International Journal of Molecular Sciences

3543 KiB

Open AccessReview

by Ziyuan Meng, Quanxia Lv, Jun Lu, Houzong Yao, Xiaoqing Lv, Feng Jiang, Aiping Lu and Ge Zhang

Int. J. Mol. Sci. 2016, 17(5), 796; https://doi.org/10.3390/ijms17050796 - 23 May 2016

Cited by 75 | Viewed by 11835

Abstract

Paclitaxel is an anti-tumor agent with remarkable anti-tumor activity and wide clinical uses. However, it is also faced with various challenges especially for its poor water solubility and low selectivity for the target. To overcome these disadvantages of paclitaxel, approaches using small molecule [...] Read more.

Paclitaxel is an anti-tumor agent with remarkable anti-tumor activity and wide clinical uses. However, it is also faced with various challenges especially for its poor water solubility and low selectivity for the target. To overcome these disadvantages of paclitaxel, approaches using small molecule modifications and macromolecule modifications have been developed by many research groups from all over the world. In this review, we discuss the different strategies especially prodrug strategies that are currently used to make paclitaxel more effective. Full article

(This article belongs to the Special Issue Translational Molecular Medicine & Molecular Drug Discovery)

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3831 KiB

Open AccessArticle

Cold-Induced Browning Dynamically Alters the Expression Profiles of Inflammatory Adipokines with Tissue Specificity in Mice

by Xiao Luo, Ru Jia, Qiangling Zhang, Bo Sun and Jianqun Yan

Int. J. Mol. Sci. 2016, 17(5), 795; https://doi.org/10.3390/ijms17050795 - 23 May 2016

Cited by 21 | Viewed by 6007

Abstract

Cold exposure or β₃-adrenoceptor agonist treatment induces the adipose tissues remodeling, relevant for beige adipogenesis within white adipose tissue (WAT). It remains unclear whether this process influences inflammatory adipokines expression in adipose tissues. We determine the temporal profile of cold or [...] Read more.

Cold exposure or β₃-adrenoceptor agonist treatment induces the adipose tissues remodeling, relevant for beige adipogenesis within white adipose tissue (WAT). It remains unclear whether this process influences inflammatory adipokines expression in adipose tissues. We determine the temporal profile of cold or β₃-adrenoceptor agonist (CL316,243)-induced changes in the expression of inflammatory adipokines in adipose tissues in mice or primary mice adipocytes. Male C57BL/6J mice at eight weeks old were exposed to 4 °C for 1–5 days. Interscapular brown adipose tissue (iBAT), inguinal subcutaneous WAT (sWAT) and epididymal WAT (eWAT) were harvested for gene and protein expression analysis. In addition, cultured primary mice brown adipocyte (BA) and white adipocyte (WA) treated with or without CL316,243 were harvested for gene expression analysis. The inflammatory adipokines expressed significantly higher in WAT than BAT at baseline. They were rapidly changed in iBAT, while down-regulated in sWAT and up-regulated in eWAT during the cold acclimation. Upon CL316,243 treatment, detected inflammatory adipokines except Leptin were transiently increased in both BA and WA. Our in vivo and in vitro data demonstrate that the browning process alters the inflammatory adipokines expression in adipose tissues, which is acutely responded to in iBAT, dynamically decreased in sWAT whilst increased in eWAT for compensation. Full article

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

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Inflammatory adipokines expression levels exhibit depot-specificity in mice adipose tissues at baseline (in room temperature without any treatment). Quantitative PCR analysis of anti-inflammatory (A) and pro-inflammatory (B) adipokines genes expression in iBAT, sWAT and eWAT at baseline. The data show the fold changes of the expressions for the target genes in interscapular brown adipose tissue (iBAT), inguinal subcutaneous WAT (sWAT) and epididymal WAT (eWAT) of RT (room temperature) mice (n = 6 for each group). Values are mean ± S.E.M. and expression of genes is corrected for the housekeeping gene Cyclophilin. (* p < 0.05, ** p < 0.01, *** p < 0.001 vs. the expression level of iBAT, while # p < 0.05 vs. the expression level of sWAT.) Full article ">Figure 2
Cold exposure differentially alters the expression of inflammatory adipokines among adipose tissues. Quantitative PCR analysis of anti-inflammatory (A) and pro-inflammatory (B) adipokines genes expression in iBAT, sWAT and eWAT of control mice and mice exposed to Cold (4 °C) up to 5 day. The data show the fold changes of the expression for the target genes in iBAT, sWAT and eWAT of RT and Cold mice (n = 6 for each group). Values are mean ± S.E.M. and expression of genes is corrected for the housekeeping gene Cyclophilin. All the data were normalized to the expression in RT mice respectively to show the cold-induced gene expression differences. Erected bars: genes up-regulated and inverted bars: genes down-regulated compared to the RT mice. (* p < 0.05, ** p < 0.01, *** p < 0.001). Full article ">Figure 3
Cold exposure changes Adiponectin and Leptin protein levels in adipose tissues in mice. (A) Western blot analysis for Adiponectin using total protein isolated from iBAT, sWAT and eWAT of RT and Cold mice. (n = 6 for each group); (B) Quantification of Western blot analysis. Protein content is expressed relative to the control and represents three independent experiments with triplicate observations in each experiment. Volume is the sum of all pixel intensities within a band. All data are normalized to β-tubulin and are expressed as mean ± S.E.M. (* p < 0.05, ** p < 0.01, *** p < 0.001); (C) Plasma Adiponectin and Leptin levels in RT and Cold mice were determined by ELISA. (n = 6 per group) All data are presented as mean ± S.E.M. * p < 0.05, ** p < 0.01 for the Cold compared to the RT group. Full article ">Figure 4
Browning transition with increase of browning markers in the cultured mature Brown Adipocyte (BA) and White Adipocyte (WA) after CL316,243 treatment. (A) Morphology of the brown and white adipocytes before (Day 0) and after (Day 9) the differentiation. The mature adipocytes after nine days of differentiation were used for CL316,243 treatment. Scale bar: 200 µm; (B) Quantitative PCR analysis of Ucp-1 and Pgc-1α gene expression in cultured mice brown and white adipocytes after CL316,243 treatment up to 24 h. The data show the fold changes of the expression for the target genes in BA and WA at Hour 0. Values are mean ± S.E.M. from three independent experiments. The expression of genes is corrected for the housekeeping gene Cyclophilin (*** p < 0.001). Full article ">Figure 5
CL316,243 treatment alters the expressions of inflammatory adipokines in BA and WA. Quantitative PCR analysis of anti-inflammatory (A) and pro-inflammatory (B) adipokines expression in cultured mice BA and WA after CL316,243 treatment up to 24 h. The data show the fold changes of the expression for the target genes in BA and WA at Hour 0. Values are mean ± S.E.M. from three independent experiments. The expression of genes is corrected for the housekeeping gene Cyclophilin (* p < 0.05, ** p < 0.01, *** p < 0.001). Full article ">

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Open AccessReview

Gibberellic Acid: A Key Phytohormone for Spikelet Fertility in Rice Grain Production

by Choon-Tak Kwon and Nam-Chon Paek

Int. J. Mol. Sci. 2016, 17(5), 794; https://doi.org/10.3390/ijms17050794 - 23 May 2016

Cited by 47 | Viewed by 9570

Abstract

The phytohormone gibberellic acid (GA) has essential signaling functions in multiple processes during plant development. In the “Green Revolution”, breeders developed high-yield rice cultivars that exhibited both semi-dwarfism and altered GA responses, thus improving grain production. Most studies of GA have concentrated on [...] Read more.

The phytohormone gibberellic acid (GA) has essential signaling functions in multiple processes during plant development. In the “Green Revolution”, breeders developed high-yield rice cultivars that exhibited both semi-dwarfism and altered GA responses, thus improving grain production. Most studies of GA have concentrated on germination and cell elongation, but GA also has a pivotal role in floral organ development, particularly in stamen/anther formation. In rice, GA signaling plays an important role in spikelet fertility; however, the molecular genetic and biochemical mechanisms of GA in male fertility remain largely unknown. Here, we review recent progress in understanding the network of GA signaling and its connection with spikelet fertility, which is tightly associated with grain productivity in cereal crops. Full article

(This article belongs to the Special Issue Metabolomics in the Plant Sciences)

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265 KiB

Open AccessReview

The Role of Proanthocyanidins Complex in Structure and Nutrition Interaction in Alfalfa Forage

by Arjan Jonker and Peiqiang Yu

Int. J. Mol. Sci. 2016, 17(5), 793; https://doi.org/10.3390/ijms17050793 - 23 May 2016

Cited by 27 | Viewed by 5616

Abstract

Alfalfa (Medicago sativa L.) is one of the main forages grown in the world. Alfalfa is a winter hardy, drought tolerant, N-fixing legume with a good longevity, high yield, high nutrient levels, high digestibility, unique structural to non-structural components ratio, high dry [...] Read more.

Alfalfa (Medicago sativa L.) is one of the main forages grown in the world. Alfalfa is a winter hardy, drought tolerant, N-fixing legume with a good longevity, high yield, high nutrient levels, high digestibility, unique structural to non-structural components ratio, high dry matter intake, and high animal productivity per hectare. However, its main limitation is its excessively rapid initial rate of protein degradation in the rumen, which results in pasture bloat and inefficient use of protein with consequent excessive excretions of nitrogen into the environment. Proanthocyanidins are secondary plant metabolites that can bind with protein and thereby reduce the rate and extent of ruminal protein degradation. However, these secondary metabolites do not accumulate in alfalfa. This review aims to firstly describe the events involved in the rapid release of protein from alfalfa and its effect on ruminant nutrition, environmental pollution, and pasture bloat; secondly, to describe occurrence, structure, functions and benefits of moderate amounts of proanthocyanidin; and finally, to describe the development of alfalfa which accumulates moderate amounts of proanthocyanidins. The emphasis of this review focuses on the role of proanthocyanidins compounds in structure and nutrition interaction in ruminant livestock systems. Full article

(This article belongs to the Section Molecular Plant Sciences)

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Open AccessCommunication

A Novel Technique to Detect EGFR Mutations in Lung Cancer

by Yuanbin Liu, Ting Lei, Zhiyu Liu, Yanbin Kuang, Jianxin Lyu and Qi Wang

Int. J. Mol. Sci. 2016, 17(5), 792; https://doi.org/10.3390/ijms17050792 - 23 May 2016

Cited by 26 | Viewed by 8737

Abstract

Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this [...] Read more.

