text: Medicine & Pharmacology http://www.mdpi.com/journal/med-pharma Latest open access articles published in Medicine & Pharmacology at http://www.mdpi.com/journal/med-pharma http://www.mdpi.com/1420-3049/21/7/826 Ziziphora species represent the prototypical example of the Lamiaceae family. The phytochemicals present in Ziziphora include monoterpenic essential oils, triterpenes and phenolic substances belonging to the flavonoids. In Kazakh traditional medicine, Ziziphora species possess several medicinal uses. In particular, Z. bungeana Lam. and Z. clinopodioides Lam. are used for the treatment of illnesses related to the cardiovascular system or to combat different infections. Unfortunately, the majority of the information about the complex Ziziphora species is only available in Russian and Chinese language, therefore, we decided gather all available information on Kazakhstan Ziziphora, namely its content compounds, medicinal uses and published patents, to draw the attention of scientists to this very interesting plant with high medicinal potential. Molecules 2016-06-25 21 7 Review 10.3390/molecules21070826 826 1420-3049 2016-06-25 doi: 10.3390/molecules21070826 Karel Šmejkal Milan Malaník Karlygash Zhaparkulova Zuriyadda Sakipova Liliya Ibragimova Galya Ibadullaeva Milan Žemlička http://www.mdpi.com/1422-0067/17/7/1013 Hyaluronic acid (HA) is used clinically to treat osteoarthritis (OA), but its pharmacological effects under hypoxic conditions remain unclear. Articular chondrocytes in patients with OA are exposed to a hypoxic environment. This study investigated whether hypoxia could potentiate the anabolic effects of exogenous HA in rat articular cartilage and whether these mechanisms involved HA receptors. HA under hypoxic conditions significantly enhanced the expression of extracellular matrix genes and proteins in explant culture, as shown by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and dimethylmethylene blue (DMMB) assays. Staining with Safranin-O and immunohistochemical staining with antibody to type II collagen were also enhanced in pellet culture. The expression of CD44 was increased by hypoxia and significantly suppressed by transfection with siRNAs targeting hypoxia-inducible factor 1 alpha (siHIF-1α). These findings indicate that hypoxia potentiates the anabolic effects of exogenous HA by a mechanism in which HIF-1α positively regulates the expression of CD44, enhancing the binding affinity for exogenous HA. The anabolic effects of exogenous HA may increase as OA progresses. International Journal of Molecular Sciences 2016-06-25 17 7 Article 10.3390/ijms17071013 1013 1422-0067 2016-06-25 doi: 10.3390/ijms17071013 Shohei Ichimaru Shuji Nakagawa Yuji Arai Tsunao Kishida Masaharu Shin-Ya Kuniaki Honjo Shinji Tsuchida Hiroaki Inoue Hiroyoshi Fujiwara Seiji Shimomura Osam Mazda Toshikazu Kubo http://www.mdpi.com/1420-3049/21/7/837 Organic-inorganic hybrid perovskite solar cells are considered as one of the most promising next-generation solar cells due to their advantages of low-cost precursors, high power conversion efficiency (PCE) and easy of processing. In the past few years, the PCEs have climbed from a few to over 20% for perovskite solar cells. Recent developments demonstrate that perovskite exhibits ambipolar semiconducting characteristics, which allows for the construction of planar heterojunction (PHJ) perovskite solar cells. PHJ perovskite solar cells can avoid the use of high-temperature sintered mesoporous metal oxides, enabling simple processing and the fabrication of flexible and tandem perovskite solar cells. In planar heterojunction materials, hole/electron transport layers are introduced between a perovskite film and the anode/cathode. The hole and electron transporting layers are expected to enhance exciton separation, charge transportation and collection. Further, the supporting layer for the perovskite film not only plays an important role in energy-level alignment, but also affects perovskite film morphology, which have a great effect on device performance. In addition, interfacial layers also affect device stability. In this review, recent progress in interfacial engineering for PHJ perovskite solar cells will be reviewed, especially with the molecular interfacial materials. The supporting interfacial layers for the optimization of perovskite films will be systematically reviewed. Finally, the challenges remaining in perovskite solar cells research will be discussed. Molecules 2016-06-25 21 7 Review 10.3390/molecules21070837 837 1420-3049 2016-06-25 doi: 10.3390/molecules21070837 Wei Yin Lijia Pan Tingbin Yang Yongye Liang http://www.mdpi.com/2304-8158/5/3/47 In recent years, besides the consumption of fresh sea urchin specimens, the demand of minimally-processed roe has grown considerably. This product has made frequent consumption in restaurants possible and frauds are becoming widespread with the partial replacement of sea urchin roe with surrogates that are similar in colour. One of the main factors that determines the quality of the roe is its colour and small differences in colour scale cannot be easily discerned by the consumers. In this study we have applied a rapid colorimetric method for reveal the fraudulent partial substitution of semi-solid sea urchin roe with liquid egg yolk. Objective assessment of whiteness (L*), redness (a*), yellowness (b*), hue (h*), and chroma (C*) was carried out with a digital spectrophotometer using the CIE L*a*b* colour measurement system. The colorimetric method highlighted statistically significant differences among sea urchin roe and liquid egg yolk that could be easily discerned quantitatively. Foods 2016-06-25 5 3 Article 10.3390/foods5030047 47 2304-8158 2016-06-25 doi: 10.3390/foods5030047 Domenico Meloni Antonio Spina Gianluca Satta Vittorio Chessa http://www.mdpi.com/1420-3049/21/7/833 Green is the future of chemistry. Catalysts with high selectivity are the key to green chemistry. Polymer-supported Raney catalysts have been found to have outstanding performance in the clean preparation of some chemicals. For example, a polyamide 6-supported Raney nickel catalyst provided a 100.0% conversion of n-butyraldehyde without producing any detectable n-butyl ether, the main byproduct in industry, and eliminated the two main byproducts (isopropyl ether and methyl-iso-butylcarbinol) in the hydrogenation of acetone to isopropanol. Meanwhile, a model for how the polymer support brought about the elimination of byproducts is proposed and confirmed. In this account the preparation and applications of polymer-supported Raney catalysts along with the corresponding models will be reviewed. Molecules 2016-06-25 21 7 Review 10.3390/molecules21070833 833 1420-3049 2016-06-25 doi: 10.3390/molecules21070833 Haibin Jiang Shuliang Lu Xiaohong Zhang Wei Dai Jinliang Qiao http://www.mdpi.com/1422-0067/17/7/1011 A response surface and Box-Behnken design approach was applied to augment polysaccharide extraction from the residue of Rhizoma gastrodiae. Statistical analysis revealed that the linear and quadratic terms for three variables during extraction exhibited obvious effects on extraction yield. The optimum conditions were determined to be a liquid-to-solid ratio of 54 mL/g, an extraction temperature of 74 °C, an extraction time of 66 min, and three extractions. These conditions resulted in a maximum Rhizoma gastrodiae polysaccharide (RGP) extraction yield of 6.11% ± 0.13%. Two homogeneous polysaccharides (RGP-1a and RGP-1b) were obtained using DEAE cellulose-52 and Sephadex G-100 columns. The preliminary characterization of RGP-1a and RGP-1b was performed using HPLC-RID, HPGPC, and FTIR. Tests of the immunological activity in vitro showed that the two polysaccharides could significantly stimulate macrophages to release NO and enhance phagocytosis in a dose-dependent manner. In particular, RGP-1b (200 μg/mL) and LPS (2 μg/mL) had almost the same influence on the NO production and phagocytic activity of RAW 264.7 macrophages (p > 0.05). All the data obtained indicate that RGP-1a and RGP-1b have the potential to be developed as a health food. International Journal of Molecular Sciences 2016-06-25 17 7 Article 10.3390/ijms17071011 1011 1422-0067 2016-06-25 doi: 10.3390/ijms17071011 Juncheng Chen Shan Tian Xiaoying Shu Hongtao Du Na Li Junru Wang http://www.mdpi.com/1422-0067/17/7/1012 A bacterial strain QH-12 isolated from activated sludge was identified as Gordonia sp. based on analysis of 16S rRNA gene sequence and was found to be capable of utilizing dibutyl phthalate (DBP) and other common phthalate esters (PAEs) as the sole carbon and energy source. The degradation kinetics of DBP under different concentrations by the strain QH-12 fit well with the modified Gompertz model (R2 > 0.98). However, strain QH-12 could not utilize the major intermediate product phthalate (phthalic acid; PA) as the sole carbon and energy source, and only a little amount of PA was detected. The QH-12 genome analysis revealed the presence of putative hydrolase/esterase genes involved in PAEs-degradation but no phthalic acid catabolic gene cluster was found, suggesting that a novel degradation pathway of PAEs was present in Gordonia sp. QH-12. This information will be valuable for obtaining a more holistic understanding on diverse genetic mechanisms of PAEs-degrading Gordonia sp. strains. International Journal of Molecular Sciences 2016-06-25 17 7 Article 10.3390/ijms17071012 1012 1422-0067 2016-06-25 doi: 10.3390/ijms17071012 Decai Jin Xiao Kong Huijun Liu Xinxin Wang Ye Deng Minghong Jia Xiangyang Yu http://www.mdpi.com/2072-6643/8/7/393 Caffeine supplementation during whole-/lower-body exercise is well-researched, yet evidence of its effect during upper-body exercise is equivocal. The current study explored the effects of caffeine on cycling/handcycling 10 km time trial (TT) performance in habitual caffeine users. Eleven recreationally trained males (mean (SD) age 24 (4) years, body mass 85.1 (14.6) kg, cycling/handcycling peak oxygen uptake ( V · peak) 42.9 (7.3)/27.6 (5.1) mL∙kg∙min−1, 160 (168) mg/day caffeine consumption) completed two maximal incremental tests and two familiarization sessions. During four subsequent visits, participants cycled/handcycled for 30 min at 65% mode-specific V · peak (preload) followed by a 10 km TT following the ingestion of 4 mg∙kg−1 caffeine (CAF) or placebo (PLA). Caffeine significantly improved cycling (2.0 (2.0)%; 16:35 vs. 16:56 min; p = 0.033) but not handcycling (1.8 (3.0)%; 24:10 vs. 24:36 min; p = 0.153) TT performance compared to PLA. The improvement during cycling can be attributed to the increased power output during the first and last 2 km during CAF. Higher blood lactate concentration (Bla) was reported during CAF compared to PLA (p < 0.007) and was evident 5 min post-TT during cycling (11.2 ± 2.6 and 8.8 ± 3.2 mmol/L; p = 0.001) and handcycling (10.6 ± 2.5 and 9.2 ± 2.9 mmol/L; p = 0.006). Lower overall ratings of perceived exertion (RPE) were seen following CAF during the preload (p < 0.05) but not post-TT. Lower peripheral RPE were reported at 20 min during cycling and at 30 min during handcycling, and lower central RPE was seen at 30 min during cycling (p < 0.05). Caffeine improved cycling but not handcycling TT performance. The lack of improvement during handcycling may be due to the smaller active muscle mass, elevated (Bla) and/or participants' training status. Nutrients 2016-06-25 8 7 Article 10.3390/nu8070393 393 2072-6643 2016-06-25 doi: 10.3390/nu8070393 Terri Graham-Paulson Claudio Perret Victoria Goosey-Tolfrey http://www.mdpi.com/1422-0067/17/7/1010 The first Pt(IV) derivative of oxaliplatin carrying a ligand for TSPO (the 18-kDa mitochondrial translocator protein) has been developed. The expression of the translocator protein in the brain and liver of healthy humans is usually low, oppositely to steroid-synthesizing and rapidly proliferating tissues, where TSPO is much more abundant. The