating NASH. The analysis of sera of patients provided these major findings: the circulating VTN fragment (i) is overexpressed in NASH patients and positively correlates with the NASH activity score (NAS); (ii) originates from the disulfide bond reduction between the V10 and the V65 subunits. In conclusion, V10 determination in the serum could represent a reliable tool for the noninvasive discrimination of NASH from simple steatosis. Full article
Open AccessArticle
The Connexin Mimetic Peptide Gap27 and Cx43-Knockdown Reveal Differential Roles for Connexin43 in Wound Closure Events in Skin Model Systems
Int. J. Mol. Sci. 2018, 19(2), 604; doi:10.3390/ijms19020604 (registering DOI) -
Abstract
In the epidermis, remodelling of Connexin43 is a key event in wound closure. However, controversy between the role of connexin channel and non-channel functions exist. We compared the impact of SiRNA targeted to Connexin43 and the connexin mimetic peptide Gap27 on scrape wound
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In the epidermis, remodelling of Connexin43 is a key event in wound closure. However, controversy between the role of connexin channel and non-channel functions exist. We compared the impact of SiRNA targeted to Connexin43 and the connexin mimetic peptide Gap27 on scrape wound closure rates and hemichannel signalling in adult keratinocytes (AK) and fibroblasts sourced from juvenile foreskin (JFF), human neonatal fibroblasts (HNDF) and adult dermal tissue (ADF). The impact of these agents, following 24 h exposure, on GJA1 (encoding Connexin43), Ki67 and TGF-β1 gene expression, and Connexin43 and pSmad3 protein expression levels, were examined by qPCR and Western Blot respectively. In all cell types Gap27 (100–100 μM) attenuated hemichannel activity. In AK and JFF cells, Gap27 (100 nM–100 μM) enhanced scrape wound closure rates by ~50% but did not influence movement in HNDF or ADF cells. In both JF and AK cells, exposure to Gap27 for 24 h reduced the level of Cx43 protein expression but did not affect the level in ADF and HNDF cells. Connexin43-SiRNA enhanced scrape wound closure in all the cell types under investigation. In HDNF and ADF, Connexin43-SiRNA enhanced cell proliferation rates, with enhanced proliferation also observed following exposure of HDNF to Gap27. By contrast, in JFF and AK cells no changes in proliferation occurred. In JFF cells, Connexin43-SiRNA enhanced TGF-β1 levels and in JFF and ADF cells both Connexin43-SiRNA and Gap27 enhanced pSmad3 protein expression levels. We conclude that Connexin43 signalling plays an important role in cell migration in keratinocytes and foreskin derived fibroblasts, however, different pathways are evoked and in dermal derived adult and neonatal fibroblasts, inhibition of Connexin43 signalling plays a more significant role in regulating cell proliferation than cell migration. Full article
Open AccessArticle
Nanoluciferase Reporter Gene System Directed by Tandemly Repeated Pseudo-Palindromic NFAT-Response Elements Facilitates Analysis of Biological Endpoint Effects of Cellular Ca2+ Mobilization
Int. J. Mol. Sci. 2018, 19(2), 605; doi:10.3390/ijms19020605 (registering DOI) -
Abstract
NFAT is a cytoplasm-localized hyper-phosphorylated transcription factor that is activated through dephosphorylation by calcineurin, a Ca2+/calmodulin-dependent phosphatase. A non-palindromic NFAT-response element (RE) found in the IL2 promoter region has been commonly used for a Ca2+-response reporter gene system, but
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NFAT is a cytoplasm-localized hyper-phosphorylated transcription factor that is activated through dephosphorylation by calcineurin, a Ca2+/calmodulin-dependent phosphatase. A non-palindromic NFAT-response element (RE) found in the IL2 promoter region has been commonly used for a Ca2+-response reporter gene system, but requirement of concomitant activation of AP-1 (Fos/Jun) often complicates the interpretation of obtained results. A new nanoluciferase (NanoLuc) reporter gene containing nine-tandem repeats of a pseudo-palindromic NFAT-RE located upstream of the IL8 promoter was designed to monitor Ca2+-induced transactivation activity of