Epidermal growth factor receptor (EGFR) gene mutations occur in multiple human cancers; therefore, the detection of EGFR mutations could lead to early cancer diagnosis. This study describes a novel EGFR mutation detection technique. Compared to direct DNA sequencing detection methods, this method is based on allele-specific amplification (ASA), recombinase polymerase amplification (RPA), peptide nucleic acid (PNA), and SYBR Green I (SYBR), referred to as the AS-RPA-PNA-SYBR (ARPS) system. The principle of this technique is based on three continuous steps: ASA or ASA combined with PNA to prevent non-target sequence amplification (even single nucleotide polymorphisms, SNPs), the rapid amplification advantage of RPA, and appropriate SYBR Green I detection (the samples harboring EGFR mutations show a green signal). Using this method, the EGFR 19Del(2) mutation was detected in 5 min, while the EGFR L858R mutation was detected in 10 min. In this study, the detection of EGFR mutations in clinical samples using the ARPS system was compatible with that determined by polymerase chain reaction (PCR) and DNA sequencing methods. Thus, this newly developed methodology that uses the ARPS system with appropriate primer sets is a rapid, reliable, and practical way to assess EGFR mutations in clinical samples. Full article

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

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Comparison of the polymerase chain reaction (PCR) and DNA sequencing data with our visualization data using the ARPS method for detection of the epidermal growth factor receptor (EGFR)19Del(2) mutation in non-small cell lung cancer (NSCLC) cells. (A) The PCR and DNA sequencing data; (B) The specificity and timely sensitivity of the AS-RPA-PNA-SYBR (ARPS) method for detection of the EGFR 19Del(2) mutation. The “+” was the positive control in the recombinase polymerase amplification (RPA) reaction kit (143 bp in size), which was detected between 5 and 20 min. The mutated EGFR 19Del(2) band was 266 bp, and 300 ng of the DNA template was used. Results of ARPS were negative in red, positive in green. Full article ">Figure 2
Comparison of the PCR and DNA sequencing data with our visualization data using the ARPS method to detect the EGFR L858R mutation in NSCLC cells. (A) The PCR and DNA sequencing data; (B) The specificity and timely sensitivity of the ARPS method for detection of the EGFR L858R mutation. The “+” was the positive control in the RPA reaction kit (143 bp in size), which was detected between 10 and 20 min. The target mutated band size was 201 bp, and 300 ng of the DNA template was used. Results of ARPS were negative in red, positive in green. Full article ">Figure 3
Sensitivity of the ARPS technique. (A) ARPS reaction using 19 Del(2) mutation-specific primers with serial dilutions of genomic DNA isolated from HCC-827 cells; (B) ARPS reaction using L858R point mutation-specific primers with serial dilutions of genomic DNA from H-1975 cells. Results of ARPS were negative in red, positive in green. Full article ">Figure 4
Comparison of the PCR and DNA sequencing data with our visualization data using the ARPS method to detect the EGFR mutation in NSCLC tissue specimens. (A) EGFR 19Del(2) mutation. The left panel shows our data, and the right panel shows the DNA sequencing data. From top to bottom, the quality control of the positive sample, the quality control of the negative sample (HCC-827 and A549 cell lines), one positive clinical tissue sample, and two negative clinical tissue samples are shown; (B) The EGFR L858R mutation. The left panel shows our data, and the right panel shows the DNA sequencing data. From top to bottom, the quality control of the positive sample, the quality control of the negative sample (H-1975 and A549 cell lines), three positive clinical tissue samples, and two negative clinical tissue samples are shown. Results of ARPS were negative in red, positive in green. Full article ">Figure 5
Primer sequences and agarose gel electrophoresis data. (A) The position of another mismatch base in the reverse primer (green letters) to detect EGFR L858R; (mutant base in red letter) (B) The agarose gel electrophoresis and ARPS results of the different mismatch bases. The band size was 201 bp, and the “+” indicates the positive control from the RPA reaction kit (143 bp). The amount of DNA used was 300 ng. Full article ">

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Open AccessReview

Circulating MicroRNAs as Biomarkers in Biliary Tract Cancers

by Pablo Letelier, Ismael Riquelme, Alfonso H. Hernández, Neftalí Guzmán, Jorge G. Farías and Juan Carlos Roa

Int. J. Mol. Sci. 2016, 17(5), 791; https://doi.org/10.3390/ijms17050791 - 23 May 2016

Cited by 36 | Viewed by 8788

Abstract

Biliary tract cancers (BTCs) are a group of highly aggressive malignant tumors with a poor prognosis. The current diagnosis is based mainly on imaging and intraoperative exploration due to brush cytology havinga low sensitivity and the standard markers, such as carcinoembryonic antigen (CEA) [...] Read more.

Biliary tract cancers (BTCs) are a group of highly aggressive malignant tumors with a poor prognosis. The current diagnosis is based mainly on imaging and intraoperative exploration due to brush cytology havinga low sensitivity and the standard markers, such as carcinoembryonic antigen (CEA) and carbohydrate 19-9 (CA19-9), not having enough sensitivity nor specificity to be used in a differential diagnosis and early stage detection. Thus, better non-invasive methods that can distinguish between normal and pathological tissue are needed. MicroRNAs (miRNAs) are small, single-stranded non-coding RNA molecules of ~20–22 nucleotides that regulate relevant physiological mechanisms and can also be involved in carcinogenesis. Recent studies have demonstrated that miRNAs are detectable in multiple body fluids, showing great stability, either free or trapped in circulating microvesicles, such as exosomes. miRNAs are ideal biomarkers that may be used in screening and prognosis in biliary tract cancers, aiding also in the clinical decisions at different stages of cancer treatment. This review highlights the progress in the analysis of circulating miRNAs in serum, plasma and bile as potential diagnostic and prognostic markers of BTCs. Full article

(This article belongs to the Special Issue MicroRNA in Various Disease States as Biomarkers)

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Open AccessArticle

Analysis of 2-(2-Phenylethyl)chromones by UPLC-ESI-QTOF-MS and Multivariate Statistical Methods in Wild and Cultivated Agarwood

by Yuanbin Li, Nan Sheng, Lingli Wang, Shijie Li, Jiannan Chen and Xiaoping Lai

Int. J. Mol. Sci. 2016, 17(5), 771; https://doi.org/10.3390/ijms17050771 - 23 May 2016

Cited by 31 | Viewed by 6873

Abstract

Agarwood is the fragrant resinous material mainly formed from species of Aquilaria. 2-(2-phenylethyl)chromones, especially the highly oxidized 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones, are the main representative compounds from agarwood. It is important to determine whether agarwood in trade is from cultivated trees or natural trees in [...] Read more.

Agarwood is the fragrant resinous material mainly formed from species of Aquilaria. 2-(2-phenylethyl)chromones, especially the highly oxidized 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones, are the main representative compounds from agarwood. It is important to determine whether agarwood in trade is from cultivated trees or natural trees in the Convention on the International Trade in Endangered Species (CITES). We characterized the 2-(2-phenylethyl)chromones in agarwood by ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry (UPLC–ESI-QTOF-MS) and differentiated wild from cultivated agarwood by metabolomic analysis. A total of 141 chromones including 50 potentially new compounds were evaluated as belonging to four structural classes (unoxidized 2-(2-phenylethyl)chromones, 5,6,7,8-tetrahydro-2-(2-phenylethyl)-chromones, bi-2-(2-phenylethyl)chromones, and tri-2-(2-phenylethyl)chromones). The metabolic difference between wild and cultivated agarwood was analyzed by component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Fourteen markers of metabolisms in wild and cultivated agarwood were constructed (e.g., 6,7-dimethoxy-2-(2-phenylethyl)chromone, 6,8-dihydroxy-2-(2-phenylethyl)chromone, 6-methoxy-2-(2-phenylethyl)chromone, etc.). These results indicated that UPLC–ESI-QTOF-MS-based metabonomics analysis in agarwood may be useful for distinguishing wild agarwood from cultivated agarwood. Full article

(This article belongs to the Special Issue Metabolomics in the Plant Sciences)

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Characteristic fragments of 2-(2-phenylethyl)chromones. Full article ">Figure 2
Proposed main fragmentation pathway of 5,6,7,8-tetrahydro-2-(2-phenylethyl)chromones. Full article ">Figure 3
The main structures of bi-2-(2-phenylethyl)chromones (A-type, B-type, and C-type). Full article ">Figure 4
(a) MS/MS spectrum of compound 117; (b) proposed fragmentation pathway of compound 117. Full article ">Figure 5
(a) MS/MS spectrum of compound 139; (b) proposed fragmentation pathway of compound 139. Full article ">Figure 6
Principal component analysis (PCA) score plot of agarwood (group 1: wild agarwood; group 2 and 3: cultivated agarwood). Full article ">Figure 7
S-plot generated by orthogonal partial least squared discriminant analysis (OPLS-DA) for the metabolites in the wild and cultivated agarwood. Full article ">

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Open AccessArticle

Variability in Immunohistochemical Detection of Programmed Death Ligand 1 (PD-L1) in Cancer Tissue Types

by Giosuè Scognamiglio, Anna De Chiara, Maurizio Di Bonito, Fabiana Tatangelo, Nunzia Simona Losito, Annamaria Anniciello, Rossella De Cecio, Crescenzo D’Alterio, Stefania Scala, Monica Cantile and Gerardo Botti

Int. J. Mol. Sci. 2016, 17(5), 790; https://doi.org/10.3390/ijms17050790 - 21 May 2016

Cited by 36 | Viewed by 7193

Abstract

In normal cell physiology, programmed death 1 (PD-1) and its ligand, PD-L1, play an immunoregulatory role in T-cell activation, tolerance, and immune-mediated tissue damage. The PD-1/PD-L1 pathway also plays a critical role in immune escape of tumor cells and has been demonstrated to [...] Read more.

In normal cell physiology, programmed death 1 (PD-1) and its ligand, PD-L1, play an immunoregulatory role in T-cell activation, tolerance, and immune-mediated tissue damage. The PD-1/PD-L1 pathway also plays a critical role in immune escape of tumor cells and has been demonstrated to correlate with a poor prognosis of patients with several types of cancer. However, recent reports have revealed that the immunohistochemical (IHC) expression of the PD-L1 in tumor cells is not uniform for the use of different antibodies clones, with variable specificity, often doubtful topographical localization, and with a score not uniquely defined. The purpose of this study was to analyze the IHC expression of PD-L1 on a large series of several human tumors to correctly define its staining in different tumor tissues. Full article

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

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Open AccessReview

Functions of miRNAs during Mammalian Heart Development

by Shun Yan and Kai Jiao

Int. J. Mol. Sci. 2016, 17(5), 789; https://doi.org/10.3390/ijms17050789 - 21 May 2016

Cited by 38 | Viewed by 5842

Abstract

MicroRNAs (miRNAs) play essential roles during mammalian heart development and have emerged as attractive therapeutic targets for cardiovascular diseases. The mammalian embryonic heart is mainly derived from four major cell types during development. These include cardiomyocytes, endocardial cells, epicardial cells, and neural crest [...] Read more.

MicroRNAs (miRNAs) play essential roles during mammalian heart development and have emerged as attractive therapeutic targets for cardiovascular diseases. The mammalian embryonic heart is mainly derived from four major cell types during development. These include cardiomyocytes, endocardial cells, epicardial cells, and neural crest cells. Recent data have identified various miRNAs as critical regulators of the proper differentiation, proliferation, and survival of these cell types. In this review, we briefly introduce the contemporary understanding of mammalian cardiac development. We also focus on recent developments in the field of cardiac miRNAs and their functions during the development of different cell types. Full article

(This article belongs to the Special Issue MicroRNA Regulation)

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Open AccessArticle

Extraction Optimization, Preliminary Characterization and Bioactivities in Vitro of Ligularia hodgsonii Polysaccharides

by Xueping Song and Jun Tang

Int. J. Mol. Sci. 2016, 17(5), 788; https://doi.org/10.3390/ijms17050788 - 21 May 2016

Cited by 21 | Viewed by 5408

Abstract

The optimization extraction, preliminary characterization and bioactivities of Ligularia hodgsonii polysaccharides were investigated. Based on single-factor experiments and orthogonal array test, the optimum extraction conditions were obtained as follows: extraction time 3 h, temperature 85 °C, water/raw material ratio 36. Further Sevag deproteinization [...] Read more.