novel Pt(IV) complex, cis,trans,cis-[Pt(ethanedioato)Cl{2-(2-(4-(6,8-dichloro-3-(2-(dipropylamino)-2-oxoethyl)imidazo[1,2-a]pyridin-2-yl)phenoxy)acetate)-ethanolato}(1R,2R-DACH)] (DACH = diaminocyclohexane), has been fully characterized by spectroscopic and spectrometric techniques and tested in vitro against human MCF7 breast carcinoma, U87 glioblastoma, and LoVo colon adenocarcinoma cell lines. In addition, affinity for TSPO (IC50 = 18.64 nM), cellular uptake (ca. 2 times greater than that of oxaliplatin in LoVo cancer cells, after 24 h treatment), and perturbation of cell cycle progression were investigated. Although the new compound was less active than oxaliplatin and did not exploit a synergistic proapoptotic effect due to the presence of the TSPO ligand, it appears to be promising in a receptor-mediated drug targeting context towards TSPO-overexpressing tumors, in particular colorectal cancer (IC50 = 2.31 μM after 72 h treatment). International Journal of Molecular Sciences 2016-06-25 17 7 Article 10.3390/ijms17071010 1010 1422-0067 2016-06-25 doi: 10.3390/ijms17071010 Salvatore Savino Nunzio Denora Rosa Iacobazzi Letizia Porcelli Amalia Azzariti Giovanni Natile Nicola Margiotta http://www.mdpi.com/1420-3049/21/7/834 Scent-marking is the most effective method of communication in the presence or absence of a signaler. These complex mixtures result in a multifaceted interaction triggered by the sense of smell. The objective was to identify volatile organic compound (VOC) composition and odors emitted by total marking fluid (MF) associated with Siberian tigers (Panthera tigris altaica). Siberian tiger, an endangered species, was chosen because its MF had never been analyzed. Solid phase microextraction (SPME) for headspace volatile collection combined with multidimensional gas chromatography-mass spectrometry-olfactometry for simultaneous chemical and sensory analyses were used. Thirty-two VOCs emitted from MF were identified. 2-acetyl-1-pyrroline, the sole previously identified compound responsible for the "characteristic" odor of P. tigris MF, was identified along with two additional compounds confirmed with standards (urea, furfural) and four tentatively identified compounds (3-methylbutanamine, (R)-3-methylcyclopentanone, propanedioic acid, and 3-hydroxybutanal) as being responsible for the characteristic aroma of Siberian tiger MF. Simultaneous chemical and sensory analyses improved characterization of scent-markings and identified compounds not previously reported in MF of other tiger species. This research will assist animal ecologists, behaviorists, and zookeepers in understanding how scents from specific MF compounds impact tiger and wildlife communication and improve management practices related to animal behavior. Simultaneous chemical and sensory analyses is applicable to unlocking scent-marking information for other species. Molecules 2016-06-25 21 7 Article 10.3390/molecules21070834 834 1420-3049 2016-06-25 doi: 10.3390/molecules21070834 Simone Soso Jacek Koziel http://www.mdpi.com/1420-3049/21/7/832 Kiwi fruit (Actinidia chinensis Planch.) seeds, present as a by-product in the food and pharmaceutical industries, remain underutilized. In this study the extraction conditions for the maximum recovery of total phenolic content (TPC) with high DPPH scavenging capacities (DPPHsc) were analyzed for kiwi fruit seed polyphenols (KSP) by response surface methodology. The optimal conditions for the highest yield of TPC (53.73 mg GAE/g DW) with 63.25% DPPHsc was found by using an extraction time of 79.65 min with an eluent containing 59.45% acetone at 38.35 °C and a 1:11.52 (w/v) solid/liquid ratio. Compared with butyl hydroxy toluene (BHT), a synthetic antioxidant, the extracted KSP showed higher DPPHsc and ferric reducing antioxidant power, but was less efficient than grape seed polyphenols extracted under the same optimum conditions. We also showed that the extracted KSP exhibited strong anti-inflammatory activities by suppressing the secretion of pro-inflammatory cytokines like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α) in lipopolysaccharides (LPS)-induced RAW 264.7 cells. High performance liquid chromatography-electrochemical detector (HPLC-ECD) analysis of the extracted KSP under optimized conditions revealed that the extract was mainly composed of five polyphenolic compounds. Our work showed the development of an optimal extraction process of the KSP, which presented excellent antioxidant and anti-inflammatory activities, indicating that kiwi fruit seeds may further be utilized as a potential source of natural biological active compounds. Molecules 2016-06-25 21 7 Article 10.3390/molecules21070832 832 1420-3049 2016-06-25 doi: 10.3390/molecules21070832 Jianjun Deng Qingqing Liu Chao Zhang Wei Cao Daidi Fan Haixia Yang http://www.mdpi.com/1420-3049/21/7/827 A metal-free, mild and efficient method for the synthesis of 2-methylquinolines was successfully developed by condensation of anilines with vinyl ethers in the presence of catalytic amount of iodine. Modification of both pyridine and benzene moieties was easily achieved by changing only the vinyl ether and aniline. In this reaction, the iodine species was revealed to show dual behavior; molecular iodine serves as an oxidant, while its reduced form, hydrogen iodide, activates the vinyl ether. The redox reaction between these iodine species enables the use of a catalytic amount of iodine in this synthetic method. Molecules 2016-06-25 21 7 Article 10.3390/molecules21070827 827 1420-3049 2016-06-25 doi: 10.3390/molecules21070827 Song Le Chisa Yasuoka Haruyasu Asahara Nagatoshi Nishiwaki http://www.mdpi.com/1422-0067/17/7/1008 Type 2 diabetes (T2D) is one of the most frequent mortality causes in western countries, with rapidly increasing prevalence. Anti-diabetic drugs are the first therapeutic approach, although many patients develop drug resistance. Most drug responsiveness variability can be explained by genetic causes. Inter-individual variability is principally due to single nucleotide polymorphisms, and differential drug responsiveness has been correlated to alteration in genes involved in drug metabolism (CYP2C9) or insulin signaling (IRS1, ABCC8, KCNJ11 and PPARG). However, most genome-wide association studies did not provide clues about the contribution of DNA variations to impaired drug responsiveness. Thus, characterizing T2D drug responsiveness variants is needed to guide clinicians toward tailored therapeutic approaches. Here, we extensively investigated polymorphisms associated with altered drug response in T2D, predicting their effects in silico. Combining different computational approaches, we focused on the expression pattern of genes correlated to drug resistance and inferred evolutionary conservation of polymorphic residues, computationally predicting the biochemical properties of polymorphic proteins. Using RNA-Sequencing followed by targeted validation, we identified and experimentally confirmed that two nucleotide variations in the CAPN10 gene—currently annotated as intronic—fall within two new transcripts in this locus. Additionally, we found that a Single Nucleotide Polymorphism (SNP), currently reported as intergenic, maps to the intron of a new transcript, harboring CAPN10 and GPR35 genes, which undergoes non-sense mediated decay. Finally, we analyzed variants that fall into non-coding regulatory regions of yet underestimated functional significance, predicting that some of them can potentially affect gene expression and/or post-transcriptional regulation of mRNAs affecting the splicing. International Journal of Molecular Sciences 2016-06-25 17 7 Communication 10.3390/ijms17071008 1008 1422-0067 2016-06-25 doi: 10.3390/ijms17071008 Valerio Costa Antonio Federico Carla Pollastro Carmela Ziviello Simona Cataldi Pietro Formisano Alfredo Ciccodicola http://www.mdpi.com/1420-3049/21/7/835 Capsaicin (trans-8-methyl-N-vanilyl-6-nonenamide) is a unique alkaloid isolated from hot chili peppers of the capsicum family. Capsaicin is an agonist of transient receptor potential vanilloid subtype 1 (TRPV1), which is expressed in nociceptive sensory neurons and a range of secretory epithelia, including salivary glands. Capsaicin has analgesic and anti-inflammatory properties in sensory neurons. Recently, increasing evidence has indicated that capsaicin also affects saliva secretion and inflammation in salivary glands. Applying capsaicin increases salivary secretion in human and animal models. Capsaicin appears to increase salivation mainly by modulating the paracellular pathway in salivary glands. Capsaicin activates TRPV1, which modulates the permeability of tight junctions (TJ) by regulating the expression and function of putative intercellular adhesion molecules in an ERK (extracelluar signal-regulated kinase) -dependent manner. Capsaicin also improved dysfunction in transplanted salivary glands. Aside from the secretory effects of capsaicin, it has anti-inflammatory effects in salivary glands. The anti-inflammatory effect of capsaicin is, however, not mediated by TRPV1, but by inhibition of the NF-κB pathway. In conclusion, capsaicin might be a potential drug for alleviating dry mouth symptoms and inflammation of salivary glands. Molecules 2016-06-25 21 7 Review 10.3390/molecules21070835 835 1420-3049 2016-06-25 doi: 10.3390/molecules21070835 Yong-Hwan Shin Jin Kim Kyungpyo Park http://www.mdpi.com/2072-6643/8/7/392 While prior research reported altered fuel utilization stemming from pre-exercise modified starch ingestion, the practical value of this starch for endurance athletes who consume carbohydrates both before and during exercise is yet to be examined. The purpose of this study was to determine the effects of ingesting a hydrothermally-modified starch supplement (HMS) before and during cycling on performance, metabolism, and gastrointestinal comfort. In a crossover design, 10 male cyclists underwent three nutritional interventions: (1) a commercially available sucrose/glucose supplement (G) 30 min before (60 g carbohydrate) and every 15 min during exercise (60 g∙h−1); (2) HMS consumed at the same time points before and during exercise in isocaloric amounts to G (Iso HMS); and (3) HMS 30 min before (60 g carbohydrate) and every 60 min during exercise (30 g·h−1; Low HMS). The exercise protocol (~3 h) consisted of 1 h at 50% Wmax, 8 × 2-min intervals at 80% Wmax, and 10 maximal sprints. There were no differences in sprint performance with Iso HMS vs. G, while both G and Iso HMS likely resulted in small performance enhancements (5.0%; 90% confidence interval = ±5.3% and 4.4%; ±3.2%, respectively) relative to Low HMS. Iso HMS and Low HMS enhanced fat oxidation (31.6%; ±20.1%; very likely (Iso); 20.9%; ±16.1%; likely (Low), and reduced carbohydrate oxidation (−19.2%; ±7.6%; most likely; −22.1%; ±12.9%; very likely) during exercise relative to G. However, nausea was increased during repeated sprints with ingestion of Iso HMS (17 scale units; ±18; likely) and Low HMS (18; ±14; likely) vs. G. Covariate analysis revealed that gastrointestinal distress was associated with reductions in performance with Low HMS vs. G (likely), but this relationship was unclear with Iso HMS vs. G. In conclusion, pre- and during-exercise ingestion of HMS increases fat oxidation relative to G. However, changes do not translate to performance improvements, possibly owing to HMS-associated increases in gastrointestinal distress, which is not attenuated by reducing the intake rate of HMS during exercise. Nutrients 2016-06-25 8 7 Article 10.3390/nu8070392 392 2072-6643 2016-06-25 doi: 10.3390/nu8070392 Daniel Baur Fernanda Vargas Christopher Bach Jordan Garvey Michael Ormsbee http://www.mdpi.com/1420-3049/21/7/831 Many naturally-occurring cellulolytic microorganisms are not readily cultivable, demanding a culture-independent approach in order to study their cellulolytic genes. Metagenomics involves the isolation of DNA from