NFAT in human embryonic kidney (HEK) 293 cells by measuring luciferase activities of NanoLuc and co-expressed firefly luciferase for normalization. Ionomycin treatment enhanced the relative luciferase activity (RLA), which was suppressed by calcineurin inhibitors. HEK293 cells that stably express human STIM1 and Orai1, components of the store-operated calcium entry (SOCE) machinery, gave a much higher RLA by stimulation with thapsigargin, an inhibitor of sarcoplasmic/endoplamic reticulum Ca2+-ATPase (SERCA). HEK293 cells deficient in a penta-EF-hand Ca2+-binding protein ALG-2 showed a higher RLA value than the parental cells by stimulation with an acetylcholine receptor agonist carbachol. The novel reporter gene system is found to be useful for applications to cell signaling research to monitor biological endpoint effects of cellular Ca2+ mobilization. Full article
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Open AccessReview
Telomere Length Maintenance in Cancer: At the Crossroad between Telomerase and Alternative Lengthening of Telomeres (ALT)
Int. J. Mol. Sci. 2018, 19(2), 606; doi:10.3390/ijms19020606 (registering DOI) -
Abstract
Eukaryotic cells undergo continuous telomere shortening as a consequence of multiple rounds of replications. During tumorigenesis, cells have to acquire telomere DNA maintenance mechanisms (TMMs) in order to counteract telomere shortening, to preserve telomeres from DNA damage repair systems and to avoid telomere-mediated
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Eukaryotic cells undergo continuous telomere shortening as a consequence of multiple rounds of replications. During tumorigenesis, cells have to acquire telomere DNA maintenance mechanisms (TMMs) in order to counteract telomere shortening, to preserve telomeres from DNA damage repair systems and to avoid telomere-mediated senescence and/or apoptosis. For this reason, telomere maintenance is an essential step in cancer progression. Most human tumors maintain their telomeres expressing telomerase, whereas a lower but significant proportion activates the alternative lengthening of telomeres (ALT) pathway. However, evidence about the coexistence of ALT and telomerase has been found both in vivo in the same cancer populations and in vitro in engineered cellular models, making the distinction between telomerase- and ALT-positive tumors elusive. Indeed, after the development of drugs able to target telomerase, the capability for some cancer cells to escape death, switching from telomerase to ALT, was highlighted. Unfortunately, to date, the mechanism underlying the possible switching or the coexistence of telomerase and ALT within the same cell or populations is not completely understood and different factors could be involved. In recent years, different studies have tried to shed light on the complex regulation network that controls the transition between the two TMMs, suggesting a role for embryonic cancer origin, epigenetic modifications, and specific genes activation—both in vivo and in vitro. In this review, we examine recent findings about the cancer-associated differential activation of the two known TMMs and the possible factors implicated in this process. Furthermore, some studies on cancers are also described that did not display any TMM. Full article
Open AccessArticle
Nanomechanical Phenotype of Melanoma Cells Depends Solely on the Amount of Endogenous Pigment in the Cells
Int. J. Mol. Sci. 2018, 19(2), 607; doi:10.3390/ijms19020607 (registering DOI) -
Abstract
Cancer cells have unique nanomechanical properties, i.e., they behave as if they were elastic. This property of cancer cells is believed to be one of the main reasons for their facilitated ability to spread and metastasize. Thus, the so-called nanomechanical phenotype of cancer
