International Journal of Molecular Sciences

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Open AccessArticle

Unraveling Molecular Differences of Gastric Cancer by Label-Free Quantitative Proteomics Analysis

by Peng Dai, Qin Wang, Weihua Wang, Ruirui Jing, Wei Wang, Fengqin Wang, Kazem M. Azadzoi, Jing-Hua Yang and Zhen Yan

Int. J. Mol. Sci. 2016, 17(1), 69; https://doi.org/10.3390/ijms17010069 - 21 Jan 2016

Cited by 32 | Viewed by 6452

Abstract

Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues [...] Read more.

Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients’ tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis. Full article

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

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Venn diagrams of total identified proteins and dysregulated proteins. (A) Total proteins identified from three cases in tumor or adjacent tissues respectively. One thousand seven hundred forty-four proteins identified appear in both tumor and adjacent tissues; (B) The number of proteins with more than twofold differential expression in three cases, respectively, and the number of proteins shared in two or three cases. Full article ">Figure 2
The hierarchical heatmap of 146 dysregulated proteins analyzed by Ingenuity Pathway Analysis (IPA). The major boxes represent specific family or category of related functions. The smaller squares within the major boxes represent the number of proteins. Each individual square represent a specific protein. Colored squares indicate protein predicted state: increasing (orange), or decreasing (blue). Darker colors indicate higher absolute Z-scores. Full article ">Figure 3
The functional annotation of dysregulated proteins was analyzed by Protein Analysis Through Evolutionary Relationships (PANTHER), Database for Annotation, Visualization and Integrated Discovery (DAVID), STRING and Reactome. (A) Protein Classes; (B) Biological Process; and (C) Molecular Function of 146 dysregulated proteins were summarized in a pie chart by PANTHER; (D) Molecular function; and (E) Biological process based on the 65 upregulated proteins were depicted in a bar graph by DAVID; (F) Pathway analysis of 146 dysregulated proteins was indicated by PANTHER, DAVID, STRING and Reactome. For each category, the percentage or p-value of dysregulated proteins is indicated. Full article ">Figure 4
Protein-protein interactions (Evidence Mode) of dysregulated proteins were predicted by STRING. (A) Protein-protein interaction network formed with 146 dysregulated proteins. The three possible systematic dynamic clusters were indicated in red circles; (B) The network predicted 65 upregulated proteins. Some important proteins dispersed and located in the keynotes were marked with a red box. Different line colors represent the types of evidence for the association. Full article ">Figure 4 Cont.
Protein-protein interactions (Evidence Mode) of dysregulated proteins were predicted by STRING. (A) Protein-protein interaction network formed with 146 dysregulated proteins. The three possible systematic dynamic clusters were indicated in red circles; (B) The network predicted 65 upregulated proteins. Some important proteins dispersed and located in the keynotes were marked with a red box. Different line colors represent the types of evidence for the association. Full article ">Figure 5
Expression levels of hnRNPs and YBX-1 in GC and adjacent tissues. (A) qRT-PCR (n = 10) results showing the mRNA expression of hnRNPs and YBX-1. The ratio below the dotted line represented down-expression in GC tissues; otherwise represented up-expression in GC tissues; (B) Western blots (n = 10) of hnRNPs and YBX-1. N represent adjacent tissue and T represent tumor tissue; (C) Grayscale scanning of western blots bands. The ratio was compared to β-actin and statistically analyzed. Significance of differences between GC and adjacent tissues are displayed by ** p-value < 0.01 or * p-value < 0.05. Full article ">Figure 6
Representative immunohistochemical staining for sectioned formalin fixed GC and adjacent tissues. Specific antibodies of Anti-hnRNPA2B1 (Santa Cruz, TX, USA), Anti-hnRNPD (Proteintech, Chicago, IL, USA), Anti-hnRNPL (Santa Cruz) and Anti-YBX-1 (Santa Cruz) were hybridized respectively. IHC results showed that the morphology of tubular glands disappeared in cancer sections when compared to adjacent tissues. Cancer sections have stronger and higher density nuclei staining, high ratios of nucleus/cytoplasmic area, different shaped nuclei including megakaryocytes and polykaryocytes (arrows). Weak cytoplasmic staining were only seen in hnRNPA2B1, hnRNPD and YBX-1 hybridized normal sections. The magnification is 400×; scale bar: 20 μm. Full article ">

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Open AccessEditorial

Acknowledgement to Reviewers of International Journal of Molecular Sciences in 2015

by International Journal of Molecular Sciences Editorial Office

Int. J. Mol. Sci. 2016, 17(1), 142; https://doi.org/10.3390/ijms17010142 - 21 Jan 2016

Viewed by 10630

Abstract

The editors of International Journal of Molecular Sciences would like to express their sincere gratitude to the following reviewers for assessing manuscripts in 2015. [...] Full article

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Open AccessReview

An Overview of Direct Somatic Reprogramming: The Ins and Outs of iPSCs

by Siddharth Menon, Siny Shailendra, Andrea Renda, Michael Longaker and Natalina Quarto

Int. J. Mol. Sci. 2016, 17(1), 141; https://doi.org/10.3390/ijms17010141 - 21 Jan 2016

Cited by 35 | Viewed by 12335

Abstract

Stem cells are classified into embryonic stem cells and adult stem cells. An evolving alternative to conventional stem cell therapies is induced pluripotent stem cells (iPSCs), which have a multi-lineage potential comparable to conventionally acquired embryonic stem cells with the additional benefits of [...] Read more.

Stem cells are classified into embryonic stem cells and adult stem cells. An evolving alternative to conventional stem cell therapies is induced pluripotent stem cells (iPSCs), which have a multi-lineage potential comparable to conventionally acquired embryonic stem cells with the additional benefits of being less immunoreactive and avoiding many of the ethical concerns raised with the use of embryonic material. The ability to generate iPSCs from somatic cells provides tremendous promise for regenerative medicine. The breakthrough of iPSCs has raised the possibility that patient-specific iPSCs can provide autologous cells for cell therapy without the concern for immune rejection. iPSCs are also relevant tools for modeling human diseases and drugs screening. However, there are still several hurdles to overcome before iPSCs can be used for translational purposes. Here, we review the recent advances in somatic reprogramming and the challenges that must be overcome to move this strategy closer to clinical application. Full article

(This article belongs to the Special Issue Pluripotent Stem Cell Technology for Disease Modeling, Drug Discovery and Regenerative Medicine)

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Totipotency: After fertilization, Embryonic Stem Cells (ESCs) maintain the ability to form all three germ layers as well as extra-embryonic tissues or placental cells and are termed as totipotent. Pluripotency: These more specialized cells of the blastocyst stage maintain the ability to self-renew and differentiate into the three germ layers and down many lineages but do not form extra-embryonic tissues or placental cells. Reprogrammed somatic cells, iPSCs, also demonstrate the ability to self-renew and differentiate into all three germ layers in vivo and in vitro. Thus, iPSCs are also considered to be pluripotent stem cells. Multipotency: Adult or somatic stem cells are undifferentiated cells found in postnatal tissues. These specialized cells are considered to be multipotent; with very limited ability to self-renew and are committed to lineage specific differentiation. Full article ">Figure 2
The direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) can be achieved through the ectopic expression of specific transcription factors: Oct-4, C-Myc, Sox-2 and Klf-4, the “Yamanaka or OSKM Factors”. Delivery of these factors can be accomplished through a variety of methods. Initial methods developed integrating retroviral or lentiviral vectors. More recent strategies utilized non-integrating methods, further categorized by the use of DNA such as plasmids, mini circles/episomal, adenovirus, Sendai virus, and non-DNA based procedures such as, mRNAs, microRNAs (miRs), small molecules and bioactive proteins. Once reprogrammed to a pluripotent state, iPSCs can be expanded in vitro and subsequently differentiated to ectoderm, mesoderm and endoderm linages for use in cell therapy, disease modeling and drug discovery. Full article ">

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Open AccessArticle

Elucidating the Diversity of Aquatic Microdochium and Trichoderma Species and Their Activity against the Fish Pathogen Saprolegnia diclina

by Yiying Liu, Christin Zachow, Jos M. Raaijmakers and Irene De Bruijn

Int. J. Mol. Sci. 2016, 17(1), 140; https://doi.org/10.3390/ijms17010140 - 21 Jan 2016

Cited by 15 | Viewed by 9201

Abstract

Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several [...] Read more.

Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several chemical control agents, novel sustainable measures are required to control Saprolegnia infections in aquaculture. Previously, fungal community analysis by terminal restriction fragment length polymorphism (T-RFLP) revealed that the Ascomycota, specifically the genus Microdochium, was an abundant fungal phylum associated with salmon eggs from a commercial fish farm. Here, phylogenetic analyses showed that most fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride species. Phylogenetic and quantitative PCR analyses showed both a quantitative and qualitative difference in Trichoderma population between diseased and healthy salmon eggs, which was not the case for the Microdochium population. In vitro antagonistic activity of the fungi against Saprolegnia diclina was isolate-dependent; for most Trichoderma isolates, the typical mycoparasitic coiling around and/or formation of papilla-like structures on S. diclina hyphae were observed. These results suggest that among the fungal community associated with salmon eggs, Trichoderma species may play a role in Saprolegnia suppression in aquaculture. Full article

(This article belongs to the Special Issue Fish Molecular Biology)