environmental sources and can be used to identify enzymes with biotechnological potential from uncultured microbes. In this study, a gene encoding an endoglucanase was cloned from red rice crop residues using a metagenomic strategy. The amino acid identity between this gene and its closest published counterparts is lower than 70%. The endoglucanase was named EglaRR01 and was biochemically characterized. This recombinant protein showed activity on carboxymethylcellulose, indicating that EglaRR01 is an endoactive lytic enzyme. The enzymatic activity was optimal at a pH of 6.8 and at a temperature of 30 °C. Ethanol production from this recombinant enzyme was also analyzed on EglaRR01 crop residues, and resulted in conversion of cellulose from red rice into simple sugars which were further fermented by Saccharomyces cerevisiae to produce ethanol after seven days. Ethanol yield in this study was approximately 8 g/L. The gene found herein shows strong potential for use in ethanol production from cellulosic biomass (second generation ethanol). Molecules 2016-06-25 21 7 Article 10.3390/molecules21070831 831 1420-3049 2016-06-25 doi: 10.3390/molecules21070831 Carlos Meneses Bruna Silva Betsy Medeiros Rodrigo Serrato David Johnston-Monje http://www.mdpi.com/1660-4601/13/7/634 Speeding is a major contributing factor to traffic crashes and frequently happens in areas where there is a mutation in speed limits, such as the transition zones that connect urban areas from rural areas. The purpose of this study is to investigate the effects of an in-vehicle audio warning system and lit speed limit sign on preventing drivers' speeding behavior in transition zones. A high-fidelity driving simulator was used to establish a roadway network with the transition zone. A total of 41 participants were recruited for this experiment, and the driving speed performance data were collected from the simulator. The experimental results display that the implementation of the audio warning system could significantly reduce drivers' operating speed before they entered the urban area, while the lit speed limit sign had a minimal effect on improving the drivers' speed control performance. Without consideration of different types of speed limit signs, it is found that male drivers generally had a higher operating speed both upstream and in the transition zones and have a larger maximum deceleration for speed reduction than female drivers. Moreover, the drivers who had medium-level driving experience had the higher operating speed and were more likely to have speeding behaviors in the transition zones than those who had low-level and high-level driving experience in the transition zones. International Journal of Environmental Research and Public Health 2016-06-25 13 7 Article 10.3390/ijerph13070634 634 1660-4601 2016-06-25 doi: 10.3390/ijerph13070634 Xuedong Yan Jiali Wang Jiawei Wu http://www.mdpi.com/1420-3049/21/7/810 Abelmoschus manihot (L.) Medic has been used for many years in Chinese traditional medicine. In this study, supercritical CO2 plus a modifier was utilized to extract flavonoids from the flowers of Abelmoschus manihot (L.) Medic. The effects of temperature (40 °C–60 °C), pressure (10–30 MPa) and different concentrations of ethanol as modifier (60%–90%, ethanol:water, v/v) on major flavonol content and the antioxidant activity of the extracts were studied by response surface methodology (RSM) using a Box-Behnken design. The flavonol content was calculated as the sum of the concentrations of seven major flavonoids, namely rutin, hyperin, isoquercetin, hibifolin, myricetin, quercetin-3′-O-glucoside and quercetin, which were simultaneously determined by a HPLC method. The antioxidant activity was evaluated by a 2,2-diphenyl-1-picrylhydarzyl (DPPH) free radical-scavenging assay. The results showed that three factors and their interactions could be well fitted to second-order polynomial models (p < 0.05). At the optimal extraction conditions for flavonol content (20 MPa, 52 °C, and 85% ethanol content), the yield of flavonoids was 41.96 mg/g and the IC50 value was 0.288 mg/mL, respectively, suggesting the extract has high antioxidant activity. Furthermore, the anti-adipogenic activity of the extract on the 3T3-L1 cell line was investigated. The results indicated that it can downregulate PPARγ and C/EBPα expression at mRNA. In summary, in this study, we have established a cost-effective method for the extraction of flavonoids from the flowers of Abelmoschus manihot (L.) Medic using supercritical fluid extraction and the extracts exhibited potent antioxidant and anti-adipogenic effects, suggesting a possible therapeutic approach for the prevention and treatment of obesity. Molecules 2016-06-25 21 7 Article 10.3390/molecules21070810 810 1420-3049 2016-06-25 doi: 10.3390/molecules21070810 Jingjing Li Juan Zhang Min Wang http://www.mdpi.com/1420-3049/21/7/830 Aerial parts of Achillea moschata Wulfen (Asteraceae) growing wild in the Italian Rhaetian Alps were investigated to describe, for the first time, their phenolic content, as well as to characterize the essential oil. Inspection of the metabolic profile combining HPLC-DAD and ESI-MS/MS data showed that the methanol extract contained glycosylated flavonoids with luteolin and apigenin as the main aglycones. Among them, the major compound was 7-O-glucosyl apigenin. Caffeoyl derivates were other phenolics identified. The essential oil obtained by steam distillation and investigated by GC/FID and GC/MS showed camphor, 1,8-cineole, and bornylacetate as the main constituents. The antioxidant capacity of three different extracts with increasing polarity and of the essential oil was evaluated by employing ABTS·+ and DPPH· radical scavenging assays. The methanolic extract was the only significantly effective sample against both synthetic radicals. All samples were also tested against Gram-positive (Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus) and Gram-negative (Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa) bacterial species using the disk diffusion assay. The non-polar extracts (dichloromethane and petroleum ether) and the essential oil possessed a broad spectrum of antimicrobial activity expressed according to inhibition zone diameter (8–24 mm). Molecules 2016-06-25 21 7 Article 10.3390/molecules21070830 830 1420-3049 2016-06-25 doi: 10.3390/molecules21070830 Sara Vitalini Moira Madeo Aldo Tava Marcello Iriti Lisa Vallone Pinarosa Avato Clementina Cocuzza Paolo Simonetti Maria Argentieri http://www.mdpi.com/2072-6651/8/7/194 Tetanus is caused by the tetanus neurotoxin (TeNT) and is one of the most dreaded diseases especially in the developing countries. The current vaccine against tetanus is based on an inactivated tetanus toxin, which is effective but has many drawbacks. In our previous study, we developed a recombinant tetanus vaccine based on protein TeNT-Hc, with clear advantages over the toxoid vaccine in terms of production, characterization, and homogeneity. In this study, the titers, growth extinction, and persistence of specific antibodies induced by the two types of vaccine in mice, rats, and cynomolgus monkeys were compared. The booster vaccination efficacy of the two types of vaccines at different time points and protection mechanism in animals were also compared. The recombinant tetanus vaccine induced persistent and better antibody titers and strengthened the immunity compared with the commercially available toxoid vaccine in animals. Our results provide a theoretical basis for the development of a safe and effective recombinant tetanus vaccine to enhance the immunity of adolescents and adults as a substitute for the current toxoid vaccine. Toxins 2016-06-25 8 7 Article 10.3390/toxins8070194 194 2072-6651 2016-06-25 doi: 10.3390/toxins8070194 Rui Yu Ting Fang Shuling Liu Xiaohong Song Changming Yu Jianmin Li Ling Fu Lihua Hou Junjie Xu Wei Chen http://www.mdpi.com/1420-3049/21/7/829 Green tea catechins exhibit high diversity of biological effects including antioncogenic properties, and there is enormous interest in their potential use in the treatment of a number of pathologies. It is recognized that the mechanism underlying the activity of catechins relay in part in processes related to the membrane, and many studies revealed that the ability of catechins to interact with lipids plays a probably necessary role in their mechanism of action. We present in this work the characterization of the interaction between an antitumoral synthetically modified catechin (3-O-(3,4,5-trimethoxybenzoyl)-(−)-catechin, TMCG) and dimiristoylphosphatidyl-ethanolamine (DMPE) membranes using an array of biophysical techniques which include differential scanning calorimetry, X-ray diffraction, infrared spectroscopy, atomic force microscopy, and molecular dynamics simulations. We found that TMCG incorporate into DMPE bilayers perturbing the thermotropic transition from the gel to the fluid state forming enriched domains which separated into different gel phases. TMCG does not influence the overall bilayer assembly of phosphatidylethanolamine systems but it manages to influence the interfacial region of the membrane and slightly decrease the interlamellar repeat distance of the bilayer. TMCG seems to be located in the interior of the phosphatidylethanolamine bilayer with the methoxy groups being in the deepest position and some portion of the molecule interacting with the water interface. We believe that the reported interactions are significant not only from the point of view of the known antitumoral effect of TMCG, but also might contribute to understanding the basic molecular mechanism of the biological effects of the catechins found at the membrane level. Molecules 2016-06-24 21 7 Article 10.3390/molecules21070829 829 1420-3049 2016-06-24 doi: 10.3390/molecules21070829 Francisco Casado José Teruel Santiago Casado Antonio Ortiz José Rodríguez-López Francisco Aranda http://www.mdpi.com/1422-0067/17/7/1007 Oral cancer is a common cancer with poor prognosis. We evaluated the expression of PBK/TOPK (PDZ-binding kinase/T-LAK cell-originated protein kinase) and its prognostic significance in oral cancer. PBK/TOPK expression was measured by immunohistochemical staining of samples from 287 patients with oral cancer. The association between PBK/TOPK expression and clinicopathological features was analyzed. The prognostic value of PBK/TOPK for overall survival was determined by Kaplan-Meier analysis and Cox proportional hazard models. A high PBK/TOPK expression level was correlated with long overall survival. The prognostic role of PBK/TOPK expression was significant in young patients (p < 0.05), patients with smoking habits (p < 0.05), and late stage disease (p < 0.05). Our results suggest that PBK/TOPK expression is enhanced in oral cancer. High PBK/TOPK expression, either alone or in subgroups according to clinicopathological features, may serve as a favorable prognostic marker for patients with oral cancer. International Journal of Molecular Sciences 2016-06-24 17 7 Article 10.3390/ijms17071007 1007 1422-0067 2016-06-24 doi: 10.3390/ijms17071007 Chin-Fang Chang Sung-Lang Chen Wen-Wei Sung Ming-Ju Hsieh Hui-Ting Hsu Li-Hsin Chen Mu-Kuan Chen Jiunn-Liang Ko Chih-Jung Chen Ming-Chih Chou http://www.mdpi.com/1422-0067/17/7/1005 Stem cells are responsible for the organ and tissue development, growth and maintenance from embryonic stage up to late adult life.