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Cancer cells have unique nanomechanical properties, i.e., they behave as if they were elastic. This property of cancer cells is believed to be one of the main reasons for their facilitated ability to spread and metastasize. Thus, the so-called nanomechanical phenotype of cancer cells is viewed as an important indicator of the cells’ metastatic behavior. One of the most highly metastatic cancer cells are melanoma cells, which have a very unusual property: they can synthesize the pigment melanin in large amounts, becoming heavily pigmented. So far, the role of melanin in melanoma remains unclear, particularly the impact of the pigment on metastatic behavior of melanoma cells. Importantly, until recently the potential mechanical role of melanin in melanoma metastasis was completely ignored. In this work, we examined melanoma cells isolated from hamster tumors containing endogenous melanin pigment. Applying an array of advanced microscopy and spectroscopy techniques, we determined that melanin is the dominating factor responsible for the mechanical properties of melanoma cells. Our results indicate that the nanomechanical phenotype of melanoma cells may be a reliable marker of the cells’ metastatic behavior and point to the important mechanical role of melanin in the process of metastasis of melanoma. Full article
Open AccessArticle
Presence of TERT Promoter Mutations is a Secondary Event and Associates with Elongated Telomere Length in Myxoid Liposarcomas
Int. J. Mol. Sci. 2018, 19(2), 608; doi:10.3390/ijms19020608 (registering DOI) -
Abstract
The occurrence of TERT promoter mutations has been well described in soft tissue sarcomas (STS). However, the biological role of these mutations as well as their impact on telomere length in STS is still unclear. We analyzed 116 patient samples diagnosed with 22
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The occurrence of TERT promoter mutations has been well described in soft tissue sarcomas (STS). However, the biological role of these mutations as well as their impact on telomere length in STS is still unclear. We analyzed 116 patient samples diagnosed with 22 distinct histological subtypes of bone and STS for the occurrence of TERT promoter mutations by Sanger sequencing. We observed TERT promoter mutations at an overall frequency of 9.5% distributed over 7 different sarcoma subtypes. Except for one chondrosarcoma case harboring a C250T mutation, all other mutations were detected at location C228T. By far the far highest frequency of TERT promoter mutations was found in myxoid liposarcoma (MLS) (4 out of 9 cases studied, i.e., 44%). Assessment of telomere length from tumor biopsies revealed that TERT promoter-mutated MLSs had significantly fewer shortened telomeres in comparison to TERT wildtype MLSs. Based on the frequency of TERT promoter mutations and the elongated telomere length in mutated compared to wildtype MLS, we hypothesize that occurrence of TERT promoter mutations has a pivotal role in the disease progression as a secondary genetic event at a time when tumor cells face the need for telomere elongation to allow further proliferation. Full article
Open AccessArticle
The Significance of MMP-1 in EGFR-TKI–Resistant Lung Adenocarcinoma: Potential for Therapeutic Targeting
Int. J. Mol. Sci. 2018, 19(2), 609; doi:10.3390/ijms19020609 (registering DOI) -
Abstract
Epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI) resistance is one of the most important problems in lung cancer therapy. Lung adenocarcinoma with EGFR-TKI resistance was reported to have higher abilities of invasion and migration than cancers sensitive to EGFR-TKI, but the function of
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Epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI) resistance is one of the most important problems in lung cancer therapy. Lung adenocarcinoma with EGFR-TKI resistance was reported to have higher abilities of invasion and migration than cancers sensitive to EGFR-TKI, but the function of matrix metalloproteinases (MMPs) has not been explored in EGFR-TKI–resistant lung adenocarcinoma. This study aims to clarify the significance of MMP-1 in EGFR-TKI–resistant lung adenocarcinoma. From the results of in vitro studies of migration and invasion assays using EGFR-TKI–sensitive and –resistant cell lines and phosphorylation antibody arrays using EGF and rapamycin, we first demonstrate that overexpression of MMP-1, which might follow activation of a mammalian target of rapamycin (mTOR) pathway, plays an important role in the migration and invasion abilities of EGFR-TKI–resistant lung adenocarcinoma. Additionally, immunohistochemical studies using 89 cases of lung adenocarcinoma demonstrate that high expression of MMP-1 is significantly correlated with poor prognosis and factors such as smoking history and the subtype of invasive mucinous adenocarcinoma. These are consistent with the results of this in vitro study. To conclude, this study provides insights into the development of a possible alternative therapy manipulating MMP-1 and the mTOR signaling pathway in EGFR-TKI–resistant lung adenocarcinoma. Full article