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Isolation of salmon egg-associated fungi and oomycetes on potato dextrose agar plates. One or two salmon eggs from a Saprolegnia-infected batch (diseased, replicate No. 1–6) or a healthy batch (replicate No. 7–12) were placed onto the agar plates to allow fungal outgrowth. Full article ">Figure 2
Phylogenetic tree of ITS rRNA sequences of nine Microdochium isolates from salmon eggs and reference strains. The phylogenetic analyses were conducted in Mega 5 using the Kimura 2-parameter method [<a href="#B48-ijms-17-00140" class="html-bibr">48</a>] to compute evolutionary distances. The bootstrap values indicated at the nodes are based on 1000 bootstrap replicates. Branch values lower than 50% are hidden. Closed and open circles indicate Microdochium isolates from Saprolegnia-infected (diseased) or healthy salmon egg samples, respectively. Red, blue and black colors indicate strains from terrestrial/plant, aquatic and unknown sources of isolation, respectively. The scale bar indicates an evolutionary distance of 0.01 nucleotide substitution per sequence position. Twenty-five ITS sequences of good quality and at least 550 bp of reference strains of Microdochium were downloaded from GenBank; their strain names are preceded by the accession numbers. Full article ">Figure 3
Detection of total fungi, Microdochium and Trichoderma in salmon egg samples by quantitative PCR. Total fungal community was detected using ITS4-ITS9 primers, Microdochium lycopodinum/Microdochium phragmitis species were detected by MPF-MPR primers, and Trichoderma species were detected by ITS1TrF-ITS4TrR primers. The concentration of DNA template of each sample was normalized at 5 ± 1 ng·μL−1. Closed and open circles indicate DNA samples extracted from Saprolegnia-infected (diseased) or healthy samples, respectively. The lane on the left is the size marker and the band size (bp) is indicated next to each band. Full article ">Figure 4
In vitro activities of Microdochium and Trichoderma isolates. (a) Dual culture of Microdochium isolate 749F1 or Trichoderma isolate 762F1b with Saprolegnia diclina 1152F4 on 1/5th strength potato dextrose agar (1/5PDA). S. diclina and the fungal isolates were pre-grown on 1/5PDA. A hyphal plug of S. diclina was inoculated on the left side of the fresh 1/5PDA and a hyphal plug of Microdochium isolate 749F1 or Trichoderma isolate 762F1b was inoculated on the right side. The dual cultures were incubated for six days at 20–25 °C. The black arrows indicate S. diclina plugs and the white arrows indicate Microdochium or Trichoderma plugs; (b) Microscopic pictures of the hyphal interaction between Trichoderma and S. diclina. The black arrows indicate hyphae of S. diclina. The white arrows indicate the coiling of Trichoderma hyphae around S. diclina hyphae (top pictures) or the formation of papilla-like structure of Trichoderma hyphae around S. diclina hyphae (bottom pictures). Scale bars represent 10 μm. Full article ">Figure 5
Phylogenetic tree of ITS rRNA sequences of nine Trichoderma isolates and reference strains. The phylogenetic analyses were conducted in Mega 5 using the Tamura 3-parameter method [<a href="#B70-ijms-17-00140" class="html-bibr">70</a>] to compute evolutionary distances. The bootstrap values indicated at the nodes are based on 1000 bootstrap replicates. Branch values lower than 50% are hidden. Closed and open circles indicate isolates from Saprolegnia-infected (diseased) or healthy samples, respectively. Red, blue and black colors indicate strains from terrestrial/plant, aquatic and unknown sources of isolation, respectively. The scale bar indicates an evolutionary distance of 0.01 nucleotide substitution per sequence position. Outer labels describe section names based on the list of species in ISTH website [<a href="#B64-ijms-17-00140" class="html-bibr">64</a>] and only sections contained at least five strains are indicated. 104 ITS sequences of good quality and at least 550 bp of reference strains of Trichoderma were downloaded from GenBank; their corresponding strain names are preceded by the accession numbers. Strain names followed by “(T)” indicate type strains. Full article ">Figure 6
Phylogenetic tree of tef1 sequences of nine Trichoderma viride isolates and reference strains. The phylogenetic analyses were conducted in Mega 5 using the Tamura-Nei method [<a href="#B71-ijms-17-00140" class="html-bibr">71</a>] to compute evolutionary distances. The bootstrap values indicated at the nodes are based on 1000 bootstrap replicates. Branch values lower than 50% are hidden. Closed and open circles indicate isolates from Saprolegnia-infected (diseased) or healthy samples, respectively. Red, blue and black colors indicate strains from terrestrial/plant, aquatic and unknown sources of isolation, respectively. The scale bar indicates an evolutionary distance of 0.01 nucleotide substitution per sequence position. 11 sequences of good quality and at least 1200 bp of reference strains of Trichoderma were downloaded from GenBank; their strain names are preceded by the accession numbers. Strain name followed by “(T)” indicates type strain. Full article ">

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Open AccessReview

Cadmium Protection Strategies—A Hidden Trade-Off?

by Adolf Michael Sandbichler and Martina Höckner

Int. J. Mol. Sci. 2016, 17(1), 139; https://doi.org/10.3390/ijms17010139 - 21 Jan 2016

Cited by 81 | Viewed by 8341

Abstract

Cadmium (Cd) is a non-essential transition metal which is introduced into the biosphere by various anthropogenic activities. Environmental pollution with Cd poses a major health risk and Cd toxicity has been extensively researched over the past decades. This review aims at changing the [...] Read more.

Cadmium (Cd) is a non-essential transition metal which is introduced into the biosphere by various anthropogenic activities. Environmental pollution with Cd poses a major health risk and Cd toxicity has been extensively researched over the past decades. This review aims at changing the perspective by discussing protection mechanisms available to counteract a Cd insult. Antioxidants, induction of antioxidant enzymes, and complexation of Cd to glutathione (GSH) and metallothionein (MT) are the most potent protective measures to cope with Cd-induced oxidative stress. Furthermore, protection mechanisms include prevention of endoplasmic reticulum (ER) stress, mitophagy and metabolic stress, as well as expression of chaperones. Pre-exposure to Cd itself, or co-exposure to other metals or trace elements can improve viability under Cd exposure and cells have means to reduce Cd uptake and improve Cd removal. Finally, environmental factors have negative or positive effects on Cd toxicity. Most protection mechanisms aim at preventing cellular damage. However, this might not be possible without trade-offs like an increased risk of carcinogenesis. Full article

(This article belongs to the Special Issue Metal Metabolism in Animals)

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Open AccessReview

The TEAD Family and Its Oncogenic Role in Promoting Tumorigenesis

by Yuhang Zhou, Tingting Huang, Alfred S. L. Cheng, Jun Yu, Wei Kang and Ka Fai To

Int. J. Mol. Sci. 2016, 17(1), 138; https://doi.org/10.3390/ijms17010138 - 21 Jan 2016

Cited by 144 | Viewed by 15904

Abstract

The TEAD family of transcription factors is necessary for developmental processes. The family members contain a TEA domain for the binding with DNA elements and a transactivation domain for the interaction with transcription coactivators. TEAD proteins are required for the participation of coactivators [...] Read more.

The TEAD family of transcription factors is necessary for developmental processes. The family members contain a TEA domain for the binding with DNA elements and a transactivation domain for the interaction with transcription coactivators. TEAD proteins are required for the participation of coactivators to transmit the signal of pathways for the downstream signaling processes. TEADs also play an important role in tumor initiation and facilitate cancer progression via activating a series of progression-inducing genes, such as CTGF, Cyr61, Myc and Gli2. Recent studies have highlighted that TEADs, together with their coactivators, promote or even act as the crucial parts in the development of various malignancies, such as liver, ovarian, breast and prostate cancers. Furthermore, TEADs are proposed to be useful prognostic biomarkers due to the ideal correlation between high expression and clinicopathological parameters in gastric, breast, ovarian and prostate cancers. In this review, we summarize the functional role of TEAD proteins in tumorigenesis and discuss the key role of TEAD transcription factors in the linking of signal cascade transductions. Improved knowledge of the TEAD proteins will be helpful for deep understanding of the molecular mechanisms of tumorigenesis and identifying ideal predictive or prognostic biomarkers, even providing clinical translation for anticancer therapy in human cancers. Full article

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

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Open AccessReview

The Clinical Significance of Phosphorylated Heat Shock Protein 27 (HSPB1) in Pancreatic Cancer

by Mitsuru Okuno, Seiji Adachi, Osamu Kozawa, Masahito Shimizu and Ichiro Yasuda

Int. J. Mol. Sci. 2016, 17(1), 137; https://doi.org/10.3390/ijms17010137 - 21 Jan 2016

Cited by 17 | Viewed by 7750

Abstract

Pancreatic cancer is one of most aggressive forms of cancer. After clinical detection it exhibits fast metastatic growth. Heat shock protein 27 (HSP27; HSPB1) has been characterized as a molecular chaperone which modifies the structures and functions of other proteins in cells when [...] Read more.

Pancreatic cancer is one of most aggressive forms of cancer. After clinical detection it exhibits fast metastatic growth. Heat shock protein 27 (HSP27; HSPB1) has been characterized as a molecular chaperone which modifies the structures and functions of other proteins in cells when they are exposed to various stresses, such as chemotherapy. While the administration of gemcitabine, an anti-tumor drug, has been the standard treatment for patients with advanced pancreatic cancer, accumulating evidence shows that HSP27 plays a key role in the chemosensitivity to gemcitabine. In addition, phosphorylated HSP27 induced by gemcitabine has been associated with the inhibition of pancreatic cancer cell growth. In this review, we summarize the role of phosphorylated HSP27, as well as HSP27, in the regulation of chemosensitivity in pancreatic cancer. Full article

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

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Open AccessArticle

Column Selection for Biomedical Analysis Supported by Column Classification Based on Four Test Parameters

by Alina Plenis, Natalia Rekowska and Tomasz Bączek

Int. J. Mol. Sci. 2016, 17(1), 136; https://doi.org/10.3390/ijms17010136 - 21 Jan 2016

Cited by 2 | Viewed by 4378

Abstract

This article focuses on correlating the column classification obtained from the method created at the Katholieke Universiteit Leuven (KUL), with the chromatographic resolution attained in biomedical separation. In the KUL system, each column is described with four parameters, which enables estimation of the [...] Read more.

This article focuses on correlating the column classification obtained from the method created at the Katholieke Universiteit Leuven (KUL), with the chromatographic resolution attained in biomedical separation. In the KUL system, each column is described with four parameters, which enables estimation of the F_KUL value characterising similarity of those parameters to the selected reference stationary phase. Thus, a ranking list based on the F_KUL value can be calculated for the chosen reference column, then correlated with the results of the column performance test. In this study, the column performance test was based on analysis of moclobemide and its two metabolites in human plasma by liquid chromatography (LC), using 18 columns. The comparative study was performed using traditional correlation of the F_KUL values with the retention parameters of the analytes describing the column performance test. In order to deepen the comparative assessment of both data sets, factor analysis (FA) was also used. The obtained results indicated that the stationary phase classes, closely related according to the KUL method, yielded comparable separation for the target substances. Therefore, the column ranking system based on the F_KUL-values could be considered supportive in the choice of the appropriate column for biomedical analysis. Full article

(This article belongs to the Section Physical Chemistry, Theoretical and Computational Chemistry)

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Open AccessReview

Photodynamic Therapy in Non-Gastrointestinal Thoracic Malignancies

by Biniam Kidane, Dhruvin Hirpara and Kazuhiro Yasufuku

Int. J. Mol. Sci. 2016, 17(1), 135; https://doi.org/10.3390/ijms17010135 - 21 Jan 2016

Cited by 9 | Viewed by 6608

Abstract

Photodynamic therapy has a role in the management of early and late thoracic malignancies. It can be used to facilitate minimally-invasive treatment of early endobronchial tumours and also to palliate obstructive and bleeding effects of advanced endobronchial tumours. Photodynamic therapy has been used [...] Read more.

Photodynamic therapy has a role in the management of early and late thoracic malignancies. It can be used to facilitate minimally-invasive treatment of early endobronchial tumours and also to palliate obstructive and bleeding effects of advanced endobronchial tumours. Photodynamic therapy has been used as a means of downsizing tumours to allow for resection, as well as reducing the extent of resection necessary. It has also been used successfully for minimally-invasive management of local recurrences, which is especially valuable for patients who are not eligible for radiation therapy. Photodynamic therapy has also shown promising results in mesothelioma and pleural-based metastatic disease. As new generation photosensitizers are being developed and tested and methodological issues continue to be addressed, the role of photodynamic therapy in thoracic malignancies continues to evolve. Full article

(This article belongs to the Special Issue Advances in Photodynamic Therapy)

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Photodynamic therapy (PDT) for radiographically occult early stage lung cancer. (A) A 65-year-old male presented with abnormal sputum cytology and normal chest X-ray; (B) Bronchoscopy revealed an endobronchial abnormality at the orifice of the left upper division anterior segmental bronchus (arrow). Biopsy was positive for squamous cell carcinoma; and (C) Bronchoscopy six months post PDT (120 J/cm2) shows complete response with no residual tumour. Full article ">Figure 2
PDT for central type lung cancer. (A) Endobronchial tumour (squamous cell carcinoma) completely obstructing the left upper lobe bronchus and extending into the left main stem bronchus; (B) complete response to PDT with only scarring on follow up bronchoscopy one year post PDT. Full article ">Figure 3
PDT for recurrent lung cancer. (A) A 70-year-old male with history of left upper lobectomy for stage IA T1aN0M0 squamous cell carcinoma. Bronchoscopy performed 10 years post resection for the investigation of abnormal sputum cytology revealed abnormal bronchial mucosa in the left lower lobe basal lateral segmental bronchus; (B) Narrow band imaging shows dotted vessels within the abnormal mucosa; (C) Autofluorescence bronchoscopy shows abnormal fluorescence in the same area; and (D) Follow-up bronchoscopy six months post PDT (180 J/cm2) shows only scarring from the treatment with complete response. Full article ">

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Open AccessReview

Structure Prediction: New Insights into Decrypting Long Noncoding RNAs

by Kun Yan, Yasir Arfat, Dijie Li, Fan Zhao, Zhihao Chen, Chong Yin, Yulong Sun, Lifang Hu, Tuanmin Yang and Airong Qian

Int. J. Mol. Sci. 2016, 17(1), 132; https://doi.org/10.3390/ijms17010132 - 21 Jan 2016

Cited by 51 | Viewed by 8100

Abstract

Long noncoding RNAs (lncRNAs), which form a diverse class of RNAs, remain the least understood type of noncoding RNAs in terms of their nature and identification. Emerging evidence has revealed that a small number of newly discovered lncRNAs perform important and complex biological [...] Read more.