[...] International Journal of Molecular Sciences 2016-06-24 17 7 Editorial 10.3390/ijms17071005 1005 1422-0067 2016-06-24 doi: 10.3390/ijms17071005 Wilhelm Bloch http://www.mdpi.com/1422-0067/17/7/1001 Traditional therapeutic methods for skin wounds have many disadvantages, and new wound dressings that can facilitate the healing process are thus urgently needed. Platelet-rich plasma (PRP) contains multiple growth factors (GFs) and shows a significant capacity to heal soft tissue wounds. However, these GFs have a short half-life and deactivate rapidly; we therefore need a sustained delivery system to overcome this shortcoming. In this study, poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (PDLLA-PEG-PDLLA: PLEL) hydrogel was successfully created as delivery vehicle for PRP GFs and was evaluated systematically. PLEL hydrogel was injectable at room temperature and exhibited a smart thermosensitive in situ gel-formation behavior at body temperature. In vitro cell culture showed PRP-loaded PLEL hydrogel (PRP/PLEL) had little cytotoxicity, and promoted EaHy926 proliferation, migration and tube formation; the factor release assay additionally indicated that PLEL realized the controlled release of PRP GFs for as long as 14 days. When employed to treat rodents' full-thickness skin defects, PRP/PLEL showed a significantly better ability to raise the number of both newly formed and mature blood vessels compared to the control, PLEL and PRP groups. Furthermore, the PRP/PLEL-treated group displayed faster wound closure, better reepithelialization and collagen formation. Taken together, PRP/PLEL provides a promising strategy for promoting angiogenesis and skin wound healing, which extends the potential of this dressing for clinical application. International Journal of Molecular Sciences 2016-06-24 17 7 Article 10.3390/ijms17071001 1001 1422-0067 2016-06-24 doi: 10.3390/ijms17071001 Manle Qiu Daoyun Chen Chaoyong Shen Ji Shen Huakun Zhao Yaohua He http://www.mdpi.com/1422-0067/17/7/1004 Plant-specific GRAS transcription factors play important roles in regulating growth, development, and stress responses. Castor beans (Ricinus communis) are important non-edible oilseed plants, cultivated worldwide for its seed oils and its adaptability to growth conditions. In this study, we identified and characterized a total of 48 GRAS genes based on the castor bean genome. Combined with phylogenetic analysis, the castor bean GRAS members were divided into 13 distinct groups. Functional divergence analysis revealed the presence of mostly Type-I functional divergence. The gene structures and conserved motifs, both within and outside the GRAS domain, were characterized. Gene expression analysis, performed in various tissues and under a range of abiotic stress conditions, uncovered the potential functions of GRAS members in regulating plant growth development and stress responses. The results obtained from this study provide valuable information toward understanding the potential molecular mechanisms of GRAS proteins in castor beans. These findings also serve as a resource for identifying the genes that allow castor beans to grow in stressful conditions and to enable further breeding and genetic improvements in agriculture. International Journal of Molecular Sciences 2016-06-24 17 7 Article 10.3390/ijms17071004 1004 1422-0067 2016-06-24 doi: 10.3390/ijms17071004 Wei Xu Zexi Chen Naeem Ahmed Bing Han Qinghua Cui Aizhong Liu http://www.mdpi.com/1422-0067/17/7/1003 Folate-mediated one-carbon metabolism (FMOCM) is linked to DNA synthesis, methylation, and cell proliferation. Vitamin B6 (B6) is a cofactor, and genetic polymorphisms of related key enzymes, such as serine hydroxymethyltransferase (SHMT), methionine synthase reductase (MTRR), and methionine synthase (MS), in FMOCM may govern the bioavailability of metabolites and play important roles in the maintenance of genomic stability and cell viability (GSACV). To evaluate the influences of B6, genetic polymorphisms of these enzymes, and gene–nutrient interactions on GSACV, we utilized the cytokinesis-block micronucleus assay (CBMN) and PCR-restriction fragment length polymorphism (PCR-RFLP) techniques in the lymphocytes from female breast cancer cases and controls. GSACV showed a significantly positive correlation with B6 concentration, and 48 nmol/L of B6 was the most suitable concentration for maintaining GSACV in vitro. The GSACV indexes showed significantly different sensitivity to B6 deficiency between cases and controls; the B6 effect on the GSACV variance contribution of each index was significantly higher than that of genetic polymorphisms and the sample state (tumor state). SHMT C1420T mutations may reduce breast cancer susceptibility, whereas MTRR A66G and MS A2756G mutations may increase breast cancer susceptibility. The role of SHMT, MS, and MTRR genotype polymorphisms in GSACV is reduced compared with that of B6. The results appear to suggest that the long-term lack of B6 under these conditions may increase genetic damage and cell injury and that individuals with various genotypes have different sensitivities to B6 deficiency. FMOCM metabolic enzyme gene polymorphism may be related to breast cancer susceptibility to a certain extent due to the effect of other factors such as stress, hormones, cancer therapies, psychological conditions, and diet. Adequate B6 intake may be good for maintaining genome health and preventing breast cancer. International Journal of Molecular Sciences 2016-06-24 17 7 Article 10.3390/ijms17071003 1003 1422-0067 2016-06-24 doi: 10.3390/ijms17071003 Xiayu Wu Weijiang Xu Tao Zhou Neng Cao Juan Ni Tianning Zou Ziqing Liang Xu Wang Michael Fenech http://www.mdpi.com/1422-0067/17/7/1000 The possibility to generate patient-specific induced pluripotent stem cells (iPSCs) offers an unprecedented potential of applications in clinical therapy and medical research. Human iPSCs and their differentiated derivatives are tools for diseases modelling, drug discovery, safety pharmacology, and toxicology. Moreover, they allow for the engineering of bioartificial tissue and are promising candidates for cellular therapies. For many of these applications, the ability to genetically modify pluripotent stem cells (PSCs) is indispensable, but efficient site-specific and safe technologies for genetic engineering of PSCs were developed only recently. By now, customized engineered nucleases provide excellent tools for targeted genome editing, opening new perspectives for biomedical research and cellular therapies. International Journal of Molecular Sciences 2016-06-24 17 7 Review 10.3390/ijms17071000 1000 1422-0067 2016-06-24 doi: 10.3390/ijms17071000 Sylvia Merkert Ulrich Martin http://www.mdpi.com/1422-0067/17/7/1006 The glutamatergic system may be involved in the effects of neuroprotectant therapies. Echinacoside, a phenylethanoid glycoside extracted from the medicinal Chinese herb Herba Cistanche, has neuroprotective effects. This study investigated the effects of echinacoside on 4-aminopyridine-evoked glutamate release in rat cerebrocortical nerve terminals (synaptosomes). Echinacoside inhibited Ca2+-dependent, but not Ca2+-independent, 4-aminopyridine-evoked glutamate release in a concentration-dependent manner. Echinacoside also reduced the 4-aminopyridine-evoked increase in cytoplasmic free Ca2+ concentration but did not alter the synaptosomal membrane potential. The inhibitory effect of echinacoside on 4-aminopyridine-evoked glutamate release was prevented by ω-conotoxin MVIIC, a wide-spectrum blocker of Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but was insensitive to the intracellular Ca2+ release-inhibitors dantrolene and 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157). Furthermore, echinacoside decreased the 4-aminopyridine-induced phosphorylation of protein kinase C, and protein kinase C inhibitors abolished the effect of echinacoside on glutamate release. According to these results, we suggest that the inhibitory effect of echinacoside on evoked glutamate release is associated with reduced voltage-dependent Ca2+ entry and subsequent suppression of protein kinase C activity. International Journal of Molecular Sciences 2016-06-24 17 7 Article 10.3390/ijms17071006 1006 1422-0067 2016-06-24 doi: 10.3390/ijms17071006 Cheng Lu Tzu Lin Shu Huang Su Wang http://www.mdpi.com/1422-0067/17/7/959 MicroRNAs (miRNAs) play an important role in regulating almost all biological processes. miRNAs bind to the 3′ untranslated region (UTR) of mRNAs by sequence matching. In a previous study, we demonstrated that miR-21 was differently expressed in alpaca skin with different hair color. However, the molecular and cellular mechanisms for miR-21 to regulate the coat color are not yet completely understood. In this study, we transfected miR-21a-5p into mouse melanocytes and demonstrated its function on melanogenesis of miR-21a-5p by targeting Sox5, which inhibits melanogenesis in mouse melanocytes. The results suggested that miR-21a-5p targeted Sox5 gene based on the binding site in 3′ UTR of Sox5 and overexpression of miR-21a-5p significantly down-regulated Sox5 mRNA and protein expression. Meanwhile, mRNA and protein expression of microphthalmia transcription factor (MITF) and Tyrosinase (TYR) were up-regulated, which subsequently make the melanin production in melanocytes increased. The results suggest that miR-21a-5p regulates melanogenesis via MITF by targeting Sox5. International Journal of Molecular Sciences 2016-06-24 17 7 Article 10.3390/ijms17070959 959 1422-0067 2016-06-24 doi: 10.3390/ijms17070959 Pengchao Wang Yuanyuan Zhao Ruiwen Fan Tianzhi Chen Changsheng Dong http://www.mdpi.com/1422-0067/17/7/999 Background: Peroxisome proliferator-activated receptors (PPARs) have demonstrated a lot of important effects in the regulation of glucose and lipid metabolism and in the correct functioning of adipose tissue. Recently, many studies have evaluated a possible effect of PPARs on tumor cells. The purpose of this review is to describe the effects of PPARs, their action and their future prospective; Methods: Narrative review aimed to synthesize cutting-edge evidence retrieved from searches of computerized databases; Results: PPARs play a key role in metabolic diseases, which include several cardiovascular diseases, insulin resistance, type 2 diabetes, metabolic syndrome, impaired immunity and the increasing risk of cancer; in particular, PPARα and PPARβ/δ mainly enable energy combustion, while PPARγ contributes to energy storage by enhancing adipogenesis; Conclusion: PPAR agonists could represent interesting types of molecules that can treat not only metabolic diseases, but also inflammation and cancer. Additional research is needed for the identification of high-affinity, high-specificity agonists for the treatment of obesity, type 2 diabetes (T2DM) and other metabolic diseases. Further studies are needed also to elucidate the role of PPARs in cancer. International Journal of Molecular Sciences 2016-06-24 17 7 Review 10.3390/ijms17070999 999 1422-0067 2016-06-24 doi: 10.3390/ijms17070999 Antonio Laganà Salvatore Vitale Angela Nigro Vincenza Sofo Francesca Salmeri Paola Rossetti Agnese Rapisarda Sandro La Vignera Rosita Condorelli Gianluca Rizzo Massimo Buscema http://www.mdpi.com/1422-0067/17/7/998 Sugars, organic acids and volatiles of apricot were determined by HPLC and GC-MS during fruit development and ripening, and the key taste and aroma components were identified by integrating flavor compound contents with consumers' evaluation. Sucrose and glucose were the major sugars in apricot fruit. The contents of all sugars increased rapidly, and the accumulation pattern of sugars converted from glucose-predominated to sucrose-predominated during fruit development and ripening. Sucrose synthase (SS), sorbitol oxidase (SO) and sorbitol dehydrogenase (SDH) are under tight developmental control and they might play important roles in sugar accumulation. Almost all organic acids identified increased during early development and then decrease rapidly. During early development, fruit mainly accumulated quinate and malate, with the increase of citrate after maturation, and quinate, malate and citrate were the predominant organic acids at the ripening stage. The odor activity values (OAV) of aroma volatiles showed that 18 aroma compounds were the characteristic components of apricot fruit. Aldehydes and terpenes decreased significantly during the whole development period, whereas