Open AccessArticle
The Antioxidant Content and Protective Effect of Argan Oil and Syzygium aromaticum Essential Oil in Hydrogen Peroxide-Induced Biochemical and Histological Changes
Int. J. Mol. Sci. 2018, 19(2), 610; doi:10.3390/ijms19020610 (registering DOI) -
Abstract
Oxidative stress is an important etiology of chronic diseases and many studies have shown that natural products might alleviate oxidative stress-induced pathogenesis. The study aims to evaluate the effect of Argan oil and Syzygium aromaticum essential oil on hydrogen peroxide (H2O
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Oxidative stress is an important etiology of chronic diseases and many studies have shown that natural products might alleviate oxidative stress-induced pathogenesis. The study aims to evaluate the effect of Argan oil and Syzygium aromaticum essential oil on hydrogen peroxide (H2O2)-induced liver, brain and kidney tissue toxicity as well as biochemical changes in wistar rats. The antioxidant content of Argan oil and Syzygium aromaticum essential oil was studied with the use of gas chromatography. The animals received daily by gavage, for 21 days, either distilled water, Syzygium aromaticum essential oil, Argan oil, H2O2 alone, H2O2 and Syzygium aromaticum essential oil, or H2O2 and Argan oil. Blood samples were withdrawn on day 21 for the biochemical blood tests, and the kidney, liver and brain tissue samples were prepared for histopathology examination. The results showed that the content of antioxidant compounds in Syzygium aromaticum essential oil is higher than that found in Argan oil. H2O2 increased level of blood urea, liver enzymes, total cholesterol, Low Density Lipoprotein (LDL-C), Triglycerides (TG) and Very Low Density Lipoprotein (VLDL), and decreased the total protein, albumin and High Density Lipoprotein-cholesterol (HDL-C). There was no significant effect on blood electrolyte or serum creatinine. The histopathology examination demonstrated that H2O2 induces dilatation in the central vein, inflammation and binucleation in the liver, congestion and hemorrhage in the brain, and congestion in the kidney. The H2O2-induced histopathological and biochemical changes have been significantly alleviated by Syzygium aromaticum essential oil or Argan oil. It is concluded that the Argan oil and especially the mixture of Argan oil with Syzygium aromaticum essential oil can reduce the oxidative damage caused by H2O2, and this will pave the way to investigate the protective effects of these natural substances in the diseases attributed to the high oxidative stress. Full article
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Open AccessReview
Secreted and Tissue miRNAs as Diagnosis Biomarkers of Malignant Pleural Mesothelioma
Int. J. Mol. Sci. 2018, 19(2), 595; doi:10.3390/ijms19020595 (registering DOI) -
Abstract
Malignant pleural mesothelioma (MPM) is a rare but aggressive tumor that originates in the pleura, is diagnosed in advanced stages and has a poor prognosis. Accurate diagnosis of MPM is often difficult and complex, and the gold standard diagnosis test is based on