The optimization extraction, preliminary characterization and bioactivities of Ligularia hodgsonii polysaccharides were investigated. Based on single-factor experiments and orthogonal array test, the optimum extraction conditions were obtained as follows: extraction time 3 h, temperature 85 °C, water/raw material ratio 36. Further Sevag deproteinization and dialysis yielded the dialyzed Ligularia hodgsonii polysaccharides (DLHP, 19.2 ± 1.4 mg/g crude herb). Compositional analysis, size-exclusion chromatography connected with multi-angle laser light-scattering and refractive index (SEC-MALLS-RI), Fourier transform infrared (FT-IR) and ¹H nuclear magnetic resonance (NMR) spectroscopy were employed for characterization of the polysaccharides. DLHP was found to have a major component with a weight-average molecular weight of 1.17 × 10⁵ Da, mainly comprising of glucose, galactose, arabinose, mannose, rhamnose, glucuronic acid and galacturonic acid. By in vitro antioxidant activity assays, DLHP presented remarkable scavenging capacities towards 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydroxyl radicals, and ferrous ions chelating ability. Moreover, it exhibited appreciable anti-hyperglycemic activity as demonstrated by differential inhibition of α-glucosidase and α-amylase. The results indicated that DLHP could potentially be a resource for antioxidant and hypoglycemic agents. Full article

(This article belongs to the Section Bioactives and Nutraceuticals)

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Effects of extraction temperature (A); extraction time (B); water volume to raw material weight (W/M ratio) (C); and extraction frequency (D) on the polysaccharide yield. Full article ">Figure 2
(A) Size-exclusion chromatography (SEC) chromatograms of dialyzed Ligularia hodgsonii polysaccharides (DLHP) detected by SEC-MALLS-RI at 25 °C. A1 (red line) and A2 (blue line) represent the signals from light scattering (LS) at 90° and differential refractive index (dRI), respectively; and (B) HPLC profiles of 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatives of seven monosaccharide standards (B1) and DLHP (B2): 1, Mannose; 2, Galacturonic acid; 3, Rhamnose; 4, Glucuronic acid; 5, Glucose; 6, Galactose; and 7, Arabinose. Full article ">Figure 3
Fourier Transform Infrared (FT-IR) spectrum of DLHP. Full article ">Figure 4
1H-NMR spectrum of DLHP. Full article ">Figure 5
Scavenging effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (A); 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical (B); and hydroxyl radical (C); metal chelating activity (D); and reductive potential (E) of DLHP, LHP and water extract. Full article ">Figure 6
The inhibitory activities of DLHP, LHP and water extract on: α-glucosidase (A); and α-amylase (B). Full article ">

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Open AccessArticle

Functional and Biochemical Characterization of Three Recombinant Human Glucose-6-Phosphate Dehydrogenase Mutants: Zacatecas, Vanua-Lava and Viangchan

by Saúl Gómez-Manzo, Jaime Marcial-Quino, America Vanoye-Carlo, Hugo Serrano-Posada, Abigail González-Valdez, Víctor Martínez-Rosas, Beatriz Hernández-Ochoa, Edgar Sierra-Palacios, Rosa Angélica Castillo-Rodríguez, Miguel Cuevas-Cruz, Eduardo Rodríguez-Bustamante and Roberto Arreguin-Espinosa

Int. J. Mol. Sci. 2016, 17(5), 787; https://doi.org/10.3390/ijms17050787 - 21 May 2016

Cited by 23 | Viewed by 7693

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation [...] Read more.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in humans causes severe disease, varying from mostly asymptomatic individuals to patients showing neonatal jaundice, acute hemolysis episodes or chronic nonspherocytic hemolytic anemia. In order to understand the effect of the mutations in G6PD gene function and its relation with G6PD deficiency severity, we report the construction, cloning and expression as well as the detailed kinetic and stability characterization of three purified clinical variants of G6PD that present in the Mexican population: G6PD Zacatecas (Class I), Vanua-Lava (Class II) and Viangchan (Class II). For all the G6PD mutants, we obtained low purification yield and altered kinetic parameters compared with Wild Type (WT). Our results show that the mutations, regardless of the distance from the active site where they are located, affect the catalytic properties and structural parameters and that these changes could be associated with the clinical presentation of the deficiency. Specifically, the structural characterization of the G6PD Zacatecas mutant suggests that the R257L mutation have a strong effect on the global stability of G6PD favoring an unstable active site. Using computational analysis, we offer a molecular explanation of the effects of these mutations on the active site. Full article

(This article belongs to the Collection Computational, Structural and Spectroscopic Studies of Enzyme Mechanisms, Inhibition and Dynamics)

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1840 KiB

Open AccessReview

Marine Antimicrobial Peptides: Nature Provides Templates for the Design of Novel Compounds against Pathogenic Bacteria

by Annarita Falanga, Lucia Lombardi, Gianluigi Franci, Mariateresa Vitiello, Maria Rosaria Iovene, Giancarlo Morelli, Massimiliano Galdiero and Stefania Galdiero

Int. J. Mol. Sci. 2016, 17(5), 785; https://doi.org/10.3390/ijms17050785 - 21 May 2016

Cited by 103 | Viewed by 10513

Abstract

The discovery of antibiotics for the treatment of bacterial infections brought the idea that bacteria would no longer endanger human health. However, bacterial diseases still represent a worldwide treat. The ability of microorganisms to develop resistance, together with the indiscriminate use of antibiotics, [...] Read more.

The discovery of antibiotics for the treatment of bacterial infections brought the idea that bacteria would no longer endanger human health. However, bacterial diseases still represent a worldwide treat. The ability of microorganisms to develop resistance, together with the indiscriminate use of antibiotics, is mainly responsible for this situation; thus, resistance has compelled the scientific community to search for novel therapeutics. In this scenario, antimicrobial peptides (AMPs) provide a promising strategy against a wide array of pathogenic microorganisms, being able to act directly as antimicrobial agents but also being important regulators of the innate immune system. This review is an attempt to explore marine AMPs as a rich source of molecules with antimicrobial activity. In fact, the sea is poorly explored in terms of AMPs, but it represents a resource with plentiful antibacterial agents performing their role in a harsh environment. For the application of AMPs in the medical field limitations correlated to their peptide nature, their inactivation by environmental pH, presence of salts, proteases, or other components have to be solved. Thus, these peptides may act as templates for the design of more potent and less toxic compounds. Full article

(This article belongs to the Special Issue Drug Delivery and Antimicrobial Agents)

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Open AccessArticle

Novel Zinc(II) Complexes [Zn(atc-Et)₂] and [Zn(atc-Ph)₂]: In Vitro and in Vivo Antiproliferative Studies

by Erica De O. Lopes, Carolina G. de Oliveira, Patricia B. da Silva, Carlos E. Eismann, Carlos A. Suárez, Amauri A. Menegário, Clarice Q. F. Leite, Victor M. Deflon and Fernando R. Pavan

Int. J. Mol. Sci. 2016, 17(5), 781; https://doi.org/10.3390/ijms17050781 - 21 May 2016

Cited by 22 | Viewed by 6143

Abstract

Cisplatin and its derivatives are the main metallodrugs used in cancer therapy. However, low selectivity, toxicity and drug resistance are associated with their use. The zinc(II) (Zn^II) thiosemicarbazone complexes [Zn(atc-Et)₂] (1) and [Zn(atc-Ph)₂] (2 [...] Read more.

Cisplatin and its derivatives are the main metallodrugs used in cancer therapy. However, low selectivity, toxicity and drug resistance are associated with their use. The zinc(II) (Zn^II) thiosemicarbazone complexes [Zn(atc-Et)₂] (1) and [Zn(atc-Ph)₂] (2) (atc-R: monovalent anion of 2-acetylpyridine N4-R-thiosemicarbazone) were synthesized and fully characterized in the solid state and in solution via elemental analysis, Fourier transform infrared (FTIR), ultraviolet-visible (UV-Vis) and proton nuclear magnetic resonance (¹H NMR) spectroscopy, conductometry and single-crystal X-ray diffraction. The cytotoxicity of these complexes was evaluated in the HepG2, HeLa, MDA-MB-231, K-562, DU 145 and MRC-5 cancer cell lines. The strongest antiproliferative results were observed in MDA-MB-231 and HepG2 cells, in which these complexes displayed significant selective toxicity (3.1 and 3.6, respectively) compared with their effects on normal MRC-5 cells. In vivo studies were performed using an alternative model (Artemia salina L.) to assure the safety of these complexes, and the results were confirmed using a conventional model (BALB/c mice). Finally, tests of oral bioavailability showed maximum plasma concentrations of 3029.50 µg/L and 1191.95 µg/L for complexes 1 and 2, respectively. According to all obtained results, both compounds could be considered as prospective antiproliferative agents that warrant further research. Full article

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

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Molecular structure determined for [Zn(atc-Et)2] (1) (left) and [Zn(atc-Ph)2] (2) (right) with atom labels. Full article ">Figure 2
Percentage survival of BALB/c mice over 14 days of drug treatment at the concentration of 500 mg/kg body weight. Full article ">Figure 3
Percent survival of BALB/c mice over 14 days of drug treatment at the concentration of 1000 mg/kg body weight. Full article ">Figure 4
Relative weight (mean ratio of organ weight to body weight of BALB/c mice) and standard deviation (SD). R: right ; L: left. Full article ">Figure 5
Quantification of the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity levels in BALB/c mice. U/L: units per liter. Full article ">Figure 6
Time-concentration profiles of ZnII complexes in plasma after a single administration via oral gavage at a dose of 300 mg/Kg body weight. Full article ">Figure 7
Time-concentration profile of cisplatin in plasma after a single administration via oral gavage at a dose of 300 mg/Kg body weight. Full article ">Scheme 1
Synthesis of the complexes. Full article ">

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Open AccessArticle

An Eco-Friendly Ultrasound-Assisted Synthesis of Novel Fluorinated Pyridinium Salts-Based Hydrazones and Antimicrobial and Antitumor Screening

by Nadjet Rezki, Salsabeel A. Al-Sodies, Mohamed R. Aouad, Sanaa Bardaweel, Mouslim Messali and El Sayed H. El Ashry

Int. J. Mol. Sci. 2016, 17(5), 766; https://doi.org/10.3390/ijms17050766 - 21 May 2016

Cited by 28 | Viewed by 5834

Abstract

The present work reports an efficient synthesis of fluorinated pyridinium salts-based hydrazones under both conventional and eco-friendly ultrasound procedures. The synthetic approach first involves the preparation of halogenated pyridinium salts through the condensation of isonicotinic acid hydrazide (1) with p-fluorobenzaldehyde [...] Read more.