Long noncoding RNAs (lncRNAs), which form a diverse class of RNAs, remain the least understood type of noncoding RNAs in terms of their nature and identification. Emerging evidence has revealed that a small number of newly discovered lncRNAs perform important and complex biological functions such as dosage compensation, chromatin regulation, genomic imprinting, and nuclear organization. However, understanding the wide range of functions of lncRNAs related to various processes of cellular networks remains a great experimental challenge. Structural versatility is critical for RNAs to perform various functions and provides new insights into probing the functions of lncRNAs. In recent years, the computational method of RNA structure prediction has been developed to analyze the structure of lncRNAs. This novel methodology has provided basic but indispensable information for the rapid, large-scale and in-depth research of lncRNAs. This review focuses on mainstream RNA structure prediction methods at the secondary and tertiary levels to offer an additional approach to investigating the functions of lncRNAs. Full article

(This article belongs to the Collection Regulation by Non-coding RNAs)

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The graphical abstract of this review. Full article ">Figure 2
Mechanisms of lncRNA action in transcriptional regulation. (a) Transcription of the lncRNA SRG1 inhibits the expression of the SER3 gene by interfering with the binding of RNA polymerase II to DNA; (b) The expression of the p15 antisense RNA, the lncRNA of a tumor suppressor gene, results in the silencing of the p15 gene through the induction of heterochromatin formation, which persisted after the p15 antisense RNA was turned off; (c) lncRNA binds to the major DHFR promoter and IIB, a general transcriptional factor, to form a stable and specific complex to dissociate the preinitiation complex from the major DHFR promoter; (d) As a response to stress, the RNA-binding protein TLS, under allosteric modulation via lncRNA upstream of CCND1, binds to chromatin-binding protein (CBP) and inhibits CBP/P300 HAT activities on CCND1; (e) The lncRNA Evf2, a crucial co-enhancer of regulatory proteins involved in transcription, cooperates with the Dlx2 protein to activate the Dlx5/6 enhancer in a target gene; (f) In response to heat shock, the lncRNA HSR1 (heat shock RNA-1) promotes the trimerization of HSF1 (heat-shock transcription factor 1), and consequently the translation factor EIF interacts with HSR1 and HSF1 to forms a complex to facilitate the expression of heat-shock protein (HSP); (g) NFAT is nuclear factor of activated T cells. The lncRNA NRON (noncoding repressor of NFAT) may form a complex with importin proteins to regulate the subcellular localization of NFAT. The knockdown of NRON increases the expression and activity of NFAT; (h) The lncRNA metastasis-associated lung adenocarcinoma transcript 1(MALAT1) has been shown to be abnormally expressed in many human cancers. The nascent MALAT1 transcript is cleaved by RNase P to produce the 3′ end of the mature MALAT1 transcript and the 5′ end of the small RNA; (i) Several studies have elucidated that some lncRNAs can act as microRNA sponges to competitively bind to microRNAs and decrease microRNA-induced tumorsphere differentiation. Full article ">

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Open AccessArticle

VLDL from Metabolic Syndrome Individuals Enhanced Lipid Accumulation in Atria with Association of Susceptibility to Atrial Fibrillation

by Hsiang-Chun Lee, Hsin-Ting Lin, Liang-Yin Ke, Chi Wei, Yi-Lin Hsiao, Chih-Sheng Chu, Wen-Ter Lai, Shyi-Jang Shin, Chu-Huang Chen, Sheng-Hsiung Sheu and Bin-Nan Wu

Int. J. Mol. Sci. 2016, 17(1), 134; https://doi.org/10.3390/ijms17010134 - 20 Jan 2016

Cited by 14 | Viewed by 6312

Abstract

Metabolic syndrome (MetS) represents a cluster of metabolic derangements. Dyslipidemia is an important factor in MetS and is related to atrial fibrillation (AF). We hypothesized that very low density lipoproteins (VLDL) in MetS (MetS-VLDL) may induce atrial dilatation and vulnerability to AF. VLDL [...] Read more.

Metabolic syndrome (MetS) represents a cluster of metabolic derangements. Dyslipidemia is an important factor in MetS and is related to atrial fibrillation (AF). We hypothesized that very low density lipoproteins (VLDL) in MetS (MetS-VLDL) may induce atrial dilatation and vulnerability to AF. VLDL was therefore separated from normal (normal-VLDL) and MetS individuals. Wild type C57BL/6 male mice were divided into control, normal-VLDL (nVLDL), and MetS-VLDL (msVLDL) groups. VLDL (15 µg/g) and equivalent volumes of saline were injected via tail vein three times a week for six consecutive weeks. Cardiac chamber size and function were measured by echocardiography. MetS-VLDL significantly caused left atrial dilation (control, n = 10, 1.64 ± 0.23 mm; nVLDL, n = 7, 1.84 ± 0.13 mm; msVLDL, n = 10, 2.18 ± 0.24 mm; p < 0.0001) at week 6, associated with decreased ejection fraction (control, n = 10, 62.5% ± 7.7%, vs. msVLDL, n = 10, 52.9% ± 9.6%; p < 0.05). Isoproterenol-challenge experiment resulted in AF in young msVLDL mice. Unprovoked AF occurred only in elderly msVLDL mice. Immunohistochemistry showed excess lipid accumulation and apoptosis in msVLDL mice atria. These findings suggest a pivotal role of VLDL in AF pathogenesis for MetS individuals. Full article

(This article belongs to the Section Biochemistry)

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Metabolic syndrome-very low density lipoproteins (MetS-VLDL) is cytotoxic and provokes oxidative stress, with greater internalization in HL-1 atrial myocytes. (A) HL-1 cells were treated with normal-VLDL or MetS-VLDL at different test concentrations (3.125, 6.25, 12.5 and 25 mg/dL) for 24 h. OD values at 450 nm indicating viability were significantly lower in the msVLDL group (* p < 0.05; n = 4 for each group); (B) HL-1 cells treated with MetS-VLDL showed significantly reduced cell viability (% of control) with a concentration of 25 mg/mL (** p < 0.01; n = 4); (C) DCF fluorescence (excitation at 480 nm and emission at 520 nm) indicated total cytosolic oxidant activity (values of % control; n = 3 for each group). MetS-VLDL significantly increased oxidative stress at 25 mg/dL (* p < 0.05; ** p < 0.01); (D) Representative images for control, nVLDL, msVLDL, and msVLDL with VLDL receptor (VLDLR) antibody (msVLDL + Ab) groups (n = 3 for each group); (E) Internalization of DiI-labeled VLDL particles (red) increased in size and number in MetS-VLDL treated HL-1 cells (msVLDL) compared to normal-VLDL treated cells (nVLDL) (* p < 0.05, n = 3). Pre-treatment with VLDLR Ab for 24 h reduced internalization (* p < 0.05). Full article ">Figure 2
Both VLDLs caused LV dilation but only MetS-VLDL caused left atrial dilation. (A) Echocardiography of murine heart. Left atrium (LA) and left ventricle (LV) were identified in B-mode; (B) M-mode images for measurements of diameters of aortic root (AO), LA and LV. LA was significantly enlarged in the MetS-VLDL injection group (msVLDL) (n = 6) but not in the normal-VLDL injection group (nVLDL) (n = 7) or the control group (n = 5); (C) Significant LA enlargement developed as early as 4–6 weeks after injection in the msVLDL group. LV dilatation developed significantly until 6 weeks. (msVLDL vs. control, $ p < 0.05; msVLDL vs. nVLDL, # p < 0.05; nVLDL vs. control, * p < 0.05); (D) No significant difference in body weight of the groups. Full article ">Figure 3
Isoproterenol-induced and unprovoked atrial fibrillation (AF) were observed only in msVLDL mice. (A–D) Representative tracings of young mice after ISO injection show abnormalities including normal regular sinus rhythm in the control group (n = 5), premature atrial complex (PAC) in the nVLDL group, premature ventricular complex (PVC *) and AF (absence of clear P waves and irregular RR intervals) in the msVLDL group (n = 5); (E) Heart rate responses after isoproterenol injection were not different among groups; (F) For elderly mice, spontaneous, unprovoked AF was noted in the msVLDL group (n = 6) with an incidence of 50%. PAC was observed in one mouse in the nVLDL group (n = 5). All control mice (n = 5) had sinus rhythm. $ p < 0.001 for msVLDL vs. control and # p < 0.001 for msVLDL vs. nVLDL. Full article ">Figure 4
Apoptosis in atrial tissue of msVLDL mice. Representative in situ terminal deoxynucleotidyl transferase (TUNEL) staining of atrial tissues from control (left), nVLDL (middle), and msVLDL (right) (n = 3 for each groups). Normal nuclei with DAPI staining appear blue. Condensed or fragmented nuclei appeared bright green and indicate cells undergoing apoptosis. Arrows indicate apoptotic atrial myocytes in the msVLDL group. The scale bars indicate 100 µm. Full article ">Figure 5
Greater lipid accumulation in atrial tissue of msVLDL mice. (A) Representative Oil-Red-O-stained sections of atrial tissues from control (left), nVLDL (middle), and msVLDL (right). Each red rectangle indicates the area to be magnified (20×). Tiny red lipid droplets in controls were few but the number increased in nVLDL and msVLDL atria. Some lipid droplets increased in size in the msVLDL; (B) Lipid droplets were significantly increased in the VLDL groups, especially in the msVLDL group (** p < 0.01, *** p < 0.001; n = 3 for each group). Full article ">Figure 6
Potential mechanism by which VLDL promotes AF in MetS. In MetS, the biochemical properties of VLDL are changed. MetS-VLDL can induce cellular reactive oxygen species, atrial myocyte cytotoxicity, and excess lipid accumulation resulting in subsequent gene dysregulation corresponding to metabolic derangement. Structural and potentially electrical remodeling initiated by MetS-VLDL in concert contribute to AF vulnerability and development. Full article ">

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Open AccessReview

Iron Homeostasis in Health and Disease

by Raffaella Gozzelino and Paolo Arosio

Int. J. Mol. Sci. 2016, 17(1), 130; https://doi.org/10.3390/ijms17010130 - 20 Jan 2016

Cited by 249 | Viewed by 19829

Abstract

Iron is required for the survival of most organisms, including bacteria, plants, and humans. Its homeostasis in mammals must be fine-tuned to avoid iron deficiency with a reduced oxygen transport and diminished activity of Fe-dependent enzymes, and also iron excess that may catalyze [...] Read more.