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Malignant pleural mesothelioma (MPM) is a rare but aggressive tumor that originates in the pleura, is diagnosed in advanced stages and has a poor prognosis. Accurate diagnosis of MPM is often difficult and complex, and the gold standard diagnosis test is based on qualitative analysis of markers in pleural tissue by immunohistochemical staining. Therefore, it is necessary to develop quantitative and non-subjective alternative diagnostic tools. MicroRNAs are non-coding RNAs that regulate essential cellular mechanisms at the post-transcriptional level. Recent evidence indicates that miRNA expression in tissue and body fluids is aberrant in various tumors, revealing miRNAs as promising diagnostic biomarkers. This review summarizes evidence regarding secreted and tissue miRNAs as biomarkers of MPM and the biological characteristics associated with their potential diagnostic value. In addition to studies regarding miRNAs with potential diagnostic value for MPM, studies that aimed to identify the miRNAs involved in molecular mechanisms associated with MPM development are described with an emphasis on relevant aspects of the experimental designs that may influence the accuracy, consistency and real diagnostic value of currently reported data. Full article
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Open AccessReview
The Biased G-Protein-Coupled Receptor Agonism Bridges the Gap between the Insulin Receptor and the Metabolic Syndrome
Int. J. Mol. Sci. 2018, 19(2), 575; doi:10.3390/ijms19020575 (registering DOI) -
Abstract
Insulin signaling, as mediated through the insulin receptor (IR), plays a critical role in metabolism. Aberrations in this signaling cascade lead to several pathologies, the majority of which are classified under the umbrella term “metabolic syndrome”. Although many of these pathologies are associated
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Insulin signaling, as mediated through the insulin receptor (IR), plays a critical role in metabolism. Aberrations in this signaling cascade lead to several pathologies, the majority of which are classified under the umbrella term “metabolic syndrome”. Although many of these pathologies are associated with insulin resistance, the exact mechanisms are not well understood. One area of current interest is the possibility of G-protein-coupled receptors (GPCRs) influencing or regulating IR signaling. This concept is particularly significant, because GPCRs have been shown to participate in cross-talk with the IR. More importantly, GPCR signaling has also been shown to preferentially regulate specific downstream signaling targets through GPCR agonist bias. A novel study recently demonstrated that this GPCR-biased agonism influences the activity of the IR without the presence of insulin. Although GPCR-IR cross-talk has previously been established, the notion that GPCRs can regulate the activation of the IR is particularly significant in relation to metabolic syndrome and other pathologies that develop as a result of alterations in IR signaling. As such, we aim to provide an overview of the physiological and pathophysiological roles of the IR within metabolic syndrome and its related pathologies, including cardiovascular health, gut microflora composition, gastrointestinal tract functioning, polycystic ovarian syndrome, pancreatic cancer, and neurodegenerative disorders. Furthermore, we propose that the GPCR-biased agonism may perhaps mediate some of the downstream signaling effects that further exacerbate these diseases for which the mechanisms are currently not well understood. Full article
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Open AccessArticle
AOX1-Subfamily Gene Members in Olea europaea cv. “Galega Vulgar”—Gene Characterization and Expression of Transcripts during IBA-Induced in Vitro Adventitious Rooting
Int. J. Mol. Sci. 2018, 19(2), 597; doi:10.3390/ijms19020597 (registering DOI) -
Abstract
Propagation of some Olea europaea L. cultivars is strongly limited due to recalcitrant behavior in adventitious root formation by semi-hardwood cuttings. One example is the cultivar ”Galega vulgar”. The formation of adventitious roots is considered a morphological response to stress. Alternative oxidase (AOX)