The present work reports an efficient synthesis of fluorinated pyridinium salts-based hydrazones under both conventional and eco-friendly ultrasound procedures. The synthetic approach first involves the preparation of halogenated pyridinium salts through the condensation of isonicotinic acid hydrazide (1) with p-fluorobenzaldehyde (2) followed by the nucleophilic alkylation of the resulting N-(4-fluorobenzylidene)isonicotinohydrazide (3) with a different alkyl iodide. The iodide counteranion of 5–10 was subjected to an anion exchange metathesis reaction in the presence of an excess of the appropriate metal salts to afford a new series of fluorinated pyridinium salts tethering a hydrazone linkage 11–40. Ultrasound irradiation led to higher yields in considerably less time than the conventional methods. The newly synthesized ILs were well-characterized with FT-IR, ¹H NMR, ¹³C NMR, ¹¹B, ¹⁹F, ³¹P and mass spectral analyses. The ILs were also screened for their antimicrobial and antitumor activities. Within the series, the salts tethering fluorinated counter anions 11–13, 21–23, 31–33 and 36–38 were found to be more potent against all bacterial and fungal strains at MIC 4–8 µg/mL. The in vitro antiproliferative activity was also investigated against four tumor cell lines (human ductal breast epithelial tumor T47D, human breast adenocarcinoma MCF-7, human epithelial carcinoma HeLa and human epithelial colorectal adenocarcinoma Caco-2) using the MTT assay, which revealed that promising antitumor activity was exhibited by compounds 5, 12 and 14. Full article

(This article belongs to the Special Issue Ionic Liquids 2016 and Selected Papers from ILMAT III)

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Open AccessArticle

Expression Patterns and Functional Novelty of Ribonuclease 1 in Herbivorous Megalobrama amblycephala

by Han Liu and Weimin Wang

Int. J. Mol. Sci. 2016, 17(5), 786; https://doi.org/10.3390/ijms17050786 - 20 May 2016

Cited by 5 | Viewed by 5106

Abstract

Ribonuclease 1 (RNase1) is an important digestive enzyme that has been used to study the molecular evolutionary and plant-feeding adaptation of mammals. However, the expression patterns and potential biological function of RNase1 in herbivorous fish is not known. Here, we identified RNase1 from [...] Read more.

Ribonuclease 1 (RNase1) is an important digestive enzyme that has been used to study the molecular evolutionary and plant-feeding adaptation of mammals. However, the expression patterns and potential biological function of RNase1 in herbivorous fish is not known. Here, we identified RNase1 from five fish species and illuminated the functional diversification and expression of RNase1 in herbivorous Megalobrama amblycephala. The five identified fish RNase1 genes all have the signature motifs of the RNase A superfamily. No expression of Ma-RNase1 was detected in early developmental stages but a weak expression was detected at 120 and 144 hours post-fertilization (hpf). Ma-RNase1 was only expressed in the liver and heart of one-year-old fish but strongly expressed in the liver, spleen, gut, kidney and testis of two-year-old fish. Moreover, the immunostaining localized RNase1 production to multiple tissues of two-year-old fish. A biological functional analysis of the recombinant protein demonstrated that M. amblycephala RNase1 had a relatively strong ribonuclease activity at its optimal pH 6.1, which is consistent with the pH of its intestinal microenvironment. Collectively, these results clearly show that Ma-RNase1 protein has ribonuclease activity and the expression patterns of Ma-RNase1 are dramatically different in one year and two-year-old fish, suggesting the functional differentiation during fish growing. Full article

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

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Multiple protein sequences alignment of RNase1 in fishes and mammals. Dashes indicate alignment gaps. Yellow underlines showed the signal peptides. The putative isoelectric point (pI) and molecular weight (Mw) indicated the isoelectric point and molecular weight (kDa) of the mature peptide, respectively. The conversed CKXXNTF motif is shown in a box. The eight structural cysteines (active-site residues) are marked with triangles and the two red triangles indicated alteration in fishes. Asterisks show the three catalytic resides. Full article ">Figure 2
Ribbon representations of 3D structures of four fish RNase1 and two mammals RNase A. The blue, light blue and green ribbons represent the three α-helices and ribbons with others colors represent the six β-strands. Full article ">Figure 3
Phylogenetic tree documenting relationships among various vertebrates RNase1. Neighbor-joining tree constructed with protein sequences by MEGA 5.1. Numbers at branches indicate the bootstrap probabilities with 1000 replicates. Full article ">Figure 4
The expression patterns of RNase1 genes in different developmental stages and tissues of M. amblycephala. B-actin was used as reference gene. (A) Two-year-old fish (the muscle tissue was the same as in the control); (B) One-year-old fish (the muscle tissue was the same as in the control); (C) early different developmental stages of M. amblycephala (0 hpf egg was the same as in the control). cDNAs were used for PCR from total RNA samples obtained from the heart (H), liver (L), spleen (S), kidney (K), brain (B), muscle (M), gut (G) and testis (T) of one year and two-year-old fish, 0 (fertilized egg), 12 (late gastrula stage), 27 (heart appearance), 40 (hatching), 72 (gill circulation), 120 (air bladder formation), 144 (intestine appearance) hours post-fertilization (hpf) embryos/larvae. Differences were determined by one-way analysis of variance (ANOVA). Statistically significant differences from the control group are marked as ** p <0.01. Full article ">Figure 5
Expression of Ma-RNase1 in different tissues of M. amblycephala. Immunohistochemistry demonstrates Ma-RNase1 production (positive cells labeled in brown showing as white arrows) in the liver (A), spleen (B) and gut (C,D) tissues of two-year-old M. amblycephala. The negative cell nucleus labeled in blue and positive nucleus labeled in brown. Full article ">Figure 6
Immunofluorescence analysis demonstrates RNase1 production in the liver, gut and spleen tissues of two-year-old M. amblycephala. Immunofluorescence staining identifying cells with RNase1 expression in red and nuclei in blue. Full article ">Figure 7
Ribonucleolytic activities of M. amblycephala recombinant protein RNase1. Comparison the ribonucleolytic activities of Ma-RNase1 and bovine RNase A at different amounts (A); RNase activity against yeast tRNA at different pH levels (B). Full article ">

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Open AccessArticle

Impact of Faba Bean-Seed Rhizobial Inoculation on Microbial Activity in the Rhizosphere Soil during Growing Season

by Anna Siczek and Jerzy Lipiec

Int. J. Mol. Sci. 2016, 17(5), 784; https://doi.org/10.3390/ijms17050784 - 20 May 2016

Cited by 28 | Viewed by 6637

Abstract

Inoculation of legume seeds with Rhizobium affects soil microbial community and processes, especially in the rhizosphere. This study aimed at assessing the effect of Rhizobium inoculation on microbial activity in the faba bean rhizosphere during the growing season in a field experiment on [...] Read more.

Inoculation of legume seeds with Rhizobium affects soil microbial community and processes, especially in the rhizosphere. This study aimed at assessing the effect of Rhizobium inoculation on microbial activity in the faba bean rhizosphere during the growing season in a field experiment on a Haplic Luvisol derived from loess. Faba bean (Vicia faba L.) seeds were non-inoculated (NI) or inoculated (I) with Rhizobium leguminosarum bv. viciae and sown. The rhizosphere soil was analyzed for the enzymatic activities of dehydrogenases, urease, protease and acid phosphomonoesterase, and functional diversity (catabolic potential) using the Average Well Color Development, Shannon-Weaver, and Richness indices following the community level physiological profiling from Biolog EcoPlate™. The analyses were done on three occasions corresponding to the growth stages of: 5–6 leaf, flowering, and pod formation. The enzymatic activities were higher in I than NI (p < 0.05) throughout the growing season. However, none of the functional diversity indices differed significantly under both treatments, regardless of the growth stage. This work showed that the functional diversity of the microbial communities was a less sensitive tool than enzyme activities in assessment of rhizobial inoculation effects on rhizosphere microbial activity. Full article

(This article belongs to the Special Issue Molecular Signals in Nodulation Control)

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Enzymatic activity. Different lowercase letters indicate significant differences (p < 0.05). NI, non-inoculated; I, inoculated with Rhizobium; T1, 5–6 leaf stage; T2, flowering; T3, pod formation stage. Full article ">Figure 2
Dendrogram of the bond distances between the carbon utilizations patterns of the substrates on the Biolog EcoPlatesTM. Grouping was conducted according to the Sneath’s criterion (66%); NI, non-inoculated; I, inoculated with Rhizobium; T1, 5–6 leaf stage; T2, flowering; T3, pod formation stage; n = 3. Red frames indicate group treatments with similar carbon utilization. Full article ">Figure 3
Biolog EcoPlateTM carbon substrates utilization intensity diagram. NI, non-inoculated; I, inoculated with Rhizobium; T1, 5–6 leaf stage; T2, flowering; T3, pod formation stage; n = 3. Full article ">Figure 4
Utilization of categorized substrate by the microbial communities. Errors bars indicate the standard deviations of the mean; * indicates significant difference in polymers utilization between NI T2 and I T2; and + indicates difference in carboxylic acids utilization between NI T3 and I T3 (p < 0.05); n = 3. NI, non-inoculated; I, inoculated with Rhizobium; T1, 5–6 leaf stage; T2, flowering; T3, pod formation stage. Full article ">

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Open AccessArticle

Quantitative Metabolomic Analysis of Urinary Citrulline and Calcitroic Acid in Mice after Exposure to Various Types of Ionizing Radiation

by Maryam Goudarzi, Siddheshwar Chauthe, Steven J. Strawn, Waylon M. Weber, David J. Brenner and Albert J. Fornace

Int. J. Mol. Sci. 2016, 17(5), 782; https://doi.org/10.3390/ijms17050782 - 20 May 2016

Cited by 12 | Viewed by 5976

Abstract

With the safety of existing nuclear power plants being brought into question after the Fukushima disaster and the increased level of concern over terrorism-sponsored use of improvised nuclear devices, it is more crucial to develop well-defined radiation injury markers in easily accessible biofluids [...] Read more.

With the safety of existing nuclear power plants being brought into question after the Fukushima disaster and the increased level of concern over terrorism-sponsored use of improvised nuclear devices, it is more crucial to develop well-defined radiation injury markers in easily accessible biofluids to help emergency-responders with injury assessment during patient triage. Here, we focused on utilizing ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify and quantitate the unique changes in the urinary excretion of two metabolite markers, calcitroic acid and citrulline, in mice induced by different forms of irradiation; X-ray irradiation at a low dose rate (LDR) of 3.0 mGy/min and a high dose rate (HDR) of 1.1 Gy/min, and internal exposure to Cesium-137 (¹³⁷Cs) and Strontium-90 (⁹⁰Sr). The multiple reaction monitoring analysis showed that, while exposure to ¹³⁷Cs and ⁹⁰Sr induced a statistically significant and persistent decrease, similar doses of X-ray beam at the HDR had the opposite effect, and the LDR had no effect on the urinary levels of these two metabolites. This suggests that the source of exposure and the dose rate strongly modulate the in vivo metabolomic injury responses, which may have utility in clinical biodosimetry assays for the assessment of exposure in an affected population. This study complements our previous investigations into the metabolomic profile of urine from mice internally exposed to ⁹⁰Sr and ¹³⁷Cs and to X-ray beam radiation. Full article