Iron is required for the survival of most organisms, including bacteria, plants, and humans. Its homeostasis in mammals must be fine-tuned to avoid iron deficiency with a reduced oxygen transport and diminished activity of Fe-dependent enzymes, and also iron excess that may catalyze the formation of highly reactive hydroxyl radicals, oxidative stress, and programmed cell death. The advance in understanding the main players and mechanisms involved in iron regulation significantly improved since the discovery of genes responsible for hemochromatosis, the IRE/IRPs machinery, and the hepcidin-ferroportin axis. This review provides an update on the molecular mechanisms regulating cellular and systemic Fe homeostasis and their roles in pathophysiologic conditions that involve alterations of iron metabolism, and provides novel therapeutic strategies to prevent the deleterious effect of its deficiency/overload. Full article

(This article belongs to the Special Issue Metal Metabolism in Animals)

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Open AccessArticle

Computational Analysis of Structure-Based Interactions for Novel H₁-Antihistamines

by Yinfeng Yang, Yan Li, Yanqiu Pan, Jinghui Wang, Feng Lin, Chao Wang, Shuwei Zhang and Ling Yang

Int. J. Mol. Sci. 2016, 17(1), 129; https://doi.org/10.3390/ijms17010129 - 19 Jan 2016

Cited by 18 | Viewed by 8557

Abstract

As a chronic disorder, insomnia affects approximately 10% of the population at some time during their lives, and its treatment is often challenging. Since the antagonists of the H₁ receptor, a protein prevalent in human central nervous system, have been proven as [...] Read more.

As a chronic disorder, insomnia affects approximately 10% of the population at some time during their lives, and its treatment is often challenging. Since the antagonists of the H₁ receptor, a protein prevalent in human central nervous system, have been proven as effective therapeutic agents for treating insomnia, the H₁ receptor is quite possibly a promising target for developing potent anti-insomnia drugs. For the purpose of understanding the structural actors affecting the antagonism potency, presently a theoretical research of molecular interactions between 129 molecules and the H₁ receptor is performed through three-dimensional quantitative structure-activity relationship (3D-QSAR) techniques. The ligand-based comparative molecular similarity indices analysis (CoMSIA) model (Q² = 0.525, R²_ncv = 0.891, R²_pred = 0.807) has good quality for predicting the bioactivities of new chemicals. The cross-validated result suggests that the developed models have excellent internal and external predictability and consistency. The obtained contour maps were appraised for affinity trends for the investigated compounds, which provides significantly useful information in the rational drug design of novel anti-insomnia agents. Molecular docking was also performed to investigate the mode of interaction between the ligand and the active site of the receptor. Furthermore, as a supplementary tool to study the docking conformation of the antagonists in the H₁ receptor binding pocket, molecular dynamics simulation was also applied, providing insights into the changes in the structure. All of the models and the derived information would, we hope, be of help for developing novel potent histamine H₁ receptor antagonists, as well as exploring the H₁-antihistamines interaction mechanism. Full article

(This article belongs to the Special Issue Big Data Analysis and QSAR/QSPR Research in Chemistry, Bio-Medical, and Network Sciences)

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Open AccessReview

Lung Regeneration: Endogenous and Exogenous Stem Cell Mediated Therapeutic Approaches

by Khondoker M. Akram, Neil Patel, Monica A. Spiteri and Nicholas R. Forsyth

Int. J. Mol. Sci. 2016, 17(1), 128; https://doi.org/10.3390/ijms17010128 - 19 Jan 2016

Cited by 55 | Viewed by 17871

Abstract

The tissue turnover of unperturbed adult lung is remarkably slow. However, after injury or insult, a specialised group of facultative lung progenitors become activated to replenish damaged tissue through a reparative process called regeneration. Disruption in this process results in healing by fibrosis [...] Read more.

The tissue turnover of unperturbed adult lung is remarkably slow. However, after injury or insult, a specialised group of facultative lung progenitors become activated to replenish damaged tissue through a reparative process called regeneration. Disruption in this process results in healing by fibrosis causing aberrant lung remodelling and organ dysfunction. Post-insult failure of regeneration leads to various incurable lung diseases including chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis. Therefore, identification of true endogenous lung progenitors/stem cells, and their regenerative pathway are crucial for next-generation therapeutic development. Recent studies provide exciting and novel insights into postnatal lung development and post-injury lung regeneration by native lung progenitors. Furthermore, exogenous application of bone marrow stem cells, embryonic stem cells and inducible pluripotent stem cells (iPSC) show evidences of their regenerative capacity in the repair of injured and diseased lungs. With the advent of modern tissue engineering techniques, whole lung regeneration in the lab using de-cellularised tissue scaffold and stem cells is now becoming reality. In this review, we will highlight the advancement of our understanding in lung regeneration and development of stem cell mediated therapeutic strategies in combating incurable lung diseases. Full article

(This article belongs to the Special Issue Stem Cell Activation in Adult Organism)

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Epithelial-mesenchymal cross-talk and governing signalling during early development and branching morphogenesis of lung. Factors are represented only at sites where the expression is most abundant. Fibroblast growth factor-10 (FGF-10) is highly expressed in the distal mesenchyme and acts as a chemotactic focus for the epithelium during lung budding. FGF-10 also regulates Sox9 expression in the distal epithelial progenitors and induces bone morphogenetic protein (BMP)-4 expression. Sox2 expression in the proximal epithelium is under regulation of histone deacetylases 1/2 (HDAC1/2) signalling. FGF-10 expression in the mesenchyme is regulated by Wnt/β-catenin signalling (red arrow). A high concentration of BMP-4 signal also serves to locally inhibit endoderm proliferation, thereby inducing the lateral outgrowth of new airway branches. Sonic hedgehog (Shh) at the distal tips functions to downregulate FGF-10 expression in the mesenchyme, which limits local budding. Transforming growth factor-β (TGF-β) signalling also prevents local budding, by decreasing endodermal proliferation and by stimulating synthesis of matrix components at branch points. Solid arrows indicate sources from and influences on cells/molecules; dotted arrow indicates direction of patterning. Full article ">Figure 2
Endogenous stem cells in the airways and alveoli with their differentiation in the postnatal lung. (A) The trachea and bronchi of the rodent and human lung are lined with multiple epithelial lineages. Basal cells are located in this region and can generate secretory club and ciliated cell lineages. Notch signalling is crucial for differentiation of basal cells and also suppresses ciliated cell differentiation. HDAC1 and HDAC2 are essential for secretory epithelial regeneration; (B) The bronchiolar lining epithelium of rodents lacks basal cells but contains variant club cells, secretory cells and ciliated cells; (C) Progenitor cell populations and their differentiated progeny in the lung alveolus. The alveolar epithelium consists of alveolar epithelial cell AECI and AECII cells. AECII cells can generate AECI cells during homeostasis and after injury. Generation of alveolar epithelium by other cells, such as BASCs has yet to be supported by lineage tracing. The AECII to AECI differentiation signalling is not clear (marked by “?”), but AECII self-renewal and proliferation occurs via EGFR/KRAS signalling. Curved arrows indicate differentiation and line arrows indicate molecular or physical stimulus. Full article ">

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Open AccessReview

The Complex Relationship between Metals and Carbonic Anhydrase: New Insights and Perspectives

by Maria Giulia Lionetto, Roberto Caricato, Maria Elena Giordano and Trifone Schettino

Int. J. Mol. Sci. 2016, 17(1), 127; https://doi.org/10.3390/ijms17010127 - 19 Jan 2016

Cited by 64 | Viewed by 8098

Abstract

Carbonic anhydrase is a ubiquitous metalloenzyme, which catalyzes the reversible hydration of CO₂ to HCO₃⁻ and H⁺. Metals play a key role in the bioactivity of this metalloenzyme, although their relationships with CA have not been completely clarified [...] Read more.

Carbonic anhydrase is a ubiquitous metalloenzyme, which catalyzes the reversible hydration of CO₂ to HCO₃⁻ and H⁺. Metals play a key role in the bioactivity of this metalloenzyme, although their relationships with CA have not been completely clarified to date. The aim of this review is to explore the complexity and multi-aspect nature of these relationships, since metals can be cofactors of CA, but also inhibitors of CA activity and modulators of CA expression. Moreover, this work analyzes new insights and perspectives that allow translating new advances in basic science on the interaction between CA and metals to applications in several fields of research, ranging from biotechnology to environmental sciences. Full article

(This article belongs to the Special Issue Metal Metabolism in Animals)

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Open AccessArticle

Statin Therapy and the Development of Cerebral Amyloid Angiopathy—A Rodent in Vivo Approach

by Björn Reuter, Alexander Venus, Saskia Grudzenski, Patrick Heiler, Lothar Schad, Matthias Staufenbiel, Michael G. Hennerici and Marc Fatar

Int. J. Mol. Sci. 2016, 17(1), 126; https://doi.org/10.3390/ijms17010126 - 19 Jan 2016

Cited by 5 | Viewed by 7069

Abstract

Background: Cerebral amyloid angiopathy (CAA) is characterized by vascular deposition of amyloid β (Aβ) with a higher incidence of cerebral microbleeds (cMBs) and spontaneous hemorrhage. Since statins are known for their benefit in vascular disease we tested for the effect on CAA. Methods: [...] Read more.

Background: Cerebral amyloid angiopathy (CAA) is characterized by vascular deposition of amyloid β (Aβ) with a higher incidence of cerebral microbleeds (cMBs) and spontaneous hemorrhage. Since statins are known for their benefit in vascular disease we tested for the effect on CAA. Methods: APP23-transgenic mice received atorvastatin-supplemented food starting at the age of eight months (n = 13), 12 months (n = 7), and 16 months (n = 6), respectively. Controls (n = 16) received standard food only. At 24 months of age cMBs were determined with T2*-weighted 9.4T magnetic resonance imaging and graded by size. Results: Control mice displayed an average of 35 ± 18.5 cMBs (mean ± standard deviation), compared to 29.3 ± 9.8 in mice with eight months (p = 0.49), 24.9 ± 21.3 with 12 months (p = 0.26), and 27.8 ± 15.4 with 16 months of atorvastatin treatment (p = 0.27). In combined analysis treated mice showed lower absolute numbers (27.4 ± 15.6, p = 0.16) compared to controls and also after adjustment for cMB size (p = 0.13). Conclusion: Despite to a non-significant trend towards fewer cMBs our results failed to provide evidence for beneficial effects of long-term atorvastatin treatment in the APP23-transgenic mouse model of CAA. A higher risk for bleeding complications was not observed. Full article

(This article belongs to the Special Issue Amyloid-beta and Neurological Diseases)

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(A) Number of cerebral microbleeds (cMBs) in MRI and APP23-tg mice treated with atorvastatin for eight months (n = 6), 12 months (n = 7), and 16 months (n = 13), respectively. Compared to controls (n = 17) and in pooled analysis no significant differences between the groups were observed; (B) Histologically-assessed numbers of cMBs per slide in untreated mice (n = 6) did not differ significantly from those in mice treated for 16 months with atorvastatin (n = 7); (C) In these mice total numbers of thioflavin S positive vessels, graded by severity of amyloid β (Aβ) deposits, and after calculation of a cerebral amyloid angiopathy (CAA) severity score showed no significant difference between the groups. Full article ">Figure 2
Time schedule of the study. APP23-tg mice received either standard food over the whole study period (controls, n = 17 with brain imaging and histology) or atorvastatin-supplemented food starting at the age of eight months (n = 13), 12 months (n = 7), and 16 months (n = 6), respectively. Full article ">Figure 3
T2* weighted and time of flight magnetic resonance angiography (TOF-MRA) image of brain vessels and vessel associated cMBs in an APP23-tg mouse obtained by 9.4 T animal scanner. (A) Representative T2* weighted image with two hypointense areas (arrows); and (B) brepresentative TOF-MRA image of corresponding brain section from the same animal with a hypointense signal representing a cMB and a hyperintense signal representing a brain vessel. Full article ">Figure 4
T2* weighted image of cMBs in an APP23-tg mouse obtained with 9.4 T animal scanner. Representative T2* weighted image of an APP23-tg mouse shows multiple cMBs which were graded by size (left image). Representative graded cMBs are shown belonging to group A ≤ 100 µm (red arrow), B = 150 ≤ x ≤ 200 µm and C > 200 µm (right images). Full article ">Figure 5
Fluorescence analysis of affected brain vessels demonstrating Aβ burden. Representative images of Aβ affected thioflavin S stained vessels in APP-tg mice. Vessels were graded into score 1, 2 or 3 depending on degree of Aβ burden. Blue = DAPI (4′,6-Diamidin-2-Phenylindol); red = STL (biotinylated Solanum Tuberosum (Potato) Lectin); green = thioflavin S. Full article ">

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Open AccessArticle

Influence of Bxpel1 Gene Silencing by dsRNA Interference on the Development and Pathogenicity of the Pine Wood Nematode, Bursaphelenchus xylophilus

by Xiu-Wen Qiu, Xiao-Qin Wu, Lin Huang and Jian-Ren Ye

Int. J. Mol. Sci. 2016, 17(1), 125; https://doi.org/10.3390/ijms17010125 - 19 Jan 2016

Cited by 20 | Viewed by 5559

Abstract

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To [...] Read more.