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Propagation of some Olea europaea L. cultivars is strongly limited due to recalcitrant behavior in adventitious root formation by semi-hardwood cuttings. One example is the cultivar ”Galega vulgar”. The formation of adventitious roots is considered a morphological response to stress. Alternative oxidase (AOX) is the terminal oxidase of the alternative pathway of the plant mitochondrial electron transport chain. This enzyme is well known to be induced in response to several biotic and abiotic stress situations. This work aimed to characterize the alternative oxidase 1 (AOX1)-subfamily in olive and to analyze the expression of transcripts during the indole-3-butyric acid (IBA)-induced in vitro adventitious rooting (AR) process. OeAOX1a (acc. no. MF410318) and OeAOX1d (acc. no. MF410319) were identified, as well as different transcript variants for both genes which resulted from alternative polyadenylation events. A correlation between transcript accumulation of both OeAOX1a and OeAOX1d transcripts and the three distinct phases (induction, initiation, and expression) of the AR process in olive was observed. Olive AOX1 genes seem to be associated with the induction and development of adventitious roots in IBA-treated explants. A better understanding of the molecular mechanisms underlying the stimulus needed for the induction of adventitious roots may help to develop more targeted and effective rooting induction protocols in order to improve the rooting ability of difficult-to-root cultivars. Full article
Open AccessArticle
AWRK6, A Synthetic Cationic Peptide Derived from Antimicrobial Peptide Dybowskin-2CDYa, Inhibits Lipopolysaccharide-Induced Inflammatory Response
Int. J. Mol. Sci. 2018, 19(2), 600; doi:10.3390/ijms19020600 (registering DOI) -
Abstract
Lipopolysaccharides (LPS) are major outer membrane components of Gram-negative bacteria and produce strong inflammatory responses in animals. Most antibiotics have shown little clinical anti-endotoxin activity while some antimicrobial peptides have proved to be effective in blocking LPS. Here, the anti-LPS activity of the
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Lipopolysaccharides (LPS) are major outer membrane components of Gram-negative bacteria and produce strong inflammatory responses in animals. Most antibiotics have shown little clinical anti-endotoxin activity while some antimicrobial peptides have proved to be effective in blocking LPS. Here, the anti-LPS activity of the synthetic peptide AWRK6, which is derived from antimicrobial peptide dybowskin-2CDYa, has been investigated in vitro and in vivo. The positively charged α-helical AWRK6 was found to be effective in blocking the binding of LBP (LPS binding protein) with LPS in vitro using ELISA. In a murine endotoxemia model, AWRK6 offered satisfactory protection efficiency against endotoxemia death, and the serum levels of LPS, IL-1β, IL-6, and TNF-α were found to be attenuated using ELISA. Further, histopathological analysis suggested that AWRK6 could improve the healing of liver and lung injury in endotoxemia mice. The results of real-time PCR and Western blotting showed that AWRK6 significantly reversed LPS-induced TLR4 overexpression and IκB depression, as well as the enhanced IκB phosphorylation. Additionally, AWRK6 did not produce any significant toxicity in vivo and in vitro. In summary, AWRK6 showed efficacious protection from LPS challenges in vivo and in vitro, by blocking LPS binding to LBP, without obvious toxicity, providing a promising strategy against LPS-induced inflammatory responses. Full article
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Open AccessArticle
The Cyanthin Diterpenoid and Sesterterpene Constituents of Hericium erinaceus Mycelium Ameliorate Alzheimer’s Disease-Related Pathologies in APP/PS1 Transgenic Mice
Int. J. Mol. Sci. 2018, 19(2), 598; doi:10.3390/ijms19020598 (registering DOI) -
Abstract
Hericium erinaceus was used in traditional Chinese medicine for physiologically beneficial medicines. Recently, it has become a candidate in causing positive brain health-related activities. We previously reported that Hericium erinaceus mycelium ameliorates Alzheimer’s disease (AD)-related pathologies. To reveal the role of the cyanthin