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

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The overall urinary metabolomic profile in mice after exposure to four types of radiation. (A) singular value decomposition-based principle component analysis (PCA) constructed in R showing the distinct separation of the metabolomic profiles of control mice (red circles) from that of mice exposed to ionizing radiation (IR) using the ions present in at least 70% of the samples. This PCA also highlights the similarities in the metabolomic signatures of the 4 exposure types; X-ray irradiation at 1.1 Gy/min (HDR) (dark blue circles), X-ray irradiation at 3.0 mGy/min (LDR) (yellow circles), internal 137Cs (purple circles), and internal 90Sr (light blue circles). While the metabolomic signatures of 90Sr and 137Cs exposures match each other more closely, they do overlap with HDR and LDR X-ray irradiations; (B) Venn diagram showing the distribution of statistically significant metabolic pathways among the different exposure types. This diagram shows the number of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways with statistically significant perturbations after the above four IR exposures. The blue box highlights the common KEGG pathways, which had statistically significant changes in the urinary excretion of at least one of their intermediates in all four exposure scenarios. This figure shows overall metabolic perturbations (increase or decrease) in these pathways induced by all exposure types. Full article ">Figure 2
Representative UPLC-MS/MS chromatograms of endogenous urinary citrulline detected at 91.5 ng/mL and citrulline-d4 at 150 ng/mL (Internal Standard). The retention time is presented on the x-axis and the y-axis represent the ion count for the channel. The mass charge ratio of the parent and daughter ions, as well as the ion count for each channel is shown. The peaks represent the most abundant ions that were used for quantification. Multiple reaction monitoring (MRM) transition chromatogram of (A) m/z 176.1→158.9 for citrulline and (B) m/z 180.1→74.1 for spiked neat internal standard citrulline-d4 used for quantitation. Full article ">Figure 3
Representative UPLC-MS/MS chromatograms of calcitroic acid standard spiked in urine detected at 250 ng/mL and 1α,25-dihydroxyvitamin-D3-d3 at 500 ng/mL (Internal Standard). The retention time is presented on the x-axis and the y-axis represent the ion count for the channel. The mass charge ratio of the parent and daughter ions, as well as the ion count for each channel, is shown. The peaks represent the most abundant ions that were used for quantification. MRM transition chromatogram of (A) m/z 375.4→357.3 for calcitroic acid and (B) m/z transition 419.2→305.0 for spiked neat internal standard 1α,25-dihydroxyvitamin-D3-d3 used for quantitation. Note that quantitation only involved peak area at 1.20 min and excluded that of the peak at 0.73 min. Full article ">Figure 4
(A) changes in the urinary concentration of citrulline from mice after various exposure types. The y-axis represents log2 fold change after exposure and x-axis represents each of the three exposure senarios; 90Sr exposure at days 7–9 after exposure at cumulative dose of 2.0 Gy, 137Cs at day 5 after exposure at cumulative dose of 4.1 Gy, and X-ray at day 5 post 4.4 Gy exposure at high dose rate (HDR) of 1.1 Gy/min and low dose rate (LDR) of 0.00309 Gy/min. 90Sr and to a lesser extent 137Cs exposure induced a decrease in the urinary levels of citrulline, while X-ray exposure (HDR) had an opposite effect. No statistically significant change in the urinary concentration of calcitroic acid was detected after LDR exposure; (B) box plot representation of urinary concentration of citrulline after 90Sr-exposure. The calculated mean ± standard error of the mean (SEM) for the control mice was 326.3 ± 24.6 ng/mL, for days 7–9 at cumulative skeleton dose of 2.0 Gy was 182.8 ± 10.7 ng/mL, and for days 25–30 at 5.0 Gy was 176.1 ± 18.8 ng/mL. Each study group contained at least six samples. Concentration fold change was calculated by the log2 ratio of urinary concentration of citrulline in mice exposed to 90Sr to that of citrulline in control mice at each time-point; (C) ROC analysis of citrulline at time-point days 7–9 after 90Sr-exposure at cumulative average dose of 2.0 Gy compared to controls shows the robustness of the biomarker classification. The predictive power of the biomarker at cutoff of 268.7 ng/mL is at 83.3% specificity and 100% sensitivity. (****) denotes the statistical significance in terms of p-value (p-value < 0.0001) of the change in concentration of citrulline after exposure to 90Sr in mice at each of the two time-points. Full article ">Figure 5
(A) changes in the urinary concentration of calcitroic acid from mice after various exposure types. The y-axis represents log2 fold change after exposure and x-axis represents each of the three exposure senarios; 90Sr exposure at days 7–9 after exposure at cumulative dose of 2.0 Gy, 137Cs at day 5 after exposure at cumulative dose of 4.1 Gy, and X-ray at day 5 post 4.4 Gy exposure at high dose rate (HDR) of 1.1 Gy/min min and low dose rate (LDR) of 0.00309 Gy/min. 90Sr and to a lesser extent 137Cs exposure induced a decrease in the urinary levels of calcitroic acid, while X-ray exposure (HDR) had an opposite effect. No statistically significant change in the urinary concentration of calcitroic acid was detected after LDR exposure; (B) box plot representation of urinary concentration of calcitroic acid from mice after 90Sr-exposure. The calculated mean ± SEM for the control mice was 13.6 ± 0.96 μg/mL, for days 7–9 at cumulative skeleton dose of 2.0 Gy was 8.4 ± 0.46 μg/mL, and for days 25–30 at 5.0 Gy was 7.5 ± 0.90 μg/mL. Each study group contained at least 6 samples; (B) concentration fold change was calculated by the log2 ratio of urinary concentration of calcitroic acid in mice exposed to 90Sr to that of calcitroic acid in control mice at each time-point. This figure clearly shows a persistent and statistically significant decrease in the urinary concentration of calcitroic acid after 90Sr-exposure; (C) ROC analysis of calcitroic at time-point days 7–9 after exposure at cumulative average dose of 2.0 Gy compared to controls shows that the sensitivity and specificity at cutoff of 11.35 μg/mL are 100% and 83.3% respectively. (***) denotes the statistical significance in terms of p-value (p-value = 0.0002) of the change in concentration of calcitroic acid after exposure at each of the two time-points. Full article ">Figure 6
Overall metabolism of citrulline in the urea cycle and of calcitroic acid secretion into the bile. Citrulline is an important intermediate of the urea cycle in the liver and is primarily synthesized from arginine and glutamine in the enterocytes. Citrulline is essential for the synthesis of arginine, which in turn produces nitric oxide. On the other hand, calcitroic acid has a less defined biological role. Calcitroic acid is an end product of 1α,25-(OH)2D3 metabolism through a C-24 oxidation by mitochondrial CYP24 enzyme and is secreted into the bile [<a href="#B11-ijms-17-00782" class="html-bibr">11</a>]. Osteoblasts, kidneys and intestine metabolize 1α,25-(OH)2D3 through this oxidation pathway, which is an important regulatory pathway for dihydroxyvitamin-D3. Full article ">Figure 7
Overview of the untargeted and the quantitative studies. Urine samples were collected at indicated time points from mice exposed to various IR sources; internal 90Sr, internal 137Cs, X-ray HDR, and X-ray LDR The samples were initially subjected to untargeted metabolomic profiling using Waters Xevo G2-S UPLC-QToF (Waters, Milford, MA, USA). The acquired data were deconvoluted and analyzed using various bioinformatics tools. A panel of statistically significant metabolite markers were selected based on their clear IR responses, robustness of responses, magnitude of responses, and biological importance. Citrulline and calcitroif acid matched these criteria and thus were selected for quantitative assessment using Waters tandem quadrupole TQ-S. Multiple reaction monitoring methods were developed with the help of appropriate internal standards. The results of such quantitative study are promising as they have the potential to be part of functional assays with clinical applications to assist medical workers assess exposure in a population after a radiological and/or nuclear event. Full article ">

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Open AccessArticle

Angiotensin II Stimulation of DPP4 Activity Regulates Megalin in the Proximal Tubules

by Annayya Aroor, Marcin Zuberek, Cornel Duta, Alex Meuth, James R. Sowers, Adam Whaley-Connell and Ravi Nistala

Int. J. Mol. Sci. 2016, 17(5), 780; https://doi.org/10.3390/ijms17050780 - 20 May 2016

Cited by 32 | Viewed by 6207

Abstract

Proteinuria is a marker of incipient kidney injury in many disorders, including obesity. Previously, we demonstrated that megalin, a receptor endocytotic protein in the proximal tubule, is downregulated in obese mice, which was prevented by inhibition of dipeptidyl protease 4 (DPP4). Obesity is [...] Read more.

Proteinuria is a marker of incipient kidney injury in many disorders, including obesity. Previously, we demonstrated that megalin, a receptor endocytotic protein in the proximal tubule, is downregulated in obese mice, which was prevented by inhibition of dipeptidyl protease 4 (DPP4). Obesity is thought to be associated with upregulation of intra-renal angiotensin II (Ang II) signaling via the Ang II Type 1 receptor (AT₁R) and Ang II suppresses megalin expression in proximal tubule cells in vitro. Therefore, we tested the hypothesis that Ang II will suppress megalin protein via activation of DPP4. We used Ang II (200 ng/kg/min) infusion in mice and Ang II (10⁻⁸ M) treatment of T35OK-AT₁R proximal tubule cells to test our hypothesis. Ang II-infused mouse kidneys displayed increases in DPP4 activity and decreases in megalin. In proximal tubule cells, Ang II stimulated DPP4 activity concurrent with suppression of megalin. MK0626, a DPP4 inhibitor, partially restored megalin expression similar to U0126, a mitogen activated protein kinase (MAPK)/extracellular regulated kinase (ERK) kinase kinase (MEK) 1/2 inhibitor and AG1478, an epidermal growth factor receptor (EGFR) inhibitor. Similarly, Ang II-induced ERK phosphorylation was suppressed with MK0626 and Ang II-induced DPP4 activity was suppressed by U0126. Therefore, our study reveals a cross talk between AT₁R signaling and DPP4 activation in the regulation of megalin and underscores the significance of targeting DPP4 in the prevention of obesity related kidney injury progression. Full article

(This article belongs to the Special Issue Advances in Chronic Kidney Disease)

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Ang II infusion activates the renin-angiotensin system (RAS) and dipeptidyl peptidase 4 (DPP4) and suppresses megalin protein levels in mice: (A) Quantification of differential mRNA expression of RAS in the kidney (Ai–Aiv) and depiction of actual bands that were used for quantification (Av); (B) DPP4 activity in the kidney expressed as relative light units (RLUs); and (C) megalin protein expression by immunoblot of kidney lysates. n = 3–4; * p < 0.05; AGT: Angiotensinogen; AT1AR: Angiotensin type 1A receptor; AT1BR: Angiotensin type 1B receptor; AT2R: Angiotensin type 2 receptor; 18s: 18s ribosomal RNA; Con: Saline-infused mice; Ang II: Ang II-infused mice (200 ng/kg/min). Full article ">Figure 2
DPP4 enzymatic activity and protein levels in T35OK-AT1R proximal tubule cells after acute stimulation with Ang II: (A) DPP4 activity after stimulation (30 min) with Ang II (10−8 M) and blockade (60 min prior to Ang II) with olmesartan (10−6 M) or MK0626 (5 × 10−6 M); and (B) DPP4 protein levels after stimulation (30 min) with Ang II and blockade with olmesartan and MK0626. n = 3–5; * p < 0.05; Olme: Olmesartan; MK0626: Rodent DPP4 inhibitor (Merck & Co., Inc.). Full article ">Figure 3
Ang II regulates megalin protein expression via DPP4 activation. (A) Megalin protein expression by immunoblot in T35OK-AT1R proximal tubule cells. Proximal tubule cells were stimulated with Ang II (10−8 M) for 24 h and pre-treated 1 h with olmesartan (10−6 M), AG1478 (10−5 M), U0126 (10−5 M) and MK0626 (5 × 10−5 M); (B) ERK1/2 activation was assessed by pThr202Tyr204-ERK1/2 increase. Proximal tubule cells were stimulated with Ang II (10−8 M) for 30 min and the ratio of pThr202Tyr204-ERK1/2 to total ERK1/2 ratio was calculated. Proximal tubule cells were pre-treated for 1 h with olmesartan (10−6 M), AG1478 (10−5 M), U0126 (10−5 M) and MK0626 (5 × 10−5 M); (C) DPP4 activity in conditions of chronic Ang II stimulation. T35OK-AT1R proximal tubule cells were stimulated for 24 h with Ang II (10−8 M) and olmesartan (10−6 M), AG1478 (10−5 M), U0126 (10−5 M) and MK0626 (5 × 10−5 M) were tested for blockade of Ang II-mediated increase in DPP4 activity. n = 3–6; * p < 0.05 when compared to control; † p < 0.05 when compared to Ang II. Full article ">

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Open AccessEditorial

International Journal of Molecular Sciences 2016 Best Paper Award

by International Journal of Molecular Sciences Editorial Office

Int. J. Mol. Sci. 2016, 17(5), 777; https://doi.org/10.3390/ijms17050777 - 20 May 2016

Cited by 7 | Viewed by 8231

Abstract

The Editors of the International Journal of Molecular Sciences have established the Best Paper Award to recognize the most outstanding articles published in the areas of molecular biology, molecular physics and chemistry that have been published in the International Journal of Molecular Sciences.[...] [...] Read more.