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p < 0.001) after treatment with dsRNAi compared with that using a control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD. Full article

(This article belongs to the Section Biochemistry)

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Open AccessReview

Molecular Regulation of Adipogenesis and Potential Anti-Adipogenic Bioactive Molecules

by Dorothy Moseti, Alemu Regassa and Woo-Kyun Kim

Int. J. Mol. Sci. 2016, 17(1), 124; https://doi.org/10.3390/ijms17010124 - 19 Jan 2016

Cited by 506 | Viewed by 17830

Abstract

Adipogenesis is the process by which precursor stem cells differentiate into lipid laden adipocytes. Adipogenesis is regulated by a complex and highly orchestrated gene expression program. In mammalian cells, the peroxisome proliferator-activated receptor γ (PPARγ), and the CCAAT/enhancer binding proteins (C/EBPs) such as [...] Read more.

Adipogenesis is the process by which precursor stem cells differentiate into lipid laden adipocytes. Adipogenesis is regulated by a complex and highly orchestrated gene expression program. In mammalian cells, the peroxisome proliferator-activated receptor γ (PPARγ), and the CCAAT/enhancer binding proteins (C/EBPs) such as C/EBPα, β and δ are considered the key early regulators of adipogenesis, while fatty acid binding protein 4 (FABP4), adiponectin, and fatty acid synthase (FAS) are responsible for the formation of mature adipocytes. Excess accumulation of lipids in the adipose tissue leads to obesity, which is associated with cardiovascular diseases, type II diabetes and other pathologies. Thus, investigating adipose tissue development and the underlying molecular mechanisms is vital to develop therapeutic agents capable of curbing the increasing incidence of obesity and related pathologies. In this review, we address the process of adipogenic differentiation, key transcription factors and proteins involved, adipogenic regulators and potential anti-adipogenic bioactive molecules. Full article

(This article belongs to the Section Biochemistry)

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Open AccessArticle

Genipin Derivatives Protect RGC-5 from Sodium Nitroprusside-Induced Nitrosative Stress

by Rikang Wang, Jiaqiang Zhao, Lei Zhang, Lizhi Peng, Xinyi Zhang, Wenhua Zheng and Heru Chen

Int. J. Mol. Sci. 2016, 17(1), 117; https://doi.org/10.3390/ijms17010117 - 19 Jan 2016

Cited by 5 | Viewed by 5376

Abstract

CHR20 and CHR21 are a pair of stable diastereoisomers derived from genipin. These stereoisomers are activators of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). In the rat retinal ganglion (RGC-5) cell model these compounds are non-toxic. Treatment of RGC-5 [...] Read more.

CHR20 and CHR21 are a pair of stable diastereoisomers derived from genipin. These stereoisomers are activators of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). In the rat retinal ganglion (RGC-5) cell model these compounds are non-toxic. Treatment of RGC-5 with 750 μM of sodium nitroprusside (SNP) produces nitrosative stress. Both genipin derivatives, however, protect these cells against SNP-induced apoptic cell death, although CHR21 is significantly more potent than CHR20 in this regard. With Western blotting we showed that the observed neuroprotection is primarily due to the activation of protein kinase B (Akt)/eNOS and extracellular signal-regulated kinase (ERK1/2) signaling pathways. Therefore, LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or PD98059 (a MAPK-activating enzyme inhibitor) abrogated the protective effects of CHR20 and CHR21. Altogether, our results show that in our experimental setup neuroprotection by the diasteromeric pair is mediated through the PI3K/Akt/eNOS and ERK1/2 signaling pathways. Further studies are needed to establish the potential of these compounds to prevent ntric oxide (NO)-induced toxicity commonly seen in many neurodegenerative diseases. Full article

(This article belongs to the Special Issue Neuroprotective Strategies 2015)

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Open AccessArticle

Technological Implications of Modifying the Extent of Cell Wall-Proanthocyanidin Interactions Using Enzymes

by Ana Belén Bautista-Ortín, Rim Ben Abdallah, Liliana Del Rocío Castro-López, María Dolores Jiménez-Martínez and Encarna Gómez-Plaza

Int. J. Mol. Sci. 2016, 17(1), 123; https://doi.org/10.3390/ijms17010123 - 18 Jan 2016

Cited by 27 | Viewed by 5926

Abstract

The transference and reactivity of proanthocyanidins is an important issue that affects the technological processing of some fruits, such as grapes and apples. These processes are affected by proanthocyanidins bound to cell wall polysaccharides, which are present in high concentrations during the processing [...] Read more.

The transference and reactivity of proanthocyanidins is an important issue that affects the technological processing of some fruits, such as grapes and apples. These processes are affected by proanthocyanidins bound to cell wall polysaccharides, which are present in high concentrations during the processing of the fruits. Therefore, the effective extraction of proanthocyanidins from fruits to their juices or derived products will depend on the ability to manage these associations, and, in this respect, enzymes that degrade these polysaccharides could play an important role. The main objective of this work was to test the role of pure hydrolytic enzymes (polygalacturonase and cellulose) and a commercial enzyme containing these two activities on the extent of proanthocyanidin-cell wall interactions. The results showed that the modification promoted by enzymes reduced the amount of proanthocyanidins adsorbed to cell walls since they contributed to the degradation and release of the cell wall polysaccharides, which diffused into the model solution. Some of these released polysaccharides also presented some reactivity towards the proanthocyanidins present in a model solution. Full article

(This article belongs to the Special Issue Phenolics and Polyphenolics 2015)

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Open AccessArticle

Metformin Restores Parkin-Mediated Mitophagy, Suppressed by Cytosolic p53

by Young Mi Song, Woo Kyung Lee, Yong-ho Lee, Eun Seok Kang, Bong-Soo Cha and Byung-Wan Lee

Int. J. Mol. Sci. 2016, 17(1), 122; https://doi.org/10.3390/ijms17010122 - 16 Jan 2016

Cited by 71 | Viewed by 10227

Abstract

Metformin is known to alleviate hepatosteatosis by inducing 5’ adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the [...] Read more.

Metformin is known to alleviate hepatosteatosis by inducing 5’ adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1) ad libitum feeding of a standard chow diet; (2) intraperitoneal injections of metformin 300 mg/kg; and (3) 3 g/day caloric restriction (CR). HepG2 cells were treated with palmitate (PA) plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER) stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins. Full article

(This article belongs to the Special Issue Modulators of Endoplasmic Reticulum Stress)

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Metformin induced mitophagy. Electron microscopy was performed on hepatocytes from ob/ob mice under ad libitum feeding of chow diet (A-a), caloric restriction (A-b), and ad libitum feeding of chow diet with 300 mg/kg metformin treatment (A-c). In the ad libitum group, spheroid mitochondria were readily detected. However, in the metformin-treated group, autophagic double membranes were detected, indicating mitophagy; (B) co-localization between a marker of autophagosomes (LC3) and a mitochondrial marker (MitoTracker Red) was assessed in HepG2 cells stably transfected with green fluorescent protein (GFP)-LC3. Although there was little co-localization between mitochondria and autophagosomes in the 0.25 mM palmitate-treated HepG2 cells, LC3-labeled structures were seen surrounding the fragmented mitochondria in cells treated with 0.5 mM metformin and 0.25 mM palmitate. Scale bar = 20 μm. Full article ">Figure 2
Metformin restored the Parkin-mediated mitophagy inhibited by glucolipotoxicity-induced cytosolic p53. Western blotting analysis was performed to assess cytosolic p53 levels with the cytosolic fractions isolated from palmitate- (A-a) or metformin- (A-b) and 35 mM glucose-treated HepG2 cells; the expression of cytosolic p53 protein was assessed following 0.5 mM metformin and/or 0.25 mM palmitate with 35 mM glucose treatment of HepG2 cells (B-a) or in hepatocytes from ob/ob mice with ad libitum feeding of chow diet, caloric restriction, and ad libitum feeding of chow diet with 300 mg/kg metformin treatment (B-b); (C) Expression of Parkin (green) on mitochondria (MitoTracker Red) was observed with a confocal microscopy. The bottom panels show enlarged views of the boxed areas. The yellow dots indicate the mitochondria that co-localize with Parkin. The cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria depending on the conditions used in the HepG2 cells; a co-immunoprecipitation assay was conducted to investigate the interaction between p53 and Parkin, depending on the conditions used in the HepG2 cells (D-a) or in hepatocytes from mice (D-b). GAPDH or COX IV was used for normalization. Values displayed are mean ± SEM (n = 3 independent experiments, respectively). Scale bar: white = 20 μm, yellow = 10 μm. Asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate significant differences. Abbreviation: Mfn1, mitofusin1. Full article ">Figure 2 Cont.
Metformin restored the Parkin-mediated mitophagy inhibited by glucolipotoxicity-induced cytosolic p53. Western blotting analysis was performed to assess cytosolic p53 levels with the cytosolic fractions isolated from palmitate- (A-a) or metformin- (A-b) and 35 mM glucose-treated HepG2 cells; the expression of cytosolic p53 protein was assessed following 0.5 mM metformin and/or 0.25 mM palmitate with 35 mM glucose treatment of HepG2 cells (B-a) or in hepatocytes from ob/ob mice with ad libitum feeding of chow diet, caloric restriction, and ad libitum feeding of chow diet with 300 mg/kg metformin treatment (B-b); (C) Expression of Parkin (green) on mitochondria (MitoTracker Red) was observed with a confocal microscopy. The bottom panels show enlarged views of the boxed areas. The yellow dots indicate the mitochondria that co-localize with Parkin. The cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria depending on the conditions used in the HepG2 cells; a co-immunoprecipitation assay was conducted to investigate the interaction between p53 and Parkin, depending on the conditions used in the HepG2 cells (D-a) or in hepatocytes from mice (D-b). GAPDH or COX IV was used for normalization. Values displayed are mean ± SEM (n = 3 independent experiments, respectively). Scale bar: white = 20 μm, yellow = 10 μm. Asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate significant differences. Abbreviation: Mfn1, mitofusin1. Full article ">Figure 3
Metformin restored Parkin-mediated mitophagy inhibited by glucolipotoxicity-induced ER stress. The expression of ER stress markers was assessed following 0.5 mM metformin and/or 0.25 mM palmitate with 35 mM glucose treatment of HepG2 cells (A-a) or in hepatocytes from ob/ob mice with ad libitum feeding of chow diet, caloric restriction, or ad libitum feeding of chow diet with 300 mg/kg metformin treatment (A-b) using Western blotting analysis. Values displayed are mean ± SEM (n = 3 independent experiments, respectively); (B) the expression of ER stress markers and p53 protein was assessed in response to thapsigargin in HepG2 cells; (C-a) cell viability was assessed in HepG2 cells exposed to 0.1 μM thapsigargin and/or 0.5 mM metformin using CCK-8. Values displayed are mean ± SEM (n = 5 independent experiments); (C-b) the expression of an ER stress marker was also examined under these conditions; (C-c) the cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria, depending on the conditions used. Values displayed are mean ± SEM (n = 3 independent experiments, respectively). GAPDH or COX IV was used for normalization. Asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate significant differences. Abbreviation: Mfn1, mitofusin1; CCK-8, cell counting kit-8. Full article ">Figure 3 Cont.
Metformin restored Parkin-mediated mitophagy inhibited by glucolipotoxicity-induced ER stress. The expression of ER stress markers was assessed following 0.5 mM metformin and/or 0.25 mM palmitate with 35 mM glucose treatment of HepG2 cells (A-a) or in hepatocytes from ob/ob mice with ad libitum feeding of chow diet, caloric restriction, or ad libitum feeding of chow diet with 300 mg/kg metformin treatment (A-b) using Western blotting analysis. Values displayed are mean ± SEM (n = 3 independent experiments, respectively); (B) the expression of ER stress markers and p53 protein was assessed in response to thapsigargin in HepG2 cells; (C-a) cell viability was assessed in HepG2 cells exposed to 0.1 μM thapsigargin and/or 0.5 mM metformin using CCK-8. Values displayed are mean ± SEM (n = 5 independent experiments); (C-b) the expression of an ER stress marker was also examined under these conditions; (C-c) the cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria, depending on the conditions used. Values displayed are mean ± SEM (n = 3 independent experiments, respectively). GAPDH or COX IV was used for normalization. Asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate significant differences. Abbreviation: Mfn1, mitofusin1; CCK-8, cell counting kit-8. Full article ">Figure 4
A p53 inhibitor restored the Parkin-mediated mitophagy inhibited by glucolipotoxicity. (A-a) the expression of cytosolic p53 was assessed in HepG2 cells treated with 50 μM PFT-α, a p53 inhibitor, and/or 0.25 mM palmitate with 35 mM glucose using Western blotting analysis; (A-b) the cytosolic and mitochondrial fractionation experiments demonstrated Parkin translocation from the cytosol to mitochondria, depending on the conditions used. Exposure time of Western blotting was long, compared to <a href="#ijms-17-00122-f002" class="html-fig">Figure 2</a>C; (B) co-localization was assessed between a marker of autophagosomes (LC3) and a mitochondrial marker (MitoTracker Red) in HepG2 cells stably transfected with GFP-LC3, depending on the conditions used. LC3-labeled structures were observed surrounding the fragmented mitochondria in cells treated with PFT-α and palmitate, although there was little co-localization between mitochondria and autophagosomes in cells treated with 0.25 mM palmitate alone. Scale bar = 20 μm. Abbreviation: Mfn1, mitofusin1; PFT-α, pifithrin-α. Full article ">Figure 5
Summary of the working thesis of this study. See the text for details. Black and red arrows indicate the effects of glucolipotoxicity and the drugs (metformin or PFTα), respectively. Full article ">