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Hericium erinaceus was used in traditional Chinese medicine for physiologically beneficial medicines. Recently, it has become a candidate in causing positive brain health-related activities. We previously reported that Hericium erinaceus mycelium ameliorates Alzheimer’s disease (AD)-related pathologies. To reveal the role of the cyanthin diterpenoid and sesterterpene constituents on this effects, erinacine A and S were isolated and their effects on attenuating AD-related pathology in APPswe/PS1dE9 transgenic mice were investigated. A 30 day short-term administration of erinacine A and S were performed to explore the effect of each erinacine on AD-related pathology including amyloid β production and degradation, plaque formation, plaque growth, glial activation and neurogenesis deterioration. Our results indicated the benefit effects of both erinacine A and S in cerebrum of APPswe/PS1dE9 mice, including: (1) attenuating cerebral plaque loading by inhibiting plaque growth; (2) diminishing the activation of glial cells; (3) raising the level of insulin degrading enzyme; and (4) promoting hippocampal neurogenesis. Moreover, erinacine A reduced the level of insoluble amyloid β and C-terminal fragment of amyloid precursor protein which was not mediated by erinacine S. We further performed a long term administration of erinacine A and found that erinacine A recovered the impairment in the tasks including burrowing, nesting, and Morris water maze. Our data pointed out that although both erinacine A and S reduce AD pathology via reducing amyloid deposition and promoting neurogenesis, erinacine A can also inhibit amyloid β production and is worth to be further developed for AD therapeutic use. Full article
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Open AccessArticle
Inhibitory Effect of Purpurogallin on Osteoclast Differentiation in Vitro through the Downregulation of c-Fos and NFATc1
Int. J. Mol. Sci. 2018, 19(2), 601; doi:10.3390/ijms19020601 (registering DOI) -
Abstract
Purpurogallin, a benzotropolone-containing natural compound, has been reported to exhibit numerous biological and pharmacological functions, such as antioxidant, anticancer, and anti-inflammatory effects. In this study, we enzymatically synthesized purpurogallin from pyrogallol and investigated its role in receptor activator of nuclear factor-κB ligand (RANKL)-induced
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Purpurogallin, a benzotropolone-containing natural compound, has been reported to exhibit numerous biological and pharmacological functions, such as antioxidant, anticancer, and anti-inflammatory effects. In this study, we enzymatically synthesized purpurogallin from pyrogallol and investigated its role in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis. Purpurogallin attenuated the formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts from bone marrow macrophages (BMMs) without causing cytotoxicity, and suppressed upregulation of osteoclast-specific markers, including TRAP (Acp5), cathepsin K (Ctsk), and dendritic cell-specific transmembrane protein (Dcstamp). However, purpurogallin did not affect the bone resorbing function of mature osteoclasts evident by the resorption pit assay. Activation of mitogen-activated protein kinases, Akt and IkB pathways in RANK signaling were not altered by purpurogallin, whereas the expression of c-Fos and NFATc1, key transcriptional regulators in osteoclastogenesis, was dramatically inhibited by purpurogallin. Purpurogallin also significantly reduced the expression level of B lymphocyte-induced maturation protein-1 (Blimp1) gene (Prdm1). Further, downregulation of Blimp1 led to forced expression of anti-osteoclastogenic genes, including interferon regulatory factor-8 (Irf8) and B-cell lymphoma 6 (Bcl6) genes. Taken together, our data suggested that purpurogallin inhibits osteoclast differentiation via downregulation of c-Fos and NFATc1. Full article
Open AccessReview
The miRNA Mirage: How Close Are We to Finding a Non-Invasive Diagnostic Biomarker in Endometriosis? A Systematic Review
Int. J. Mol. Sci. 2018, 19(2), 599; doi:10.3390/ijms19020599 (registering DOI) -
Abstract
Background: Endometriosis is a common disorder of the reproductive age group, characterised by the presence of ectopic endometrial tissue. The disease not only causes enormous suffering to the affected women, but also brings a tremendous medical and economic burden to bear on society.