The Editors of the International Journal of Molecular Sciences have established the Best Paper Award to recognize the most outstanding articles published in the areas of molecular biology, molecular physics and chemistry that have been published in the International Journal of Molecular Sciences.[...] Full article

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1096 KiB

Open AccessArticle

Integrated Analysis of Expression Profile Based on Differentially Expressed Genes in Middle Cerebral Artery Occlusion Animal Models

by Huaqiang Zhou, Zeting Qiu, Shaowei Gao, Qinchang Chen, Si Li, Wulin Tan, Xiaochen Liu and Zhongxing Wang

Int. J. Mol. Sci. 2016, 17(5), 776; https://doi.org/10.3390/ijms17050776 - 20 May 2016

Cited by 12 | Viewed by 6458

Abstract

Stroke is one of the most common causes of death, only second to heart disease. Molecular investigations about stroke are in acute shortage nowadays. This study is intended to explore a gene expression profile after brain ischemia reperfusion. Meta-analysis, differential expression analysis, and [...] Read more.

Stroke is one of the most common causes of death, only second to heart disease. Molecular investigations about stroke are in acute shortage nowadays. This study is intended to explore a gene expression profile after brain ischemia reperfusion. Meta-analysis, differential expression analysis, and integrated analysis were employed on an eight microarray series. We explored the functions and pathways of target genes in gene ontology (GO) enrichment analysis and constructed a protein-protein interaction network. Meta-analysis identified 360 differentially expressed genes (DEGs) for Mus musculus and 255 for Rattus norvegicus. Differential expression analysis identified 44 DEGs for Mus musculus and 21 for Rattus norvegicus. Timp1 and Lcn2 were overexpressed in both species. The cytokine-cytokine receptor interaction and chemokine signaling pathway were highly enriched for the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. We have exhibited a global view of the potential molecular differences between middle cerebral artery occlusion (MCAO) animal model and sham for Mus musculus or Rattus norvegicus, including the biological process and enriched pathways in DEGs. This research helps contribute to a clearer understanding of the inflammation process and accurate identification of ischemic infarction stages, which might be transformed into a therapeutic approach. Full article

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

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Search in the public microarray data repositories identified 128 microarray datasets. We finally selected eight studies for studies according to the included and excluded criteria. Full article ">Figure 2
Protein–protein interaction (PPI) network. The confidence scores are marked in the line of interaction. (White: Mus musculus; Black: Rattus norvegicus). Full article ">Figure 3
Forest plots of Timp1 and Lcn2. Forest plots of (A) Timp1 in Mus musculus; (B) Lcn2 in Mus musculus; (C) Timp1 in Rattus norvegicus; and (D) Lcn2 in Rattus norvegicus. For each forest plot, the bar at the top shows the occurrence of combining p-values and combining effect size methods in selected genes. The dark gray box means that the result of the particular method in the selected gene is present, while light gray means that it is absent. The graph at the bottom shows the combination of effect size. The point marks the effect size, the horizontal lines denote the variance of effect size. Adjusted FDR (false discovery rate) on the right side of the graph presents the statistical significance of the difference in gene expression. Full article ">Figure 3 Cont.
Forest plots of Timp1 and Lcn2. Forest plots of (A) Timp1 in Mus musculus; (B) Lcn2 in Mus musculus; (C) Timp1 in Rattus norvegicus; and (D) Lcn2 in Rattus norvegicus. For each forest plot, the bar at the top shows the occurrence of combining p-values and combining effect size methods in selected genes. The dark gray box means that the result of the particular method in the selected gene is present, while light gray means that it is absent. The graph at the bottom shows the combination of effect size. The point marks the effect size, the horizontal lines denote the variance of effect size. Adjusted FDR (false discovery rate) on the right side of the graph presents the statistical significance of the difference in gene expression. Full article ">Figure 4
A summary of analysis workflow. RLE, relative log expression; NUSE, normalized unscaled standard errors; DEGs, differentially expressed genes. Solid line with arrow refers to directed workflow. Dash line refers to enriched gene sets of several studies. Full article ">

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Open AccessReview

The Natural Course of Non-Alcoholic Fatty Liver Disease

by Luis Calzadilla Bertot and Leon Anton Adams

Int. J. Mol. Sci. 2016, 17(5), 774; https://doi.org/10.3390/ijms17050774 - 20 May 2016

Cited by 456 | Viewed by 21622

Abstract

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease in the world, paralleling the epidemic of obesity and Type 2 diabetes mellitus (T2DM). NAFLD exhibits a histological spectrum, ranging from “bland steatosis” to the more aggressive necro-inflammatory form, [...] Read more.

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent form of chronic liver disease in the world, paralleling the epidemic of obesity and Type 2 diabetes mellitus (T2DM). NAFLD exhibits a histological spectrum, ranging from “bland steatosis” to the more aggressive necro-inflammatory form, non-alcoholic steatohepatitis (NASH) which may accumulate fibrosis to result in cirrhosis. Emerging data suggests fibrosis, rather than NASH per se, to be the most important histological predictor of liver and non-liver related death. Nevertheless, only a small proportion of individuals develop cirrhosis, however the large proportion of the population affected by NAFLD has led to predictions that NAFLD will become a leading cause of end stage liver disease, hepatocellular carcinoma (HCC), and indication for liver transplantation. HCC may arise in non-cirrhotic liver in the setting of NAFLD and is associated with the presence of the metabolic syndrome (MetS) and male gender. The MetS and its components also play a key role in the histological progression of NAFLD, however other genetic and environmental factors may also influence the natural history. The importance of NAFLD in terms of overall survival extends beyond the liver where cardiovascular disease and malignancy represents additional important causes of death. Full article

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

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Open AccessArticle

Polymeric Nanoparticle-Based Photodynamic Therapy for Chronic Periodontitis in Vivo

by Laura Marise De Freitas, Giovana Maria Fioramonti Calixto, Marlus Chorilli, Juçaíra Stella M. Giusti, Vanderlei Salvador Bagnato, Nikolaos S. Soukos, Mansoor M. Amiji and Carla Raquel Fontana

Int. J. Mol. Sci. 2016, 17(5), 769; https://doi.org/10.3390/ijms17050769 - 20 May 2016

Cited by 82 | Viewed by 9987

Abstract

Antimicrobial photodynamic therapy (aPDT) is increasingly being explored for treatment of periodontitis. Here, we investigated the effect of aPDT on human dental plaque bacteria in suspensions and biofilms in vitro using methylene blue (MB)-loaded poly(lactic-co-glycolic) (PLGA) nanoparticles (MB-NP) and red light [...] Read more.

Antimicrobial photodynamic therapy (aPDT) is increasingly being explored for treatment of periodontitis. Here, we investigated the effect of aPDT on human dental plaque bacteria in suspensions and biofilms in vitro using methylene blue (MB)-loaded poly(lactic-co-glycolic) (PLGA) nanoparticles (MB-NP) and red light at 660 nm. The effect of MB-NP-based aPDT was also evaluated in a clinical pilot study with 10 adult human subjects with chronic periodontitis. Dental plaque samples from human subjects were exposed to aPDT—in planktonic and biofilm phases—with MB or MB-NP (25 µg/mL) at 20 J/cm² in vitro. Patients were treated either with ultrasonic scaling and scaling and root planing (US + SRP) or ultrasonic scaling + SRP + aPDT with MB-NP (25 µg/mL and 20 J/cm²) in a split-mouth design. In biofilms, MB-NP eliminated approximately 25% more bacteria than free MB. The clinical study demonstrated the safety of aPDT. Both groups showed similar improvements of clinical parameters one month following treatments. However, at three months ultrasonic SRP + aPDT showed a greater effect (28.82%) on gingival bleeding index (GBI) compared to ultrasonic SRP. The utilization of PLGA nanoparticles encapsulated with MB may be a promising adjunct in antimicrobial periodontal treatment. Full article

(This article belongs to the Special Issue Drug Delivery and Antimicrobial Agents)

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Recovered CFU/mL after antimicrobial photodynamic therapy (aPDT) treatment of planktonic bacteria with free methylene blue (MB) (25 µg/mL) and MB-NP (25 µg/mL equivalent to MB) and visible light at 660 nm with an energy fluence of 20 J/cm2. Each bar is the mean values of the means from 10 samples (data from each sample were representative of four independent suspensions). Error bars denote the standard deviation of the mean. The asterisks represent the statistical difference between the groups and the control (one-way ANOVA with Tukey’s post hoc). *** p < 0.001; ns: not significant; MB: methylene blue; MB-NP: MB-loaded PLGA nanoparticles; CFU: colony forming units. Full article ">Figure 2
Recovered CFU/mL after aPDT treatment of bacteria growing in biofilms with free MB (25 µg/mL) and MB-NP (25 µg/mL equivalent to MB) and visible light at 660 nm with an energy fluence of 20 J/cm2. Each bar is the mean values of the means from 10 samples (data from each sample were representative of four independent biofilms). The asterisks represent the statistical difference between the groups and the control (one-way ANOVA with Tukey’s post hoc). *** p < 0.001; ns: not significant; MB: methylene blue; MB-NP: MB-loaded PLGA nanoparticles; CFU: colony forming units. Full article ">Figure 3
Probing pocket depths. Probing was accessed at baseline, one week, one month, and three months after treatments. Shapes represent mean values from 10 patients at each time point. Error bars represent the standard deviation. US + SRP: ultrasonic scaling and scaling and root planing; US + SRP + aPDT: US + SRP followed by antimicrobial photodynamic therapy. Full article ">Figure 4
Visible plaque and Gingival bleeding indexes. VPI (a) and GBI (b) scores were accessed at baseline, one week, one month, and three months after treatments. Shapes represent mean values from 10 patients at each time point. Error bars represent the standard deviation. US + SRP: ultrasonic scaling and scaling and root planing; US + SRP + aPDT: US + SRP followed by antimicrobial photodynamic therapy. Full article ">Figure 5
Bleeding on probing and Clinical attachment level. BOP (a) and CAL (b) scores were accessed at baseline, one week, one month, and three months after treatments. Shapes represent mean values from 10 patients at each time point. Error bars represent the standard deviation. US + SRP: ultrasonic scaling and scaling and root planing; US + SRP + aPDT: US + SRP followed by antimicrobial photodynamic therapy. Full article ">Figure 6
Scanning electron micrograph (SEM) of PLGA nanoparticles. Figure shows an SEM image of higher magnification with spherical nanoparticles of 150–250 nm in diameter. Full article ">

0 pages, 155 KiB

Open AccessRetraction

RETRACTED: Xu, et al. Tanshinone IIA Pretreatment Renders Free Flaps against Hypoxic Injury through Activating Wnt Signaling and Upregulating Stem Cell-Related Biomarkers. Int. J. Mol. Sci. 2014, 15, 18117–18130

by International Journal of Molecular Sciences Editorial Office

Int. J. Mol. Sci. 2016, 17(5), 768; https://doi.org/10.3390/ijms17050768 - 20 May 2016

Cited by 2 | Viewed by 3634

Abstract

We have been made aware that text, ﬁgures and experimental data reported in the title paper [...] Full article

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Open AccessArticle

Development of Therapeutic Chimeric Uricase by Exon Replacement/Restoration and Site-Directed Mutagenesis

by Guangrong Xie, Weizhen Yang, Jing Chen, Miaomiao Li, Nan Jiang, Baixue Zhao, Si Chen, Min Wang and Jianhua Chen

Int. J. Mol. Sci. 2016, 17(5), 764; https://doi.org/10.3390/ijms17050764 - 20 May 2016

Cited by 10 | Viewed by 5737

Abstract

The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with [...] Read more.