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Open AccessArticle

Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer

by Rosanna Aversa, Anna Sorrentino, Roberta Esposito, Maria Rosaria Ambrosio, Angela Amato, Alberto Zambelli, Alfredo Ciccodicola, Luciana D’Apice and Valerio Costa

Int. J. Mol. Sci. 2016, 17(1), 121; https://doi.org/10.3390/ijms17010121 - 16 Jan 2016

Cited by 16 | Viewed by 6470

Abstract

Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells [...] Read more.

Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations—in human breast cell lines and breast tumor biopsies—we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression. Full article

(This article belongs to the Special Issue Pre-mRNA Splicing 2015)

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Graphical abstract

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Full article ">Figure 1
(A) Overview of RNA-Seq data processing and of the computational and experimental approach used for the identification and validation of the new spliced transcripts; (B) Barplot with log2 normalized expression values (FPKM) of semaphorin/Plexin/Neuropilin—and of the adhesion markers CDH1 and CD44—according to RNA-Seq data. Full article ">Figure 2
Schematic representation of de novo identified transcripts for adhesion-related genes and their differential expression by RT-PCR in breast cell lines (a representative image is shown for each gene). (A) THBS1 new transcript; (B) RAP1GAP new transcript; (C) LGALS1 new transcript; (D) CD47 new transcript. For each new transcript, in the upper part is shown the genomic region encompassing the gene; red arrows indicate the direction of transcription. Below, the exon/intron structure of the gene is depicted. Red cross indicates the exons that are spliced out in the new transcript. The electropherogram and the nucleotide sequence above refer to the new splice junction identified. Images of PCR amplicons on agarose gel from three independent replicates experiments were acquired. For all panels, M is 100 bp DNA marker. The black arrows indicate the PCR band corresponding to the newly identified transcripts. Pixel density of each band (only for the newly identified mRNA isoforms) was quantified by ImageJ software and compared to the pixel density of the 100 bp DNA marker (at known concentration) to normalize data across replicates and cell lines. GAPDH gel bands are reported below to show that the starting amount of template was the same for all analyzed samples. Values are reported as AU = Arbitrary Units in the boxplots. Dark horizontal lines represent the mean, the box represents the 25th and 75th percentiles and the whiskers the 5th and 95th percentiles. Asterisks indicate significant p values (* p < 0.05). Full article ">Figure 3
Schematic representation of de novo identified transcripts of motility-related genes and their expression by RT-PCR in breast cell lines (a representative image is shown for each gene). (A) RHOA; (B) RHOD; (C) CASK; (D) JAG2; (E) CTTN new transcripts. Details about the gel bands and their quantification are provided in <a href="#ijms-17-00121-f002" class="html-fig">Figure 2</a> legend. Asterisks indicate significant p values (* p < 0.05; ** p < 0.01). Full article ">Figure 4
Schematic representation of de novo identified transcripts for (A) SEMA3C (B) PLXNB1 genes. Nucleotide sequenceand their expression by RT-PCR in breast cell lines are also depicted. Details about the gel bands and their quantification are provided in <a href="#ijms-17-00121-f002" class="html-fig">Figure 2</a>’s legend. Asterisks indicate significant p values (** p < 0.01). Full article ">Figure 5
(A) Scheme of the new SEMA3F transcript and expression by RT-PCR on MCF-7 cell line, showing the canonical (307 bp) and the new alternative transcript (149 bp) of SEMA3F; M is 100 bp DNA marker; (B) Boxplots with the relative expression of both annotated and new SEMA3F transcript in breast cell lines measured with qRT-PCR (n = 3); (C) On the left, immunoreactive bands—corresponding to the canonical (about 90 kDa) and the new isoform (about 70 kDa) of semaphorin 3F—detected by Western Blot on whole breast cell lines lysates. On the right, the densitometry results (AU = Arbitrary Units) by ImageJ of the Western Blot bands, after normalization with actin, are shown as bar graphs (n = 2); (D) A detail of the protein alignment of the canonical and the new semaphorin 3F protein isoform is shown. The dashed boxes represent the functional domains that are deleted in the novel protein sequence. Asterisks indicate significant p values (* p < 0.05; ** p < 0.01; *** p < 0.001). Full article ">Figure 6
Semaphorin 3F expression in BC tumor biopsies (n = 18). (A,B) qRT-PCR analysis of the new (LN626688) and the annotated (NM_004186.3) SEMA3F transcripts, respectively. Samples have been ordered according to tumor grade (G2, G3); # indicates Triple Negative biopsies; asterisks (*) indicate the sample with the lowest absolute expression, used as reference; (C) Relative ratio of ΔCt values between skipping and canonical mRNA isoforms of SEMA3F across breast cancer subtypes. TNM: pT1 (n = 7); pT2 (n = 9); pT3 (n = 2). Tumor grade: G2 (n = 11); G3 (n = 7). ER+/PR+ (n = 15); TN (triple negative samples) (n = 3); (D) Six representative images by immunohistochemistry assay on FFPE slices from breast cancer specimens are shown; N-terminal anti-Sema3F (that recognizes both the canonical and the new splicing variant) primary antibody was used. Scale Bar (in red) value = 33.76 µm. Full article ">

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Open AccessArticle

Platelet-Rich Plasma Increases the Levels of Catabolic Molecules and Cellular Dedifferentiation in the Meniscus of a Rabbit Model

by Hye-Rim Lee, Oog-Jin Shon, Se-Il Park, Han-Jun Kim, Sukyoung Kim, Myun-Whan Ahn and Sun Hee Do

Int. J. Mol. Sci. 2016, 17(1), 120; https://doi.org/10.3390/ijms17010120 - 16 Jan 2016

Cited by 33 | Viewed by 7236

Abstract

Despite the susceptibility to frequent intrinsic and extrinsic injuries, especially in the inner zone, the meniscus does not heal spontaneously owing to its poor vascularity. In this study, the effect of platelet-rich plasma (PRP), containing various growth factors, on meniscal mechanisms was examined [...] Read more.

Despite the susceptibility to frequent intrinsic and extrinsic injuries, especially in the inner zone, the meniscus does not heal spontaneously owing to its poor vascularity. In this study, the effect of platelet-rich plasma (PRP), containing various growth factors, on meniscal mechanisms was examined under normal and post-traumatic inflammatory conditions. Isolated primary meniscal cells of New Zealand white (NZW) rabbits were incubated for 3, 10, 14 and 21 days with PRP(−), 10% PRP (PRP(+)), IL(+) or IL(+)PRP(+). The meniscal cells were collected and examined using reverse-transcription polymerase chain reaction (RT-PCR). Culture media were examined by immunoblot analyses for matrix metalloproteinases (MMP) catabolic molecules. PRP containing growth factors improved the cellular viability of meniscal cells in a concentration-dependent manner at Days 1, 4 and 7. However, based on RT-PCR, meniscal cells demonstrated dedifferentiation, along with an increase in type I collagen in the PRP(+) and in IL(+)PRP(+). In PRP(+), the aggrecan expression levels were lower than in the PRP(−) until Day 21. The protein levels of MMP-1 and MMP-3 were higher in each PRP group, i.e., PRP(+) and IL(+)PRP(+), at each culture time. A reproducible 2-mm circular defect on the meniscus of NZW rabbit was used to implant fibrin glue (control) or PRP in vivo. After eight weeks, the lesions in the control and PRP groups were occupied with fibrous tissue, but not with meniscal cells. This study shows that PRP treatment of the meniscus results in an increase of catabolic molecules, especially those related to IL-1α-induced inflammation, and that PRP treatment for an in vivo meniscus injury accelerates fibrosis, instead of meniscal cartilage. Full article

(This article belongs to the Section Biochemistry)

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3003 KiB

Open AccessArticle

Cloning and Transcriptional Activity of the Mouse Omi/HtrA2 Gene Promoter

by Dan Liu, Xin Liu, Ye Wu, Wen Wang, Xinliang Ma and Huirong Liu

Int. J. Mol. Sci. 2016, 17(1), 119; https://doi.org/10.3390/ijms17010119 - 16 Jan 2016

Cited by 10 | Viewed by 6074

Abstract

HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney [...] Read more.

HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at −1205~−838 bp and −146~+93 bp, with the −838~−649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at −709~+37 bp, and eight heat shock transcription factor 1 (HSF1) sites, two Sp1 transcription factor (SP1)sites, one activator protein (AP) site, seven p53 sites, and four YY1 transcription factor(YY1) sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases. Full article

(This article belongs to the Section Biochemistry)

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Figure 1

Figure 1
Tissue specific expression of HtrA serine peptidase 2 (Omi/HtrA2). Omi/HtrA2 protein expression in young and aging mice was detected by Western blotting (A), and the relative levels were quantified using ImageJ software (B). Omi/HtrA2 mRNA expression was detected by quantitative RT-PCR (C). n = 5 per group. * p < 0.05 vs. young. Full article ">Figure 2
Verification of mouse Omi/HtrA2 promoter luciferase expression plasmids. Plasmids were verified by double enzyme digestion with KpnI and HindIII, which releases a vector fragment and a variable-sized promoter insert. M: DL2000 Marker; 1: pGL3-239; 2: pGL3-472; 3: pGL3-742; 4: pGL3-931; 5: pGL3-1298. Luc: luciferase expression plasmids. Full article ">Figure 3
Relative luciferase activity of full length and truncated mouse Omi/HtrA2 promoter plasmids. Luciferase assays were performed in NIH3T3 cells (A); H9c2 cells (B); and HEK-293 cells (C). The different letters indicate the significant differences between the plasmids (p < 0.05). Full article ">Figure 4
Chromatin Immunoprecipitation(ChIP) was performed with chromatin prepared from mouse heart. The promoter region containing the Heat shock transcription factor 1 (HSF1) and p53 site was analysed by PCR following immunoprecipitation with the HSF1 and p53 antibodies. Results of amplification for soluble chromatin before immunoprecipitation are shown as positive control (input) and negative control (IgG). Full article ">

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Open AccessArticle

Ultra-Fast Glyco-Coating of Non-Biological Surfaces

by Eleanor Williams, Katie Barr, Elena Korchagina, Alexander Tuzikov, Stephen Henry and Nicolai Bovin

Int. J. Mol. Sci. 2016, 17(1), 118; https://doi.org/10.3390/ijms17010118 - 16 Jan 2016

Cited by 13 | Viewed by 6480

Abstract

The ability to glycosylate surfaces has medical and diagnostic applications, but there is no technology currently recognized as being able to coat any surface without the need for prior chemical modification of the surface. Recently, a family of constructs called function-spacer-lipids (FSL) has [...] Read more.

The ability to glycosylate surfaces has medical and diagnostic applications, but there is no technology currently recognized as being able to coat any surface without the need for prior chemical modification of the surface. Recently, a family of constructs called function-spacer-lipids (FSL) has been used to glycosylate cells. Because it is known that lipid-based material can adsorb onto surfaces, we explored the potential and performance of cell-labelling FSL constructs to “glycosylate” non-biological surfaces. Using blood group A antigen as an indicator, the performance of a several variations of FSL constructs to modify a large variety of non-biological surfaces was evaluated. It was found the FSL constructs when optimised could in a few seconds glycosylate almost any non-biological surface including metals, glass, plastics, rubbers and other polymers. Although the FSL glycan coating was non-covalent, and therefore temporary, it was sufficiently robust with appropriate selection of spacer and surface that it could capture anti-glycan antibodies, immobilize cells (via antibody), and withstand incubation in serum and extensive buffer washing, making it suitable for diagnostic and research applications. Full article

(This article belongs to the Special Issue Glycosylation and Glycoproteins)

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1416 KiB

Open AccessArticle

Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry

by Laszlo Prokai and Stanley M. Stevens

Int. J. Mol. Sci. 2016, 17(1), 116; https://doi.org/10.3390/ijms17010116 - 16 Jan 2016

Cited by 7 | Viewed by 6461

Abstract

Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance [...] Read more.

Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae. Full article

(This article belongs to the Special Issue Fourier Transform Mass Spectrometry in Molecular Sciences)

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Full article ">Figure 1
Illustration of the sampling method: (a) About 1.3 ± 0.1 mL of aqueous 0.4% w/v malathion solution was sprayed from approximately 30 cm distance from a household spray bottle (foreground) by a single application of the lever onto a concrete wall in the background; (b) After swabbing the surface across the sprayed area using cotton-tipped applicators with their wooden handle glued in scintillation-vial caps (on the left), the swabs were screwed into the vials (on the right) to protect from sample loss and cross-contamination. Full article ">Figure 2
(a) Direct analysis in real time (DART) mass spectrum of a sample collected by swabbing from the field (<a href="#ijms-17-00116-f001" class="html-fig">Figure 1</a>) using FT-ICR with desired M/ΔM set to 100,000 FWHM at m/z 400 (Mass spectrum averaged for the entire acquisition period); actual M/ΔM (FWHM) achieved for (b) the protonated malathion (nominal m/z 331) and (c) DART fragment ion of the compound (nominal m/z 285) at set mass resolution of 100,000 at m/z 400 (The insets show the actual M/ΔM with desired mass resolution set to 500,000 at m/z 400). Full article ">Figure 3
Product ion MS/MS of protonated malathion (m/z 331) from a sample collected by swabbing from the field (<a href="#ijms-17-00116-f001" class="html-fig">Figure 1</a>) followed by DART–MS using a hybrid mass spectrometer with CID in the linear ion trap and fragment ions detected in the FT-ICR (set M/ΔM of 100,000 FWHM at m/z 400). Inset: The fragment ion m/z 285 (set M/ΔM of 500,000 FWHM at m/z 400). The fragmentation leading to the observed ion is indicated by the thick dashed line on the displayed structure of the compound. Full article ">Figure 4
Relative abundances of the measured A+1 and A+2 isotope peaks (bottom trace), as well as isotopic fine structure of the A+2 isotope peak (inset over the bottom trace labeled “Measured”) for the protonated analyte also affords the best match with those predicted for C10H20O6PS2. The isotope peak abundances and isotopic fine structures for the indicated elemental compositions listed in <a href="#ijms-17-00116-t001" class="html-table">Table 1</a>a (colored traces and insets, respectively) were predicted presuming M/ΔM of 120,000, FWHM. Full article ">

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Open AccessArticle

Cerebellar Expression of the Neurotrophin Receptor p75 in Naked-Ataxia Mutant Mouse

by Maryam Rahimi Balaei, Xiaodan Jiao, Niloufar Ashtari, Pegah Afsharinezhad, Saeid Ghavami and Hassan Marzban

Int. J. Mol. Sci. 2016, 17(1), 115; https://doi.org/10.3390/ijms17010115 - 15 Jan 2016

Cited by 13 | Viewed by 6740

Abstract

Spontaneous mutation in the lysosomal acid phosphatase 2 (Acp2) mouse (nax—naked-ataxia mutant mouse) correlates with severe cerebellar defects including ataxia, reduced size and abnormal lobulation as well as Purkinje cell (Pc) degeneration. Loss of Pcs in the nax cerebellum is compartmentalized [...] Read more.

Spontaneous mutation in the lysosomal acid phosphatase 2 (Acp2) mouse (nax—naked-ataxia mutant mouse) correlates with severe cerebellar defects including ataxia, reduced size and abnormal lobulation as well as Purkinje cell (Pc) degeneration. Loss of Pcs in the nax cerebellum is compartmentalized and harmonized to the classic pattern of gene expression of the cerebellum in the wild type mouse. Usually, degeneration starts in the anterior and posterior zones and continues to the central and nodular zones of cerebellum. Studies have suggested that the p75 neurotrophin receptor (NTR) plays a role in Pc degeneration; thus, in this study, we investigated the p75NTR pattern and protein expression in the cerebellum of the nax mutant mouse. Despite massive Pc degeneration that was observed in the nax mouse cerebellum, p75NTR pattern expression was similar to the HSP25 pattern in nax mice and comparable with wild type sibling cerebellum. In addition, immunoblot analysis of p75NTR protein expression did not show any significant difference between nax and wild type sibling (p > 0.5). In comparison with wild type counterparts, p75NTR pattern expression is aligned with the fundamental cytoarchitecture organization of the cerebellum and is unchanged in the nax mouse cerebellum despite the severe neurodevelopmental disorder accompanied with Pc degeneration. Full article

(This article belongs to the Special Issue Mechanisms of Neurodegeneration)

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Frontal sections of an adult mouse cerebellum immunostained with p75NTR, CaBP and HSP25 at P28. The lobules are shown by Roman numerals. (A,B) The cerebellar cortex immunoperoxidase stained by p75NTR shows that the Pc somata in the Purkinje cell layer and dendrites in the molecular layer are immunoreactive (arrow; black in A, and white in B). Some Pc somata expressing p75NTR are weak (A, arrowhead) or absent (B, arrowhead); (C–E) Double immunostaining of a frontal section at lobule IX and X using p75NTR (red) and CaBP (green). Stripes of immunoreactive Pcs are clear in the vermis; (F) Immunostaining of a frontal section at the cerebellum using p75NTR. Immunoreactivity is not present in the AZ and PZ. Stripes of immunoreactive Pcs are clear in the vermis of the CZ (VII) and NZ (X); (G) Immunostaining of a frontal section through the lobule VII using HSP25. Stripes of immunoreactive Pcs are clear in the vermis of the CZ. Abbreviations: ml, molecular layer; Pcl, Purkinje cell layer; gl, granular layer.Scale bars: A = 100 µm; B = 20 µm; E = 250 μm (applies to C–E); F = 500 µm; G = 250 µm. Full article ">Figure 2
Sagittal sections of P4 wt+/+ (A–C) and nax−/− (D–I) and sagittal sections of P6 wt (J) and nax (K) cerebella immunostained with p75NTR, CaBP and Pax6. The lobules are shown by Roman numerals. (A–C) The wt cerebellum shows normal lobules (A) with expression of p75NTR (red) in the entire egz (external germinal zone)and dispersing through the Pc layers. A higher magnification of “A” shown in “B” and Pax6 immunostaining is indicated egz in “C”; (D–F) The nax cerebellum shows a small cerebellum with underdeveloped lobulation (B). P75NTR expression (red) is present in the underdeveloped egz and in the developing Pcl (B). Immunostaining with Pax6 shows the thin layer of the egz in the nax cerebellum (F); (G–I) Magnified views of white box in E showing CaBP (G), p75NTR (H), and merged image (I); (J–K) The sagittal sections immunoperoxidase stained by p75NTR shows immunoreactivity of the Pc in wt (J) and nax (K) cerebella. Abbreviations: egz, external germinal zone; Pcl, Purkinje cell layer. Scale bars: D = 500 µm (applies to A and D); E = 50 µm (applies to B and E); F = 40 μm (applies to C and F); J = 50 µm; K = 50 µm. Full article ">Figure 3
Frontal sections at the cerebellum of wt (A,B) and nax (C,D) at P6 immunoperoxidase stained with p75NTR. (A,B) The wt cerebellum shows normal vermis (v) and hemisphere (h) lobulation (A) with uniform expression of p75NTR in the entire cerebellar cortex. A higher magnification of black box (b) in “A” indicates localization of p75NTR expression in Pcl and EGZ; (C,D) Underdeveloped nax cerebellum exhibits uniform p75NTR expression in putative vermis and in the hemisphere. Magnified views of black box (d) in “C” showing p75NTR in multilayer Pc and thin or lack of EGZ. The p75NTR expression shows the outline of cerebellar nuclei (cn) both in wt (A) and nax (C) sections. Abbreviations: egz, external germinal zone; Pcl, Purkinje cell layer; h, hemisphere; cn, cerebellar nuclei; gl, granular layer. Scale bars: A and C = 500 µm; D = 100 µm (applies to B and D). Full article ">Figure 4
Transverse sections of nax−/− (A–D) and wt sibling cerebella at P22 immunostained with p75NTR, CaBP and HSP25. (A,B) P75NTR immunostaining shows an array of parasagittal stripes in the CZ and NZ of the nax cerebellum, with high magnification of the CZ shown in B; (C,D) HSP25 immunostaining of the nax cerebellum shows parasagittal stripes in the CZ and NZ, D is a higher magnification of C; (E) At a high magnification, p75NTR expression is shown in the CZ of the wt+/+ sibling. Scale bars: C = 1 mm (applies to A and C); D = 500 µm (applies to B and D); E = 250 μm. Full article ">Figure 5
Western blot analysis of p75NTR expression during cerebellar development at P5, P9 and P18. This experiment was repeated over three different litters for each postnatal days in wt and nax siblings (P5, P9 and P18, wt; n = 9 and nax; n = 9). (A) Immunoblots of total cell lysate from wt sibling and nax mouse cerebellum indicate significant down-regulation in the wt sibling and also in the nax cerebellum from early postnatal development to P18. However, there is no considerable difference in p75NTR expression between the wt and nax cerebellum, despite the higher p75NTR expression in nax compared with wt. (p > 0.05). Protein loading was confirmed using GAPDH (n = 18); (B) The data in the bar graph are presented as the mean ± SEM, and statistical analysis was performed using Kruskal-Wallis test followed by Dunnett’s multiple comparison test using Mann-Whitney test. Abbreviation: ns, not significant. Full article ">Figure 5 Cont.
Western blot analysis of p75NTR expression during cerebellar development at P5, P9 and P18. This experiment was repeated over three different litters for each postnatal days in wt and nax siblings (P5, P9 and P18, wt; n = 9 and nax; n = 9). (A) Immunoblots of total cell lysate from wt sibling and nax mouse cerebellum indicate significant down-regulation in the wt sibling and also in the nax cerebellum from early postnatal development to P18. However, there is no considerable difference in p75NTR expression between the wt and nax cerebellum, despite the higher p75NTR expression in nax compared with wt. (p > 0.05). Protein loading was confirmed using GAPDH (n = 18); (B) The data in the bar graph are presented as the mean ± SEM, and statistical analysis was performed using Kruskal-Wallis test followed by Dunnett’s multiple comparison test using Mann-Whitney test. Abbreviation: ns, not significant. Full article ">