lactones and apocarotenoids significantly increased with fruit ripening. The partial least squares regression (PLSR) results revealed that β-ionone, γ-decalactone, sucrose and citrate are the key characteristic flavor factors contributing to consumer acceptance. Carotenoid cleavage dioxygenases (CCD) may be involved in β-ionone formation in apricot fruit. International Journal of Molecular Sciences 2016-06-24 17 7 Article 10.3390/ijms17070998 998 1422-0067 2016-06-24 doi: 10.3390/ijms17070998 Wanpeng Xi Huiwen Zheng Qiuyun Zhang Wenhui Li http://www.mdpi.com/1422-0067/17/7/997 NF-E2-related factor 2 (Nrf2) has been identified as a master regulatory factor in the protection of cells from oxidative and electrophilic stress. However, overexpression of Nrf2 in lung cancer may cause chemoresistance, as well as radioresistance. In this study, we examined the relationship between radioresistance and Nrf2 protein levels in H1299, A549, and H460 cells, and finally chose the A549 cell line to continue with due to its strong radioresistance and high Nrf2 protein levels. We found that the Nrf2 inhibitor, brusatol, could prevent the increase and accumulation of Nrf2 after exposure to irradiation. Additionally, following treatment with 80 nM brusatol, A549 cells became sensitive to irradiation, suffering severe DNA damage. Combination treatment with brusatol and ionizing radiation (IR) can distinctly increase the level of reactive oxygen species in A549 cells, causing a 1.8-fold increase compared with the control, and a 1.4-fold increase compared with IR alone. In fact, in the treatment with both brusatol and IR, lung cancer cell proliferation is halted, gradually leading to cell death. Because Nrf2 is closely linked to DNA damage repair, inhibiting the function of Nrf2, as in brusatol treatment, may increase the DNA damage caused by radiotherapy or chemotherapy, possibly enhancing the efficacy of chemotherapeutic drugs. Our study is the first to demonstrate brusatol's ability to enhance the responsiveness of lung cancer cells to irradiation, and its potential application as a natural sensitizer in radiotherapy. International Journal of Molecular Sciences 2016-06-24 17 7 Article 10.3390/ijms17070997 997 1422-0067 2016-06-24 doi: 10.3390/ijms17070997 Xiaohui Sun Qin Wang Yan Wang Liqing Du Chang Xu Qiang Liu http://www.mdpi.com/1660-3397/14/7/121 For a long time, fucoidan has been well known for its pharmacological activities, and recently low molecular weight fucoidan (LMF) has been used in food supplements and pharmaceutical products. In the present study, LMF was extracted from Laminaria japonica by enzyme hydrolysis. The toxicity of LMF in mouse and rat models was determined by many methods, such as total arsenic content, bacterial reverse mutation assay, chromosome aberration assay, and in vivo micronucleus assay. The present findings showed that LMF at 5000 μg/mL exhibited no mutagenicity. It also produced no formatting disruption of red blood cells in vivo. At 2000 mg/kg BW/day there were no toxicological indications. LMF is expected to be used as a safe food supplement. Marine Drugs 2016-06-24 14 7 Article 10.3390/md14070121 121 1660-3397 2016-06-24 doi: 10.3390/md14070121 Pai-An Hwang Ming-De Yan Hong-Ting Lin Kuan-Lun Li Yen-Chang Lin http://www.mdpi.com/2304-8158/5/3/46 Ayvalik is an important olive cultivar producing high quality oils in Turkey. In the present study, volatile and phenolic compositions of early-harvest extra virgin olive oil (cv. Ayvalik) were determined. The solvent-assisted flavor evaporation (SAFE) technique was used for the extraction of volatile components. The aromatic extract obtained by SAFE was representative of the olive oil odor. A total of 32 aroma compounds, including alcohols, aldehydes, terpenes, esters, and an acid, were identified in the olive oil. Aldehydes and alcohols were qualitatively and quantitatively the most dominant volatiles in the oil sample. Of these, six volatile components presented odor activity values (OAVs) greater than one, with (Z)-3-hexenal (green), hexanal (green-sweet) and nonanal (fatty-pungent) being those with the highest OAVs in olive oil. A total of 14 phenolic compounds were identified and quantified by liquid chromatography combined with a diode array detector and ion spray mass spectrometry. The major phenolic compounds were found as 3,4-DHPEA-EDA, 3,4-DHPEA-EA and p-HPEA-EDA. Foods 2016-06-24 5 3 Article 10.3390/foods5030046 46 2304-8158 2016-06-24 doi: 10.3390/foods5030046 Gamze Guclu Onur Sevindik Hasim Kelebek Serkan Selli http://www.mdpi.com/2072-6651/8/7/176 Food safety has attracted extensive attention around the world, and food-borne diseases have become one of the major threats to health. Staphylococcus aureus is a major food-borne pathogen worldwide and a frequent contaminant of foodstuffs. Staphylococcal enterotoxins (SEs) produced by some S. aureus strains will lead to staphylococcal food poisoning (SFP) outbreaks. The most common symptoms caused by ingestion of SEs within food are nausea, vomiting, diarrhea and cramps. Children will suffer SFP by ingesting as little as 100 ng of SEs, and only a few micrograms of SEs are enough to cause SPF in vulnerable populations. Therefore, it is a great challenge and of urgent need to detect and identify SEs rapidly and accurately for governmental and non-governmental agencies, including the military, public health departments, and health care facilities. Herein, an overview of SE detection has been provided through a comprehensive literature survey. Toxins 2016-06-24 8 7 Review 10.3390/toxins8070176 176 2072-6651 2016-06-24 doi: 10.3390/toxins8070176 Shijia Wu Nuo Duan Huajie Gu Liling Hao Hua Ye Wenhui Gong Zhouping Wang http://www.mdpi.com/2304-6767/4/3/22 This case report describes a 33-year-old female currently undergoing breast cancer treatment following the AC-T-T (doxorubicin hydrochloride (Adriamycin) and cyclophosphamide followed by paclitaxel (Taxol) and trastuzumab (Herceptin)) treatment regimen. Her chief complaint at the time of the emergency visit at the dental office was that she had an episode of profuse spontaneous bleeding located at the palatal gingiva in the maxilla between the left central and lateral incisor. To our knowledge, this is a novel finding related to the medications she is utilizing and should be further investigated. Dentistry Journal 2016-06-24 4 3 Case Report 10.3390/dj4030022 22 2304-6767 2016-06-24 doi: 10.3390/dj4030022 Nima Sarmast Maria Gutierrez Quevedo Howard Wang Estatio Gutierrez Herrera http://www.mdpi.com/1420-3049/21/7/821 A topological index of graph G is a numerical parameter related to G which characterizes its molecular topology and is usually graph invariant. In the field of quantitative structure-activity (QSAR)/quantitative structure-activity structure-property (QSPR) research, theoretical properties of the chemical compounds and their molecular topological indices such as the Randić connectivity index, atom-bond connectivity (ABC) index and geometric-arithmetic (GA) index are used to predict the bioactivity of different chemical compounds. A dendrimer is an artificially manufactured or synthesized molecule built up from the branched units called monomers. In this paper, the fourth version of ABC index and the fifth version of GA index of certain families of nanostar dendrimers are investigated. We derive the analytical closed formulas for these families of nanostar dendrimers. The obtained results can be of use in molecular data mining, particularly in researching the uniqueness of tested (hyper-branched) molecular graphs. Molecules 2016-06-24 21 7 Article 10.3390/molecules21070821 821 1420-3049 2016-06-24 doi: 10.3390/molecules21070821 Mohamad Husin Roslan Hasni Nabeel Arif Muhammad Imran http://www.mdpi.com/1420-3049/21/7/825 In order to discover novel eco-friendly compounds with good activity for aphid control, (E)-β-farnesene (EβF), the main component of the aphid alarm pheromone, was chosen as the lead compound. By introducing a 2-nitroimino-hexahydro-1,3,5-triazine moiety (abbreviated NHT) to replace the unstable conjugated double bond system of EβF, a series of novel EβF analogues containing the NHT moiety were synthesized via the reaction of substituted NHT rings with (E)-1-chloro-3,7-dimethylocta-2,6-diene. All the compounds were characterized by 1H-NMR, 13C-NMR, IR, and high resolution mass spectroscopy (HRMS). The bioassay results showed that all the analogues displayed different repellent and aphicidal activities against green peach aphid (Myzus persicae). Particularly, the analogue 4r exhibited obvious repellent activity (repellent proportion: 78.43%) and similar aphicidal activity against M. persicae (mortality: 82.05%) as the commercial compound pymetrozine (80.07%). A preliminary structure-activity relationship (SAR) study was also performed, which offered valuable clues for the design of further new EβF analogues. Molecules 2016-06-24 21 7 Article 10.3390/molecules21070825 825 1420-3049 2016-06-24 doi: 10.3390/molecules21070825 Yaoguo Qin Jingpeng Zhang Dunlun Song Hongxia Duan Wenhao Li Xinling Yang http://www.mdpi.com/1420-3049/21/7/824 The amicoumacins belong to a class of dihydroisocoumarin natural products and display antibacterial, antifungal, anticancer, and anti-inflammatory activities. Amicoumacins are the pro-drug activation products of a bacterial nonribosomal peptide-polyketide hybrid biosynthetic pathway and have been isolated from Gram-positive Bacillus and Nocardia species. Here, we report the stimulation of a "cryptic" amicoumacin pathway in the entomopathogenic Gram-negative bacterium Xenorhabdus bovienii, a strain not previously known to produce amicoumacins. X. bovienii participates in a multi-lateral symbiosis where it is pathogenic to insects and mutualistic to its Steinernema nematode host. Waxmoth larvae are common prey of the X. bovienii-Steinernema pair. Employing a medium designed to mimic the amino acid content of the waxmoth circulatory fluid led to the detection and characterization of amicoumacins in X. bovienii. The chemical structures of the amicoumacins were supported by 2D-NMR, HR-ESI-QTOF-MS, tandem MS, and polarimeter spectral data. A comparative gene cluster analysis of the identified X. bovienii amicoumacin pathway to that of the Bacillus subtilis amicoumacin pathway and the structurally-related Xenorhabdus nematophila xenocoumacin pathway is presented. The X. bovienii pathway encodes an acetyltransferase not found in the other reported pathways, which leads to a series of N-acetyl-amicoumacins that lack antibacterial activity. N-acetylation of amicoumacin was validated through in vitro protein biochemical studies, and the impact of N-acylation on amicoumacin's mode of action was examined through ribosomal structural analyses. Molecules 2016-06-24 21 7 Article 10.3390/molecules21070824 824 1420-3049 2016-06-24 doi: 10.3390/molecules21070824 Hyun Park Corey Perez Elena Perry Jason Crawford http://www.mdpi.com/1420-3049/21/7/828 Anilinopyrimidines are the main chemical agents for management of Botrytis cinerea. However, the drug resistance in fungi against this kind of compounds is very serious. To explore new potential fungicides against B. cinerea, a series of 4-phenyl-6-trifluoromethyl-2-amino-pyrimidine compounds (compounds III-1 to III-22) were synthesized, and their structures were confirmed by 1H-NMR, IR and MS. Most of these compounds possessed excellent fungicidal activity. The compounds III-3 and III-13 showed higher fungicidal activity than the positive control pyrimethanil on fructose gelatin agar (FGA), and compound III-3 on potato dextrose agar (PDA) indicated high activity compared to the positive control cyprodinil. In vivo greenhouse results indicated that the activity of compounds III-3, III-8, and III-11 was significantly higher than that of the fungicide pyrimethanil. Scanning electron micrography (SEM) and transmission electron micrography (TEM) were applied to illustrate the mechanism of title compounds against B. cinerea. The title compounds, especially those containing a fluorine atom at the ortho-position on the benzene ring, could maintain the antifungal activity against B. cinerea, but their mechanism of action is different from that of cyprodinil. The present study lays a good foundation for us to find more efficient reagents against B. cinerea. Molecules 2016-06-24 21 7 Article 10.3390/molecules21070828 828 1420-3049 2016-06-24 doi: 10.3390/molecules21070828 Chunhui Liu Zining Cui Xiaojing Yan Zhiqiu Qi Mingshan Ji Xinghai Li http://www.mdpi.com/1660-4601/13/7/631 Waste management, namely, collection, transport, sorting and processing, and disposal, is an issue of social concern owing to its environmental impact and effects on public health. In fact, waste management activities are carried out according to procedures that can have various negative effects on the environment and, potentially, on human health. The aim of our study was to assess the potential effects on respiratory health of this exposure in workers in the waste management and disposal field, as compared with a group of workers with no occupational exposure to outdoor pollutants. The sample consisted of a total of 124 subjects, 63 waste collectors, and 61 office clerks. Informed consent was obtained from all subjects before inclusion in the study. The entire study population underwent pulmonary function assessments with spirometry and completed two validated questionnaires for the diagnosis of rhinitis and chronic bronchitis. Statistical analyses were performed using STATA 13. Spirometry showed a statistically significant reduction in the mean Tiffenau Index values in the exposed workers, as compared with the controls, after adjusting for the confounding factors of age, BMI, and smoking habit. Similarly, the mean FEV1 values were lower in the exposed workers than in the controls, this difference being again statistically significant. The FVC differences measured in the two groups were not found to be statistically significant. We ran a cross-sectional study to investigate the respiratory health of a group of workers in the solid waste collection and disposal field as compared with a group of office workers. In agreement with most of the data in the literature, our findings support the existence of a prevalence of respiratory deficits in waste disposal workers. Our data suggest the importance of adopting preventive measures, such as wearing specific individual protection devices, to protect this particular category of workers from adverse effects on respiratory health. International Journal of Environmental Research and Public Health 2016-06-24 13 7 Article 10.3390/ijerph13070631 631 1660-4601 2016-06-24 doi: 10.3390/ijerph13070631 Luigi Vimercati Antonio Baldassarre Maria Gatti Luigi De Maria Antonio Caputi Angelica Dirodi Francesco Cuccaro Raffaello Bellino http://www.mdpi.com/2227-7382/4/3/21 Brachypodium distachyon is a suitable plant model for studying temperate cereal crops, such as wheat, barley or rice, and helpful in the study of the grain cell wall. Indeed, the most abundant hemicelluloses that are in the B. distachyon cell wall of grain are (1-3)(1-4)-β-glucans and arabinoxylans, in a ratio similar to those of cereals such as barley or oat. Conversely, these cell walls contain few pectins and xyloglucans. Cell walls play an important role in grain physiology. The modifications of cell wall polysaccharides that occur during grain development and filling are key in the determination of the size and weight of the cereal grains. The mechanisms required for cell wall assembly and remodelling are poorly understood, especially in cereals. To provide a better understanding of these processes, we purified the cell wall at three developmental stages of the B. distachyon grain. The proteins were then extracted, and a quantitative and comparative LC-MS/MS analysis was performed to investigate the protein profile changes during grain development. Over 466 cell wall proteins (CWPs) were identified and classified according to their predicted functions. This work highlights the different proteome profiles that we could relate to the main phases of grain development and to the reorganization of cell wall polysaccharides that occurs during these different developmental stages. These results provide a good springboard to pursue functional validation to better understand the role of CWPs in the assembly and remodelling of the grain cell wall of cereals. Proteomes 2016-06-24 4 3 Article 10.3390/proteomes4030021 21 2227-7382 2016-06-24 doi: 10.3390/proteomes4030021 Mathilde Francin-Allami Virginie Lollier Marija Pavlovic Hélène San Clemente Hélène Rogniaux Elisabeth Jamet Fabienne Guillon Colette Larré http://www.mdpi.com/1660-4601/13/7/633 Endophytes are ubiquitous in plants, and they may have a natural capacity to biodegrade polycyclic aromatic hydrocarbons (PAHs). In our study, a phenanthrene-degrading endophytic Paenibacillus sp. PHE-3 was isolated from P. asiatica L. grown in a PAH-contaminated site. The effects of environmental variables on phenanthrene biodegradation by strain PHE-3 were studied, and the ability of strain PHE-3 to use high molecular weight PAH (HMW-PAH) as a sole carbon source was also evaluated. Our results indicated that pH value of 4.0–8.0, temperature of 30 °C–42 °C, initial phenanthrene concentration less than 100 mg·L−1, and some additional nutrients are favorable for the biodegradation of phenanthrene by strain PHE-3. The maximum biodegradation efficiency of phenanthrene was achieved at 99.9% after 84 h cultivation with additional glutamate. Moreover, the phenanthrene biodegradation by strain PHE-3 was positively correlated with the catechol 2,3-dioxygenase activity (ρ = 0.981, p < 0.05), suggesting that strain PHE-3 had the capability of degrading HMW-PAHs. In the presence of other 2-, 3-ringed PAHs, strain PHE-3 effectively degraded HMW-PAHs through co-metabolism. The results of this study are beneficial in that the re-colonization potential and PAH degradation performance of endophytic Paenibacillus sp. PHE-3 may be applied towards reducing PAH contamination in plants. International Journal of Environmental Research and Public Health 2016-06-24 13 7 Article 10.3390/ijerph13070633 633 1660-4601 2016-06-24 doi: 10.3390/ijerph13070633 Xuezhu Zhu Li Jin Kai Sun Shuang Li Wanting Ling Xuelin Li http://www.mdpi.com/1660-4601/13/7/632 This study proposes an inventory analysis method to evaluate the greenhouse gas (GHG) emissions from Portland cement concrete pavement construction, based on a case project in the west of China. The concrete pavement construction process was divided into three phases, namely raw material production, concrete manufacture and pavement onsite construction. The GHG emissions of the three phases are analyzed by a life cycle inventory method. The CO2e is used to indicate the GHG emissions. The results show that for 1 km Portland cement concrete pavement construction, the total CO2e is 8215.31 tons. Based on the evaluation results, the CO2e of the raw material production phase is 7617.27 tons, accounting for 92.7% of the total GHG emissions; the CO2e of the concrete manufacture phase is 598,033.10 kg, accounting for 7.2% of the total GHG emissions. Lastly, the CO2e of the pavement onsite construction phase is 8396.59 kg, accounting for only 0.1% of the total GHG emissions. The main greenhouse gas is CO2 in each phase, which accounts for more than 98% of total emissions. N2O and CH4 emissions are relatively insignificant. International Journal of Environmental Research and Public Health 2016-06-24 13 7 Article 10.3390/ijerph13070632 632 1660-4601 2016-06-24 doi: 10.3390/ijerph13070632 Feng Ma Aimin Sha Panpan Yang Yue Huang http://www.mdpi.com/2072-6643/8/7/391 Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. It is characterized by a wide spectrum of hepatic changes, which may progress to non-alcoholic steatohepatitis (NASH) and cirrhosis. NAFLD is considered a hepatic manifestation of metabolic syndrome; however, mechanisms underlying the onset and progression of NAFLD are still unclear. Resident and recruited macrophages are key players in the homeostatic function of the liver and in the progression of NAFLD to NASH. Progress has been made in understanding the molecular mechanisms underlying the polarized activation of macrophages. New NAFLD therapies will likely involve modification of macrophage polarization by restraining M1 activation or driving M2 activation. Carotenoids are potent antioxidants and anti-inflammatory micronutrients that have been used to prevent and treat NAFLD. In addition to their antioxidative action, carotenoids can regulate macrophage polarization and thereby halt the progression of NASH. In this review, we summarize the molecular mechanisms of macrophage polarization and the function of liver macrophages/Kupffer cells in NAFLD. From our review, we propose that dietary carotenoids, such as β-cryptoxanthin and astaxanthin, be used to prevent or treat NAFLD through the regulation of macrophage polarization and liver homeostasis. Nutrients 2016-06-24 8 7 Review 10.3390/nu8070391 391 2072-6643 2016-06-24 doi: 10.3390/nu8070391 Yinhua Ni Fen Zhuge Mayumi Nagashimada Tsuguhito Ota http://www.mdpi.com/2076-393X/4/3/22 Interleukin-8 (IL-8, CXCL8) is a pro-inflammatory chemokine produced by various cell types to recruit leukocytes to sites of infection or tissue injury. Acquisition of IL-8 and/or its receptors CXCR1 and CXCR2 are known to be a relatively common occurrence during tumor progression. Emerging research now indicates that paracrine signaling by tumor-derived IL-8 promotes the trafficking of neutrophils and myeloid-derived suppressor cells (MDSCs) into the tumor microenvironment, which have the ability to dampen anti-tumor immune responses. Furthermore, recent studies have also shown that IL-8 produced by the tumor mass can induce tumor cells to undergo the transdifferentiation process epithelial-to-mesenchymal transition (EMT) in which tumor cells shed their epithelial characteristics and acquire mesenchymal characteristics. EMT can increase metastatic dissemination, stemness, and intrinsic resistance, including to killing by cytotoxic immune cells. This review highlights the dual potential roles that the inflammatory cytokine IL-8 plays in promoting tumor resistance by enhancing the immunosuppressive microenvironment and activating EMT, and then discusses the potential for targeting the IL-8/IL-8 receptor axis to combat these various resistance mechanisms. Vaccines 2016-06-24 4 3 Review 10.3390/vaccines4030022 22 2076-393X 2016-06-24 doi: 10.3390/vaccines4030022 Justin David Charli Dominguez Duane Hamilton Claudia Palena http://www.mdpi.com/2079-6382/5/3/22 The machinery of translation is one of the most common targets of antibiotics. The development and screening of new antibiotics usually proceeds by testing antimicrobial activity followed by laborious studies of the mechanism of action. High-throughput methods for new antibiotic screening based on antimicrobial activity have become routine; however, identification of molecular targets is usually a challenge. Therefore, it is highly beneficial to combine primary screening with the identification of the mechanism of action. In this review, we describe a collection of methods for screening translation inhibitors, with a special emphasis on methods which can be performed in a high-throughput manner. Antibiotics 2016-06-24 5 3 Review 10.3390/antibiotics5030022 22 2079-6382 2016-06-24 doi: 10.3390/antibiotics5030022 Ilya Osterman Alexey Bogdanov Olga Dontsova Petr Sergiev http://www.mdpi.com/1660-4601/13/7/630 Much attention has been paid to hospitals environments since modern pandemics have emerged. The building sector is considered to be the largest world energy consumer, so many global organizations are attempting to create a sustainable