The activity of urate oxidase was lost during hominoid evolution, resulting in high susceptibility to hyperuricemia and gout in humans. In order to develop a more “human-like” uricase for therapeutic use, exon replacement/restoration and site-directed mutagenesis were performed to obtain porcine–human uricase with higher homology to deduced human uricase (dHU) and increased uricolytic activity. In an exon replacement study, substitution of exon 6 in wild porcine uricase (wPU) gene with corresponding exon in dhu totally abolished its activity. Substitutions of exon 5, 3, and 1–2 led to 85%, 60%, and 45% loss of activity, respectively. However, replacement of exon 4 and 7–8 did not significantly change the enzyme activity. When exon 5, 6, and 3 in dhu were replaced by their counterparts in wpu, the resulting chimera H_1-2P₃H₄P_5-6H_7-8 was active, but only about 28% of wPU. Multiple sequence alignment and homology modeling predicted that mutations of E24D and E83G in H_1-2P₃H₄P_5-6H_7-8 were favorable for further increase of its activity. After site-directed mutagenesis, H_1-2P₃H₄P_5-6H_7-8 (E24D & E83G) with increased homology (91.45%) with dHU and higher activity and catalytic efficiency than the FDA-approved porcine–baboon chimera (PBC) was obtained. It showed optimum activity at pH 8.5 and 35 °C and was stable in a pH range of 6.5–11.0 and temperature range of 20–40 °C. Full article

(This article belongs to the Section Biochemistry)

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704 KiB

Open AccessArticle

Degree of Conversion and BisGMA, TEGDMA, UDMA Elution from Flowable Bulk Fill Composites

by Edina Lempel, Zsuzsanna Czibulya, Bálint Kovács, József Szalma, Ákos Tóth, Sándor Kunsági-Máté, Zoltán Varga and Katalin Böddi

Int. J. Mol. Sci. 2016, 17(5), 732; https://doi.org/10.3390/ijms17050732 - 20 May 2016

Cited by 67 | Viewed by 8534

Abstract

The degree of conversion (DC) and the released bisphenol A diglycidyl ether dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) monomers of bulk-fill composites compared to that of conventional flowable ones were assessed using micro-Raman spectroscopy and high performance liquid chromatography [...] Read more.

The degree of conversion (DC) and the released bisphenol A diglycidyl ether dimethacrylate (BisGMA), triethylene glycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) monomers of bulk-fill composites compared to that of conventional flowable ones were assessed using micro-Raman spectroscopy and high performance liquid chromatography (HPLC). Four millimeter-thick samples were prepared from SureFil SDR Flow (SDR), X-tra Base (XB), Filtek Bulk Fill (FBF) and two and four millimeter samples from Filtek Ultimate Flow (FUF). They were measured with micro-Raman spectroscopy to determine the DC% of the top and the bottom surfaces. The amount of released monomers in 75% ethanol extraction media was measured with HPLC. The differences between the top and bottom DC% were significant for each material. The mean DC values were in the following order for the bottom surfaces: SDR_4mm_20s > FUF_2mm_20s > XB_4mm_20s > FBF_4mm_20s > XB_4mm_10s > FBF_4mm_10s > FUF_4mm_20s. The highest rate in the amount of released BisGMA and TEGDMA was found from the 4 mm-thick conventional flowable FUF. Among bulk-fills, FBF showed a twenty times higher amount of eluted UDMA and twice more BisGMA; meanwhile, SDR released a significantly higher amount of TEGDMA. SDR bulk-fill showed significantly higher DC%; meanwhile XB, FBF did not reach the same level DC, as that of the 2 mm-thick conventional composite at the bottom surface. Conventional flowable composites showed a higher rate of monomer elution compared to the bulk-fills, except FBF, which showed a high amount of UDMA release. Full article

(This article belongs to the Special Issue Molecular Research on Dental Materials and Biomaterials)

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4955 KiB

Open AccessArticle

Ruthenium Complexes Induce HepG2 Human Hepatocellular Carcinoma Cell Apoptosis and Inhibit Cell Migration and Invasion through Regulation of the Nrf2 Pathway

by Yiyu Lu, Ting Shen, Hua Yang and Weiguang Gu

Int. J. Mol. Sci. 2016, 17(5), 775; https://doi.org/10.3390/ijms17050775 - 19 May 2016

Cited by 33 | Viewed by 6887

Abstract

Ruthenium (Ru) complexes are currently the focus of substantial interest because of their potential application as chemotherapeutic agents with broad anticancer activities. This study investigated the in vitro and in vivo anticancer activities and mechanisms of two Ru complexes—2,3,7,8,12,13,17,18-Octaethyl-21H,23H-porphine Ru(II) carbonyl (Ru1) and [...] Read more.

Ruthenium (Ru) complexes are currently the focus of substantial interest because of their potential application as chemotherapeutic agents with broad anticancer activities. This study investigated the in vitro and in vivo anticancer activities and mechanisms of two Ru complexes—2,3,7,8,12,13,17,18-Octaethyl-21H,23H-porphine Ru(II) carbonyl (Ru1) and 5,10,15,20-Tetraphenyl-21H,23H-porphine Ru(II) carbonyl (Ru2)—against human hepatocellular carcinoma (HCC) cells. These Ru complexes effectively inhibited the cellular growth of three human hepatocellular carcinoma (HCC) cells, with IC₅₀ values ranging from 2.7–7.3 μM. In contrast, the complexes exhibited lower toxicity towards L02 human liver normal cells with IC₅₀ values of 20.4 and 24.8 μM, respectively. Moreover, Ru2 significantly inhibited HepG2 cell migration and invasion, and these effects were dose-dependent. The mechanistic studies demonstrated that Ru2 induced HCC cell apoptosis, as evidenced by DNA fragmentation and nuclear condensation, which was predominately triggered via caspase family member activation. Furthermore, HCC cell treatment significantly decreased the expression levels of Nrf2 and its downstream effectors, NAD(P)H: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO1). Ru2 also exhibited potent in vivo anticancer efficacy in a tumor-bearing nude mouse model, as demonstrated by a time- and dose-dependent inhibition on tumor growth. The results demonstrate the therapeutic potential of Ru complexes against HCC via Nrf2 pathway regulation. Full article

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

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Open AccessArticle

Predicting MicroRNA Biomarkers for Cancer Using Phylogenetic Tree and Microarray Analysis

by Hsiuying Wang

Int. J. Mol. Sci. 2016, 17(5), 773; https://doi.org/10.3390/ijms17050773 - 19 May 2016

Cited by 26 | Viewed by 7729

Abstract

MicroRNAs (miRNAs) are shown to be involved in the initiation and progression of cancers in the literature, and the expression of miRNAs is used as an important cancer prognostic tool. The aim of this study is to predict high-confidence miRNA biomarkers for cancer. [...] Read more.

MicroRNAs (miRNAs) are shown to be involved in the initiation and progression of cancers in the literature, and the expression of miRNAs is used as an important cancer prognostic tool. The aim of this study is to predict high-confidence miRNA biomarkers for cancer. We adopt a method that combines miRNA phylogenetic structure and miRNA microarray data analysis to discover high-confidence miRNA biomarkers for colon, prostate, pancreatic, lung, breast, bladder and kidney cancers. There are 53 miRNAs selected through this method that either have potential to involve a single cancer’s development or to involve several cancers’ development. These miRNAs can be used as high-confidence miRNA biomarkers of these seven investigated cancers for further experiment validation. miR-17, miR-20, miR-106a, miR-106b, miR-92, miR-25, miR-16, miR-195 and miR-143 are selected to involve a single cancer’s development in these seven cancers. They have the potential to be useful miRNA biomarkers when the result can be confirmed by experiments. Full article

(This article belongs to the Special Issue MicroRNA Regulation)

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Open AccessArticle

A Study of Single Nucleotide Polymorphisms of the SLC19A1/RFC1 Gene in Subjects with Autism Spectrum Disorder

by Naila Al Mahmuda, Shigeru Yokoyama, Jian-Jun Huang, Li Liu, Toshio Munesue, Hideo Nakatani, Kenshi Hayashi, Kunimasa Yagi, Masakazu Yamagishi and Haruhiro Higashida

Int. J. Mol. Sci. 2016, 17(5), 772; https://doi.org/10.3390/ijms17050772 - 19 May 2016

Cited by 12 | Viewed by 6055

Abstract

Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with complex genetic etiology. Recent studies have indicated that children with ASD may have altered folate or methionine metabolism, suggesting that the folate–methionine cycle may play a key role in the etiology of [...] Read more.

Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders with complex genetic etiology. Recent studies have indicated that children with ASD may have altered folate or methionine metabolism, suggesting that the folate–methionine cycle may play a key role in the etiology of ASD. SLC19A1, also referred to as reduced folate carrier 1 (RFC1), is a member of the solute carrier group of transporters and is one of the key enzymes in the folate metabolism pathway. Findings from multiple genomic screens suggest the presence of an autism susceptibility locus on chromosome 21q22.3, which includes SLC19A1. Therefore, we performed a case-control study in a Japanese population. In this study, DNA samples obtained from 147 ASD patients at the Kanazawa University Hospital in Japan and 150 unrelated healthy Japanese volunteers were examined by the sequence-specific primer-polymerase chain reaction method pooled with fluorescence correlation spectroscopy. p < 0.05 was considered to represent a statistically significant outcome. Of 13 single nucleotide polymorphisms (SNPs) examined, a significant p-value was obtained for AA genotype of one SNP (rs1023159, OR = 0.39, 95% CI = 0.16–0.91, p = 0.0394; Fisher’s exact test). Despite some conflicting results, our findings supported a role for the polymorphism rs1023159 of the SLC19A1 gene, alone or in combination, as a risk factor for ASD. However, the findings were not consistent after multiple testing corrections. In conclusion, although our results supported a role of the SLC19A1 gene in the etiology of ASD, it was not a significant risk factor for the ASD samples analyzed in this study. Full article

(This article belongs to the Special Issue The Identification of the Genetic Components of Autism Spectrum Disorders 2017)

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Figure 1
The genomic structure of SLC19A1 (A). Bars, exons. Arrows, positions of single nucleotide polymorphisms (SNPs). Linkage disequilibrium plot of SNPs in the samples studied (B). Numbers in squares indicate D′ values. Reference Number (rs) with asterisk indicates the SNP with p < 0.05. The blocks are defined following the four-gamete rule [<a href="#B22-ijms-17-00772" class="html-bibr">22</a>]. Explanation of color scheme: If D′ < 1 and LOD (log of the likelihood odds ratio) <2, the cell color is white; if D′ = 1 and LOD < 2, the cell color is blue; if D′ < 1 and LOD ≥ 2, the cell color is shades of pink/red; if D′ = 1 and LOD ≥ 2, the cell color is bright red. Full article ">Figure 1 Cont.
The genomic structure of SLC19A1 (A). Bars, exons. Arrows, positions of single nucleotide polymorphisms (SNPs). Linkage disequilibrium plot of SNPs in the samples studied (B). Numbers in squares indicate D′ values. Reference Number (rs) with asterisk indicates the SNP with p < 0.05. The blocks are defined following the four-gamete rule [<a href="#B22-ijms-17-00772" class="html-bibr">22</a>]. Explanation of color scheme: If D′ < 1 and LOD (log of the likelihood odds ratio) <2, the cell color is white; if D′ = 1 and LOD < 2, the cell color is blue; if D′ < 1 and LOD ≥ 2, the cell color is shades of pink/red; if D′ = 1 and LOD ≥ 2, the cell color is bright red. Full article ">

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Open AccessArticle

Downregulation of Runx2 by 1,25-Dihydroxyvitamin D₃ Induces the Transdifferentiation of Osteoblasts to Adipocytes

by Jung Ha Kim, Semun Seong, Kabsun Kim, Inyoung Kim, Byung-Chul Jeong and Nacksung Kim

Int. J. Mol. Sci. 2016, 17(5), 770; https://doi.org/10.3390/ijms17050770 - 19 May 2016

Cited by 13 | Viewed by 6517

Abstract

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)₂D₃ enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The [...] Read more.

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)₂D₃ enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The positive role of 1,25(OH)₂D₃ in adipocyte differentiation was confirmed when murine osteoblasts were cultured in adipogenic medium. Additionally, 1,25(OH)₂D₃ enhanced the expression of adipocyte marker genes, but inhibited the expression of osteoblast marker genes in osteoblasts. The inhibition of osteoblast differentiation and promotion of adipocyte differentiation mediated by 1,25(OH)₂D₃ were compensated by Runx2 overexpression. Our results suggest that 1,25(OH)₂D₃ induces the transdifferentiation of osteoblasts to adipocytes via Runx2 downregulation in osteoblasts. Full article

(This article belongs to the Section Biochemistry)

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1,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits osteoblast differentiation. (a–c) Primary osteoblasts were cultured in osteogenic medium (OGM) with vehicle or increasing concentrations of 1,25(OH)2D3. (a) Cultured cells were fixed and stained with Alizarin red; (b) Alizarin red staining was quantified by densitometry at 562 nm; (c) Primary osteoblasts were cultured in osteogenic medium (OGM) containing either vehicle or 1,25(OH)2D3 (10−8 M). The mRNA levels of Runx2, alkaline phosphatase (ALP), and bone sialoprotein (BSP) were analyzed by real-time polymerase chain reaction (PCR). # p < 0.05; * p < 0.01; ** p < 0.001 as compared with controls. Full article ">Figure 2
1,25(OH)2D3 induces transdifferentiation of osteoblasts to adipocytes. (a–c) Primary osteoblasts were cultured in osteogenic medium (OGM) with vehicle or increasing concentrations of 1,25(OH)2D3. (a) Cultured cells were fixed and stained with Oil Red-O, Bar: 50 µm; (b) The number of Oil Red-O—positive cells was counted; (c) Primary osteoblasts were cultured in osteogenic medium (OGM) containing either vehicle or 1,25(OH)2D3 (10−8 M). The mRNA levels of CCAAT/enhancer binding protein-α (CEBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and adipocyte protein 2 (aP) were analyzed by real-time PCR. # p < 0.05; * p < 0.01; ** p < 0.001 as compared with controls. Full article ">Figure 3
1,25(OH)2D3 induces the transdifferentiation of osteoblasts to adipocytes at the early stage of osteoblast differentiation. (a–c) Primary osteoblasts were cultured in osteogenic medium (OGM). Cells were treated with either vehicle or 1,25(OH)2D3 (10−8 M) during the indicated time periods. (a) Cultured cells were fixed and stained with Alizarin red or Oil Red-O, Bar: 50 µm; (b) Alizarin red staining was quantified by densitometry at 562 nm; (c) The number of Oil Red-O—positive cells was counted. # p < 0.05; * p < 0.01; ** p < 0.001 as compared with controls. Full article ">Figure 4
1,25(OH)2D3 induces adipocyte differentiation in primary osteoblasts. (a–d) Primary osteoblasts were cultured in adipogenic medium (AGM) containing either vehicle or 1,25(OH)2D3 (10−8 M). (a) Cultured cells were fixed and stained with Oil Red-O, Bar: 50 µm; (b) The number of Oil Red-O—positive cells was counted; (c) The mRNA levels of Runx2, ALP, and BSP were analyzed by real-time PCR; (d) The mRNA levels of CEBP-α, PPAR-γ, and aP2 were analyzed by real-time PCR. # p < 0.05; * p < 0.01; ** p < 0.001 as compared with controls. Full article ">Figure 5
1,25(OH)2D3 induces transdifferentiation of osteoblasts to adipocytes by inhibiting Runx2 expression. (a–c) Primary osteoblasts were cultured in osteogenic medium (OGM) with vehicle, 1,25(OH)2D3, or PPi as indicated. (a) Cultured cells were fixed and stained with Alizarin red or Oil Red-O, Bar: 50 µm; (b) Alizarin red staining was quantified by densitometry at 562 nm; (c) The number of Oil Red-O–positive cells was counted; (d–f) Osteoblasts were transduced with pMX-IRES-EGFP (control) or Runx2 retrovirus. Transduced osteoblasts were cultured in osteogenic medium (OGM) with vehicle or 1,25(OH)2D3. (d) Cultured cells were fixed and stained with Alizarin red or Oil Red-O, Bar: 50 µm; (e) Alizarin red staining was quantified by densitometry at 562 nm; (f) The number of Oil Red-O—positive cells was counted; (g) C2C12 cells were transfected with a Runx2 reporter construct. Transfected cells were treated with vehicle or 1,25(OH)2D3 for 48 h. Luciferase activity was measured using a dual-luciferase reporter assay system. The data represent means and SD of triplicate samples. # p < 0.05; * p < 0.01; ** p < 0.001 as compared with controls. Full article ">

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Open AccessArticle

How Diet Intervention via Modulation of DNA Damage Response through MicroRNAs May Have an Effect on Cancer Prevention and Aging, an in Silico Study

by Felicia Carotenuto, Maria C. Albertini, Dario Coletti, Alessandra Vilmercati, Luigi Campanella, Zbigniew Darzynkiewicz and Laura Teodori

Int. J. Mol. Sci. 2016, 17(5), 752; https://doi.org/10.3390/ijms17050752 - 19 May 2016

Cited by 18 | Viewed by 6460

Abstract

The DNA damage response (DDR) is a molecular mechanism that cells have evolved to sense DNA damage (DD) to promote DNA repair, or to lead to apoptosis, or cellular senescence if the damage is too extensive. Recent evidence indicates that microRNAs (miRs) play [...] Read more.

The DNA damage response (DDR) is a molecular mechanism that cells have evolved to sense DNA damage (DD) to promote DNA repair, or to lead to apoptosis, or cellular senescence if the damage is too extensive. Recent evidence indicates that microRNAs (miRs) play a critical role in the regulation of DDR. Dietary bioactive compounds through miRs may affect activity of numerous genes. Among the most studied bioactive compounds modulating expression of miRs are epi-gallocatechin-3-gallate, curcumin, resveratrol and n3-polyunsaturated fatty acids. To compare the impact of these dietary compounds on DD/DDR network modulation, we performed a literature search and an in silico analysis by the DIANA-mirPathv3 software. The in silico analysis allowed us to identify pathways shared by different miRs involved in DD/DDR vis-à-vis the specific compounds. The results demonstrate that certain miRs (e.g., -146, -21) play a central role in the interplay among DD/DDR and the bioactive compounds. Furthermore, some specific pathways, such as “fatty acids biosynthesis/metabolism”, “extracellular matrix-receptor interaction” and “signaling regulating the pluripotency of stem cells”, appear to be targeted by most miRs affected by the studied compounds. Since DD/DDR and these pathways are strongly related to aging and carcinogenesis, the present in silico results of our study suggest that monitoring the induction of specific miRs may provide the means to assess the antiaging and chemopreventive properties of particular dietary compounds. Full article

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

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Venn diagram showing the microRNAs involved in DD/DDR (ellipse, grey) and identified as modulated by bioactive compounds: EGCG (epi-gallocatechin-3-gallate; green), CRC (curcumin; blue), RSV (resveratrol; pink) and n3-PUFAs (n3-polyunsaturated fatty acids, yellow). The common miRNAs, modulated by all four compounds, are indicated in red (miR-16, miR-25, miR-21, miR-181a, miR-146b, miR-34a). Full article ">Figure 2
Binary heat map of pathways related to the common microRNAs involved in DDR signaling and modulated by all of the compounds: EGCG, CRC, RSV, n3-PUFAs. In this plot, heat map calculation is based on binary p-values (0: not targeted, 1: targeted); all significantly targeted pathways are marked with deep red. The plot shows miRNAs targeting similar pathways and pathways being targeted by miRNAs. Full article ">Figure 3
MicroRNAs involved in DDR and modulated by EGCG versus the pathways’ heat map. In this plot, heat map calculation is based on absolute p-values. Darker colors represent lower p-values (higher significance). The plot shows miRNAs targeting similar pathway clusters and pathways being targeted by miRNA groups. Full article ">Figure 4
MicroRNAs involved in DDR and modulated by CRC versus the pathways’ heat map. In this plot, heat map calculation is based on absolute p-values. Darker colors represent lower p-values (higher significance). The plot shows microRNAs targeting similar pathway clusters and pathways being targeted by miRNA groups. Full article ">Figure 5
MicroRNAs involved in DDR and modulated by RSV versus the pathways’ heat map. In this plot, heat map calculation is based on absolute p-values. Darker colors represent lower p-values (higher significance). The plot shows microRNAs targeting similar pathway clusters and pathways being targeted by miRNA groups. Full article ">Figure 6
MicroRNAs involved in DDR and modulated by n3-PUFAs versus the pathways’ heat map. In this plot, heat map calculation is based on absolute p-values. Darker colors represent lower p-values (higher significance). The plot shows miRNAs targeting similar pathway clusters and pathways being targeted by miRNA groups. Full article ">

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Int. J. Mol. Sci., Volume 17, Issue 5 (May 2016) – 180 articles

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