464 KiB

Open AccessArticle

Herb-Induced Liver Injury in the Berlin Case-Control Surveillance Study

by Antonios Douros, Elisabeth Bronder, Frank Andersohn, Andreas Klimpel, Reinhold Kreutz, Edeltraut Garbe and Juliane Bolbrinker

Int. J. Mol. Sci. 2016, 17(1), 114; https://doi.org/10.3390/ijms17010114 - 15 Jan 2016

Cited by 31 | Viewed by 10765

Abstract

Herb-induced liver injury (HILI) has recently attracted attention due to increasing reports of hepatotoxicity associated with use of phytotherapeutics. Here, we present data on HILI from the Berlin Case-Control Surveillance Study. The study was initiated in 2000 to investigate the serious toxicity of [...] Read more.

Herb-induced liver injury (HILI) has recently attracted attention due to increasing reports of hepatotoxicity associated with use of phytotherapeutics. Here, we present data on HILI from the Berlin Case-Control Surveillance Study. The study was initiated in 2000 to investigate the serious toxicity of drugs including herbal medicines. Potential cases of liver injury were ascertained in more than 180 Departments of all 51 Berlin hospitals from October 2002 to December 2011. Drug or herb intake was assessed through a standardized face-to-face interview. Drug or herbal aetiology was assessed based on the updated Council for International Organizations of Medical Sciences scale. In ten of all 198 cases of hepatotoxicity included in the study, herbal aetiology was assessed as probable (once ayurvedic herb) or possible (Valeriana five times, Mentha piperita once, Pelargonium sidoides once, Hypericum perforatum once, Eucalyptus globulus once). Mean age was 56.4 ± 9.7 years, and the predominant pattern of liver injury was hepatocellular. No cases of acute liver failure or death were observed. This case series corroborates known risks for ayurvedic herbs, supports the suspected association between Valeriana use and liver injury, and indicates a hepatotoxic potential for herbs such as Pelargonium sidoides, Hypericum perforatum or Mentha piperita that were rarely associated with liver injury before. However, given that possible causality does not prove clinical significance, further studies in this field are needed. Full article

(This article belongs to the Special Issue Drug, Herb, and Dietary Supplement Hepatotoxicity)

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1286 KiB

Open AccessArticle

In Silico Insight into Potential Anti-Alzheimer’s Disease Mechanisms of Icariin

by Zhijie Cui, Zhen Sheng, Xinmiao Yan, Zhiwei Cao and Kailin Tang

Int. J. Mol. Sci. 2016, 17(1), 113; https://doi.org/10.3390/ijms17010113 - 15 Jan 2016

Cited by 14 | Viewed by 6169

Abstract

Herbal compounds that have notable therapeutic effect upon Alzheimer's disease (AD) have frequently been found, despite the recent failure of late-stage clinical drugs. Icariin, which is isolated from Epimedium brevicornum, is widely reported to exhibit significant anti-AD effects in in vitro and in [...] Read more.

Herbal compounds that have notable therapeutic effect upon Alzheimer's disease (AD) have frequently been found, despite the recent failure of late-stage clinical drugs. Icariin, which is isolated from Epimedium brevicornum, is widely reported to exhibit significant anti-AD effects in in vitro and in vivo studies. However, the molecular mechanism remains thus far unclear. In this work, the anti-AD mechanisms of icariin were investigated at a target network level assisted by an in silico target identification program (INVDOCK). The results suggested that the anti-AD effects of icariin may be contributed by: attenuation of hyperphosphorylation of tau protein, anti-inflammation and regulation of Ca²⁺ homeostasis. Our results may provide assistance in understanding the molecular mechanism and further developing icariin into promising anti-AD agents. Full article

(This article belongs to the Section Physical Chemistry, Theoretical and Computational Chemistry)

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1926 KiB

Open AccessArticle

Transcription Factor Sp1 Promotes the Expression of Porcine ROCK1 Gene

by Ruirui Zhang, Xiaoting Feng, Mengsi Zhan, Cong Huang, Kun Chen, Xiaoyin Tang, Tingting Kang, Yuanzhu Xiong and Minggang Lei

Int. J. Mol. Sci. 2016, 17(1), 112; https://doi.org/10.3390/ijms17010112 - 15 Jan 2016

Cited by 10 | Viewed by 6048

Abstract

Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) gene plays a crucial role in maintaining genomic stability, tumorigenesis and myogenesis. However, little is known about the regulatory elements governing the transcription of porcine ROCK1 gene. In the current study, the transcription start [...] Read more.

Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) gene plays a crucial role in maintaining genomic stability, tumorigenesis and myogenesis. However, little is known about the regulatory elements governing the transcription of porcine ROCK1 gene. In the current study, the transcription start site (TSS) was identified by 5’-RACE, and was found to differ from the predicted one. The region in ROCK1 promoter which is critical for promoter activity was investigated via progressive deletions. Site-directed mutagenesis indicated that the region from −604 to −554 bp contains responsive elements for Sp1. Subsequent experiments showed that ROCK1 promoter activity is enhanced by Sp1 in a dose-dependent manner, whereas treatment with specific siRNA repressed ROCK1 promoter activity. Electrophoretic mobility shift assay (EMSA), DNA pull down and chromatin immunoprecipitation (ChIP) assays revealed Sp1 can bind to this region. qRT-PCR and Western blotting research followed by overexpression or inhibition of Sp1 indicate that Sp1 can affect endogenous ROCK1 expression at both mRNA and protein levels. Overexpression of Sp1 can promote the expression of myogenic differentiation 1(MyoD), myogenin (MyoG), myosin heavy chain (MyHC). Taken together, we conclude that Sp1 positively regulates ROCK1 transcription by directly binding to the ROCK1 promoter region (from −604 to −532 bp) and may affect the process of myogenesis. Full article

(This article belongs to the Section Biochemistry)

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Figure 1

Figure 1
5’-Deletion analysis of the porcine ROCK1 promoter activity. Schematic representation of the progressive deletions of porcine ROCK1 5’-flanking region in pGL3-Basic vector and the relative activities of ROCK1 promoter corresponding to the progressive deletions. The predicted transcription start site (TSS, the red arrow in the figure) was set +1, differs from the TSS in NCBI database. The pGL3-control/basic vectors were used as positive/negative control, while pRL-TK was used as internal control. Data were expressed as means ± SD of three replicates. Full article ">Figure 2
Site-directed mutation of Sp1 binding sites in ROCK1-P5 fragment. (A) Schematic structure of site-directed mutagenesis in the putative Sp1 binding sites (the black slash) of porcine ROCK1 gene. LUC represents the Luciferase gene in the vectors; (B,C) Luciferase activity of site-directed mutagenesis in PK and C2C12 cells. Statistical differences of relative activities were analyzed in the same cells; ** p < 0.01, data were expressed as means ± SD of three replicates. Full article ">Figure 3
Binding of Sp1 with the ROCK1-P5 fragment was analyzed in vitro and in vivo. The first (A), the second (B) and the third (C) biotin-labeled probes were incubated with the NE of PK cells. Lane 1 was the negative control without NE; the reagents were incubated in the absence competitor probes in Lane 2 or in presence of 50× excess competitor (Lane 3)/competitor-mutant (Lane 4) probes, respectively; (D) The three probes were incubated with PK NE, respectively; (E) Proteins of PK extracted from DNA-pull down materials were detected by Western blot. The total non-denaturing proteins/Streptavidin MagneSphere® Paramagnetic Particles were taken as positive/negative control (PC/NC). The three potential Sp1 binding sites were named as Sp1.1, Sp1.2, and Sp1.3 in (D,E). The competitor/competitor-mutant probes were 50-fold excess and arrows indicated the specific DNA-protein complex bands; (F) Schematic diagram of the Sp1 binding sites in the porcine ROCK1-P5 fragment; (G) ChIP assay of Sp1 binding to porcine ROCK1-P5 fragment in PK cells. The in vivo interaction of Sp1 and Sp3 with porcine ROCK1 promoter was determined by ChIP assay, in which Normal mouse IgG was used as negative control. DNA isolated from immunoprecipitated materials was used for PCR amplification, whereas total chromatin was used as input (positive control). The antibodies used in ChIP assay were listed in the right of the figure and the corresponding amplification product obtained here was 107 bp. Full article ">Figure 4
Sp1 stimulates the expression of porcine ROCK1 gene. (A) Over-expression of Sp1 up-regulated ROCK1 luciferase activity; (B) Over-expression efficacy of Sp1; (C,D) Over-expression of Sp1 stimulated ROCK1 expression at mRNA and protein level; (E) The interferences efficacy of siRNA; (F) Suppressing Sp1 reduced the ROCK1 promoter activity; (G,H) Inhibition of Sp1 suppressed ROCK1 expression at mRNA and protein level. The amount of plasmid was kept constant by the addition of pcDNA3.1 (+) vector. The data were obtained both in PK and C2C12 cells and expressed as means ± SD of three replicates. * p < 0.05, ** p < 0.01. Full article ">Figure 5
Sp1 promotes the process of myogenesis. Over-expression of Sp1 significantly stimulated MyoD, MyoG, MyHC mRNA expression in C2C12 cells, ** p < 0.01. Full article ">

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Int. J. Mol. Sci., Volume 17, Issue 1 (January 2016) – 134 articles

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