environment in building construction by reducing energy consumption. Therefore, maintaining high standards of hygiene while reducing energy consumption has become a major task for hospitals. This study develops a decision model based on genetic algorithms and A* graph search algorithms to evaluate existing hospital environmental conditions and to recommend an optimal scheme of sustainable renovation strategies, considering trade-offs among minimal renovation cost, maximum quality improvement, and low environmental impact. Reusing vacant buildings is a global and sustainable trend. In Taiwan, for example, more and more school space will be unoccupied due to a rapidly declining birth rate. Integrating medical care with local community elder-care efforts becomes important because of the aging population. This research introduces a model that converts a simulated vacant school building into a community public hospital renovation project in order to validate the solutions made by hospital managers and suggested by the system. The result reveals that the system performs well and its solutions are more successful than the actions undertaken by decision-makers. This system can improve traditional hospital building condition assessment while making it more effective and efficient. International Journal of Environmental Research and Public Health 2016-06-24 13 7 Article 10.3390/ijerph13070630 630 1660-4601 2016-06-24 doi: 10.3390/ijerph13070630 Yi-Kai Juan Yu-Ching Cheng Yeng-Horng Perng Daniel Castro-Lacouture http://www.mdpi.com/1424-8247/9/3/34 The performance of compressed tablet drug delivery systems made using polymeric materials depend on multiple factors, such as surface properties like contact angle, surface free energy and water absorption rate, besides the release mechanisms driven by the kind of polymer used. Hence, it should be possible to establish a relationship between the surface properties and the drug release kinetics. Compressed tablets with different proportions of poly(maleic acid-alt-octadecene) potassium salt (0%, 10%, 20%, 30% and 40%) were prepared. Blends of a model drug (ampicillin trihydrate) and the polymer material were analyzed by DSC. The surface properties of the tablets were determined by the sessile drop method, while the surface energy was determined using the semi-empirical Young-Dupre, Neumann and OWRK models. The release profiles were determined simulating in vitro conditions (buffer solutions pH 1.2 and pH 7.4 with ionic strength of 1.5 M at 37 °C (310.15 K)). A kinetic analysis of the dissolution profiles using different models (zero order, first order, Higuchi and Korsmeyer-Peppas) was realized. The results showed a significant effect of the proportion of polymer in both the surface properties of the tablets and the dissolution release, indicating a relationship between the kinetic and thermodynamic properties. Pharmaceuticals 2016-06-24 9 3 Article 10.3390/ph9030034 34 1424-8247 2016-06-24 doi: 10.3390/ph9030034 Cristhian Yarce Diego Pineda Clara Correa Constain Salamanca http://www.mdpi.com/2076-0817/5/3/45 Streptococcus suis, a major porcine pathogen and an important zoonotic agent, is considered to be composed of phenotypically and genetically diverse strains. However, recent studies reported several "S. suis-like strains" that were identified as S. suis by commonly used methods for the identification of this bacterium, but were regarded as distinct species from S. suis according to the standards of several taxonomic analyses. Furthermore, it has been suggested that some S. suis-like strains can be assigned to several novel species. In this review, we discuss the current taxonomical situation of S. suis with a focus on (1) the classification history of the taxon of S. suis; (2) S. suis-like strains revealed by taxonomic analyses; (3) methods for detecting and identifying this species, including a novel method that can distinguish S. suis isolates from S. suis-like strains; and (4) current topics on the reclassification of S. suis-like strains. Pathogens 2016-06-24 5 3 Review 10.3390/pathogens5030045 45 2076-0817 2016-06-24 doi: 10.3390/pathogens5030045 Masatoshi Okura Makoto Osaki Ryohei Nomoto Sakura Arai Ro Osawa Tsutomu Sekizaki Daisuke Takamatsu http://www.mdpi.com/2227-7382/4/3/20 The extracellular matrix (ECM) has a molecular machinery composed of diverse proteins and proteoforms that combine properties of tensile strength with extensibility exhibiting growth-regulatory functions and self- and non-self-recognition. The identification of ECM proteoforms is the prerequisite towards a comprehensive understanding of biological functions accomplished by the outermost layer of the cell. Regulatory mechanisms of protein functions rely on post-translational modifications, phosphorylation in particular, affecting enzymatic activity, interaction, localization and stability. To investigate the ECM proteoforms, we have isolated the cell wall proteome and phosphoproteome of a tuberous crop, potato (Solanum tuberosum). LC-MS/MS analysis led to the identification of 38 proteins and 35 phosphoproteins of known and unknown functions. The findings may provide a better understanding of biochemical machinery and the integrated protein and phosphoprotein network of ECM for future functional studies of different developmental pathways and guidance cues in mechanosensing and integrity signaling. Proteomes 2016-06-24 4 3 Article 10.3390/proteomes4030020 20 2227-7382 2016-06-24 doi: 10.3390/proteomes4030020 Eman Elagamey Kanika Narula Arunima Sinha Pooja Aggarwal Sudip Ghosh Niranjan Chakraborty Subhra Chakraborty http://www.mdpi.com/1420-3049/21/7/823 DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), which plays an important role in inducing drug resistance against alkylating agents that modify the O6 position of guanine in DNA, is an attractive target for anti-tumor chemotherapy. A series of MGMT inhibitors have been synthesized over the past decades to improve the chemotherapeutic effects of O6-alkylating agents. In the present study, we performed a three-dimensional quantitative structure activity relationship (3D-QSAR) study on 97 guanine derivatives as MGMT inhibitors using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. Three different alignment methods (ligand-based, DFT optimization-based and docking-based alignment) were employed to develop reliable 3D-QSAR models. Statistical parameters derived from the models using the above three alignment methods showed that the ligand-based CoMFA (Qcv2 = 0.672 and Rncv2 = 0.997) and CoMSIA (Qcv2 = 0.703 and Rncv2 = 0.946) models were better than the other two alignment methods-based CoMFA and CoMSIA models. The two ligand-based models were further confirmed by an external test-set validation and a Y-randomization examination. The ligand-based CoMFA model (Qext2 = 0.691, Rpred2 = 0.738 and slope k = 0.91) was observed with acceptable external test-set validation values rather than the CoMSIA model (Qext2 = 0.307, Rpred2 = 0.4 and slope k = 0.719). Docking studies were carried out to predict the binding modes of the inhibitors with MGMT. The results indicated that the obtained binding interactions were consistent with the 3D contour maps. Overall, the combined results of the 3D-QSAR and the docking obtained in this study provide an insight into the understanding of the interactions between guanine derivatives and MGMT protein, which will assist in designing novel MGMT inhibitors with desired activity. Molecules 2016-06-23 21 7 Article 10.3390/molecules21070823 823 1420-3049 2016-06-23 doi: 10.3390/molecules21070823 Guohui Sun Tengjiao Fan Na Zhang Ting Ren Lijiao Zhao Rugang Zhong http://www.mdpi.com/1422-0067/17/7/995 Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling plays a dominant role in the pathogenesis of liver ischemia-reperfusion (IR) injury. Dexmedetomidine (Dex) protects the liver against IR injury via α2-adrenoceptor activation, but the contribution of TLR4 signaling remains unknown. The authors aimed to examine whether pretreatment with Dex produces hepatic protection and investigate the influence of Dex on TLR4/NF-κB signaling. Dex was given via intraperitoneal injection 30 min prior to orthotopic autologous liver transplantation (OALT) in rats, and three α2-adrenoceptor antagonists including atipamezole (a nonselective α2 receptor blocker), ARC-239 (a specific α2B/C blocker) and BRL-44408 (a specific α2A blocker) were injected intraperitoneally 10 min before Dex administration. Histopathologic evaluation of the liver and the measurement of serum alanine aminotransferase activity, TLR4/NF-κB expression in the liver, and pro-inflammatory factors (serum tumor necrosis factor-α, interleukin-1β and hepatic myeloperoxidase) concentrations were performed 8 h after OALT. Dex ameliorated liver injury after OALT probably by suppressing the TLR4/NF-κB pathway and decreasing inflammatory mediator levels. The protective effects of Dex were reversed by atipamezole and BRL-44408, but not by ARC-239, suggesting that these effects were mediated in part by the α2A subtype. In conclusion, Dex attenuates liver injury partly via the α2A-adrenoceptor subtype, and the mechanism is due to the suppression of the TLR4/NF-κB pathway. International Journal of Molecular Sciences 2016-06-23 17 7 Article 10.3390/ijms17070995 995 1422-0067 2016-06-23 doi: 10.3390/ijms17070995 Yiheng Wang Shan Wu Xiaofang Yu Shaoli Zhou Mian Ge Xinjin Chi Jun Cai http://www.mdpi.com/1420-3049/21/7/822 Expanded (x) and widened (y) deoxyribose nucleic acids (DNA) have an extra benzene ring incorporated either horizontally (xDNA) or vertically (yDNA) between a natural pyrimidine base and the deoxyribose, or between the 5- and 6-membered rings of a natural purine. Far-reaching applications for (x,y)DNA include nucleic acid probes and extending the natural genetic code. Since modified nucleobases must encode information that can be passed to the next generation in order to be a useful extension of the genetic code, the ability of translesion (bypass) polymerases to replicate modified bases is an active area of research. The common model bypass polymerase DNA polymerase IV (Dpo4) has been previously shown to successfully replicate and extend past a single modified nucleobase on a template DNA strand. In the current study, molecular dynamics (MD) simulations are used to evaluate the accommodation of expanded/widened nucleobases in the Dpo4 active site, providing the first structural information on the replication of (x,y)DNA. Our results indicate that the Dpo4 catalytic (palm) domain is not significantly impacted by the (x,y)DNA bases. Instead, the template strand is displaced to accommodate the increased C1'–C1' base-pair distance. The structural insights unveiled in the present work not only increase our fundamental understanding of Dpo4 replication, but also reveal the process by which Dpo4 replicates (x,y)DNA, and thereby will contribute to the optimization of high fidelity and efficient polymerases for the replication of modified nucleobases. Molecules 2016-06-23 21 7 Article 10.3390/molecules21070822 822 1420-3049 2016-06-23 doi: 10.3390/molecules21070822 Laura Albrecht Katie Wilson Stacey Wetmore http://www.mdpi.com/1422-0067/17/7/996 Chitosan (CHT) is a natural, safe, and cheap product of chitin deacetylation, widely used by several industries because of its interesting features. The availability of industrial quantities of CHT in the late 1980s enabled it to be tested in agriculture. CHT has been proven to stimulate plant growth, to protect the safety of edible products, and to induce abiotic and biotic stress tolerance in various horticultural commodities. The stimulating effect of different enzyme activities to detoxify reactive oxygen species suggests the involvement of hydrogen peroxide and nitric oxide in CHT signaling. CHT could also interact with chromatin and directly affect gene expression. Recent innovative uses of CHT