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Background: Endometriosis is a common disorder of the reproductive age group, characterised by the presence of ectopic endometrial tissue. The disease not only causes enormous suffering to the affected women, but also brings a tremendous medical and economic burden to bear on society. There is a long lag phase between the onset and diagnosis of the disease, mainly due to its non-specific symptoms and the lack of a non-invasive test. Endometriosis can only be diagnosed invasively by laparoscopy. A specific, non-invasive test to diagnose endometriosis is an unmet clinical need. The recent discovery of microRNAs (miRNAs) as modulators of gene expression, and their stability and specificity, make them an attractive candidate biomarker. Various studies on miRNAs in endometriosis have identified their cardinal role in the pathogenesis of the disease, and have proposed them as potential biomarkers in endometriosis. Rationale/Objectives: The aims of this review were to study the role of circulatory miRNAs in endometriosis, and bring to light whether circulatory miRNAs could be potential non-invasive biomarkers to diagnose the disease. Search methods: Three databases, PubMed, EMBASE, and BIOSIS were searched, using a combination of Mesh or Emtree headings and free-text terms, to identify literature relating to circulating miRNAs in endometriosis published from 1996 to 31 December 2017. Only peer-reviewed, full-text original research articles in English were included in the current review. The studies meeting the inclusion criteria were critically assessed and checked using the QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies) tool. The dysregulated miRNAs were assessed regarding the concordance between the various studies and their role in the disease. Outcomes: Nine studies were critically analysed, and 42 different miRNAs were found to be dysregulated in them, with only one common miRNA (miR-20a) differentially expressed in more than one study. miR-17-5p/20a, miR-200, miR-199a, miR-143, and miR-145 were explored for their pivotal role in the aetiopathogenesis of endometriosis. Wider implications: It is emerging that miRNAs play a central role in the pathogenesis of endometriosis and have the potential of being promising biomarkers. Circulating miRNAs as a non-invasive diagnostic tool may shorten the delay in the diagnosis of the disease, thus alleviating the suffering of women and reducing the burden on health care systems. However, despite numerous studies on circulating miRNAs in endometriosis, no single miRNA or any panel of them seems to meet the criteria of a diagnostic biomarker. The disagreement between the various studies upholds the demand of larger, well-controlled systematic validation studies with uniformity in the research approaches and involving diverse populations. Full article
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Open AccessArticle
Anti-Inflammatory and Gastroprotective Roles of Rabdosia inflexa through Downregulation of Pro-Inflammatory Cytokines and MAPK/NF-κB Signaling Pathways
Int. J. Mol. Sci. 2018, 19(2), 584; doi:10.3390/ijms19020584 (registering DOI) -
Abstract
Globally, gastric ulcer is a vital health hazard for a human. Rabdosia inflexa (RI) has been used in traditional medicine for inflammatory diseases. The present study aimed to investigate the protective effect and related molecular mechanism of RI using lipopolysaccharide (LPS)-induced inflammation in
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Globally, gastric ulcer is a vital health hazard for a human. Rabdosia inflexa (RI) has been used in traditional medicine for inflammatory diseases. The present study aimed to investigate the protective effect and related molecular mechanism of RI using lipopolysaccharide (LPS)-induced inflammation in RAW 246.7 cells and HCl/EtOH-induced gastric ulcer in mice. We applied 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), nitric oxide (NO), reactive oxygen species (ROS), histopathology, malondialdehyde (MDA), quantitative real-time polymerase chain reaction (qPCR), immunohistochemistry (IHC), and Western blot analyses to evaluate the protective role of RI. Study revealed that RI effectively attenuated LPS-promoted NO and ROS production in RAW 246.7 cells. In addition, RI mitigated gastric oxidative stress by inhibiting lipid peroxidation, elevating NO, and decreasing gastric inflammation. RI significantly halted elevated gene expression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), inducible nitric oxide synthetase (iNOS), and cyclooxygenase-2 (COX-2) in gastric tissue. Likewise, RI markedly attenuated the mitogen-activated protein kinases (MAPKs) phosphorylation, COX-2 expression, phosphorylation and degradation of inhibitor kappa B (IκBα) and activation of nuclear factor kappa B (NF-κB). Thus, experimental findings suggested that the anti-inflammatory and gastroprotective activities of RI might contribute to regulating pro-inflammatory cytokines and MAPK/NF-κB signaling pathways. Full article
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Open AccessArticle
Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii
Int. J. Mol. Sci. 2018, 19(2), 585; doi:10.3390/ijms19020585 (registering DOI) -
Abstract
Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of
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Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a “plant-like” algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN. Full article
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