include synthesis of CHT nanoparticles as a valuable delivery system for fertilizers, herbicides, pesticides, and micronutrients for crop growth promotion by a balanced and sustained nutrition. In addition, CHT nanoparticles can safely deliver genetic material for plant transformation. This review presents an overview on the status of the use of CHT in plant systems. Attention was given to the research that suggested the use of CHT for sustainable crop productivity. International Journal of Molecular Sciences 2016-06-23 17 7 Review 10.3390/ijms17070996 996 1422-0067 2016-06-23 doi: 10.3390/ijms17070996 Massimo Malerba Raffaella Cerana http://www.mdpi.com/1422-0067/17/7/993 Three polyphenols were isolated and purified from sugar beet molasses by ultrasonic-aid extraction and various chromatographic techniques, and their structures were elucidated by spectral analysis. Cytotoxicity and the molecular mechanism were measured by methyl thiazolyl tetrazolium (MTT) assay, flow cytometry, caspase-3 activity assay and Western blot assay. The results showed that gallic acid, cyanidin-3-O-glucoside chloride and epicatechin have cytotoxicity to the human colon, hepatocellular and breast cancer cells. Cyanidin-3-O-glucoside chloride showed its cytotoxicity against various tumor cell lines, particularly against colon cancer Caco-2 cells with half maximal inhibitory concentration (IC50) value of 23.21 ± 0.14 μg/mL in vitro. Cyanidin-3-O-glucoside chloride may be a potential candidate for the treatment of colon cancer. In the mechanism study, cyanidin-3-O-glucoside chloride increased the ratio of cell cycle at G0/G1 phase and reduced cyclin D1 expression on Caco-2 cells. Cyanidin-3-O-glucoside chloride decreased mutant p21 expression, and increased the ratio of Bax/Bcl-2 and the activation of caspase-3 to induce apoptosis. International Journal of Molecular Sciences 2016-06-23 17 7 Article 10.3390/ijms17070993 993 1422-0067 2016-06-23 doi: 10.3390/ijms17070993 Mingshun Chen Zhengang Zhao Shujuan Yu http://www.mdpi.com/1422-0067/17/7/994 A polyoxomolybdate inorganic-organic hybrid crystal was synthesized with deprotonatable ionic-liquid surfactant. 1-dodecylimidazolium cation was employed for its synthesis. The hybrid crystal contained δ-type octamolybdate (Mo8) isomer, and possessed alternate stacking of Mo8 monolayers and interdigitated surfactant bilayers. The crystal structure was compared with polyoxomolybdate hybrid crystals comprising 1-dodecyl-3-methylimidazolium surfactant, which preferred β-type Mo8 isomer. The less bulky hydrophilic moiety of the 1-dodecylimidazolium interacted with the δ-Mo8 anion by N–H···O hydrogen bonds, which presumably induced the formation of the δ-Mo8 anion. Anhydrous conductivity of the hybrid crystal was estimated to be 5.5 × 10−6 S·cm−1 at 443 K by alternating current (AC) impedance spectroscopy. International Journal of Molecular Sciences 2016-06-23 17 7 Article 10.3390/ijms17070994 994 1422-0067 2016-06-23 doi: 10.3390/ijms17070994 Jun Kobayashi Ryosuke Kawahara Sayaka Uchida Shinichi Koguchi Takeru Ito http://www.mdpi.com/1422-0067/17/7/797 Arterial hypertension is a common condition worldwide and an important predictor of several complicated diseases. Arterial hypertension can be triggered by many factors, including physiological, genetic, and lifestyle causes. Specifically, molecules of the renin-angiotensin-aldosterone system not only play important roles in the control of blood pressure, but they are also associated with the genesis of arterial hypertension, thus constituting a need for pharmacological interventions. Chronic high pressure generates mechanical damage along the vascular system, heart, and kidneys, which are the principal organs affected in this condition. In addition to mechanical stress, hypertension-induced oxidative stress, chronic inflammation, and the activation of reparative mechanisms lead to end-organ damage, mainly due to fibrosis. Clinical trials have demonstrated that renin-angiotensin-aldosterone system intervention in hypertensive patients lowers morbidity/mortality and inflammatory marker levels as compared to placebo patients, evidencing that this system controls more than blood pressure. This review emphasizes the detrimental effects that a renin-angiotensin-aldosterone system (RAAS) imbalance has on health considerations above and beyond high blood pressure, such as fibrotic end-organ damage. International Journal of Molecular Sciences 2016-06-23 17 7 Review 10.3390/ijms17070797 797 1422-0067 2016-06-23 doi: 10.3390/ijms17070797 Natalia Muñoz-Durango Cristóbal Fuentes Andrés Castillo Luis González-Gómez Andrea Vecchiola Carlos Fardella Alexis Kalergis http://www.mdpi.com/1999-4915/8/7/179 Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo. Viruses 2016-06-23 8 7 Review 10.3390/v8070179 179 1999-4915 2016-06-23 doi: 10.3390/v8070179 Michael Breen Aitor Nogales Steven Baker Luis Martínez-Sobrido http://www.mdpi.com/1660-4601/13/7/628 Background: Undergoing a routine medical examination may be associated with the prevalence rate of chronic diseases from a population-based household interview survey. However, this important issue has not been examined so far. Methods: Data came from the first health service household interview of Hunan province, China, in 2013. A Rao–Scott chi-square test was performed to examine the difference in prevalence rates between subgroups. Adjusted odds ratio (OR) was calculated using the PROC SURVEYLOGISTIC procedure of SAS9.1 statistical software. Results: In total, 24,282 residents of 8400 households were surveyed. A higher proportion of elderly adults had undergone a medical examination within the prior 12 months compared with young adults (≥65 years, 60%; 45–64 years, 46%; 18–44 years, 37%). After controlling for location, sex, and household income per capita, undergoing a medical examination was significantly associated with high prevalence rates of hypertension (adjusted OR: 2.0, 95% CI: 1.1–3.5) and of diabetes mellitus (adjusted OR: 3.3, 95% CI: 1.7–6.5) for young adults aged 18–44 years. The associations were not statistically significant for age groups 45–64 years and 65 years or older. Conclusion: The prevalence rates of hypertension and diabetes mellitus may be seriously underestimated for young adults not undergoing a routine medical examination in a health household interview survey. International Journal of Environmental Research and Public Health 2016-06-23 13 7 Article 10.3390/ijerph13070628 628 1660-4601 2016-06-23 doi: 10.3390/ijerph13070628 Lin Zhang Wei Zhang Lingling Zhang Danping Tian Li Li Xin Deng Jing Deng Peishan Ning Guoqing Hu http://www.mdpi.com/2411-5142/1/3/269 Physical activity for health is the topic chosen for the third editorial of this newly launched journal. The aim of this editorial is to illustrate and comment on the physical activity guidelines of the Italian Ministry of Health. Journal of Functional Morphology and Kinesiology 2016-06-23 1 3 Editorial 10.3390/jfmk1030269 269 275 2411-5142 2016-06-23 doi: 10.3390/jfmk1030269 Giuseppe Musumeci http://www.mdpi.com/1660-4601/13/7/629 Although there have been numerous studies examining the toxicity and carcinogenicity of nickel compounds in humans and animals, its molecular mechanisms of action are not fully elucidated. In our research, NIH/3T3 cells were exposed to nickel-refining fumes at the concentrations of 0, 6.25, 12.50, 25, 50 and 100 μg/mL for 24 h. Cell viability, cell apoptosis, reactive oxygen species (ROS) level, lactate dehydrogenase (LDH) assay, the level of glutathione (GSH), activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) level were detected. The exposure of NIH/3T3 cells to nickel-refining fumes significantly reduced cell viability and induced cell apoptotic death in a dose-dependent manner. Nickel-refining fumes significantly increased ROS levels and induced DNA damage. Nickel-refining fumes may induce the changes in the state of ROS, which may eventually initiate oxidative stress, DNA damage and apoptosis of NIH/3T3 cells. International Journal of Environmental Research and Public Health 2016-06-23 13 7 Article 10.3390/ijerph13070629 629 1660-4601 2016-06-23 doi: 10.3390/ijerph13070629 Yue Wang Sheng-Yuan Wang Li Jia Lin Zhang Jing-Chong Ba Dan Han Cui-Ping Yu Yong-Hui Wu http://www.mdpi.com/1420-3049/21/7/793 The α-aminoamide family of sodium ion channel blockers have exhibited analgesic effects on neuropathic pain. Here, a series of novel α-aminoamides containing an indole ring were designed and synthesized. These compounds were evaluated in mice using a formalin test and they exhibited significant anti-allodynia activities. However, the analgesic mechanism of these compounds remains unclear; a subset of the synthesized compounds can only moderately inhibit the sodium ion channel, Nav1.7, in a whole-cell patch clamp assay. Overall, these results suggest that introduction of an indole moiety to α-aminoamide derivatives can significantly improve their bioactivity and further study is warranted. Molecules 2016-06-23 21 7 Article 10.3390/molecules21070793 793 1420-3049 2016-06-23 doi: 10.3390/molecules21070793 Haotian Li Shiyong Fan Jingchao Cheng Ping Zhang Bohua Zhong Weiguo Shi http://www.mdpi.com/1420-3049/21/7/820 Adverse effects and drug resistance to the current onchopharmacologicals have increased the demand for alternative novel therapeutics. We herein introduce justicidin B, an arylnaphthalen lignan isolated from different plant origins, especially Justicia, Phyllanthus, Haplophyllum and Linum species. This cyclolignan exhibits a wide array of biological properties ranges from piscicidal to antifungal, antiviral and antibacterial activities. Activity against Trypanosoma brucei makes justicidin B a potential antiprotozoal agent for the treatment of neglected tropical diseases. Pharmacological properties like antiplatelet, anti-inflammatory and bone resorption inhibition have been also attributed to justicidin B. This compound is a potent cytotoxic substance on several cell lines, especially chronic myeloid and chronic lymphoid leukemia. Pharmacological values, natural variation, as well as biotechnological production of justicidin B by plant cell, tissue and organ culture are also described in this review. Chemical characteristics and chromatographic methods to identify justicidin B and its biosynthetic pathway have been discussed. Different approaches to the total synthesis of justicidin B are compared. This review would shed light on the role of justicidin B as an intriguing natural compound and provides a chance to optimize conditions for industrial applications. Molecules 2016-06-23 21 7 Review 10.3390/molecules21070820 820 1420-3049 2016-06-23 doi: 10.3390/molecules21070820 Shiva Hemmati Hassan Seradj http://www.mdpi.com/1660-4601/13/7/627 There have been relatively few opportunities to examine the cardiovascular effects of an extreme air pollution event in China. We aimed to examine the impact of the 2013 Eastern China Smog occurring from 2 to 9 December 2013, on outpatient visits for coronary heart diseases (CHD) in a typical hospital in Shanghai, China. We used the over-dispersed, generalized additive model to estimate the relative risk (RR) of the 2013 Eastern China Smog on the outpatient visits by comparing the smog period (2–9 December 2013; 8 days) to the non-smog period (1 November–1 December 2013, and 10 December–28 February 2014; 112 days). This model also controlled for time trends, days of the week, holidays, and meteorological factors. A stratification analysis was performed to estimate sex- and age-specific RRs. The daily average PM2.5 (fine particulate matter with an aerodynamic diameter less than 2.5 μm) concentrations during the smog period were 212 μg/m3, which were three times higher than during the non-smog period (76 μg/m3). The smog in Eastern China in 2013 was significantly associated with an increased risk of outpatient visits for CHD. For example, the RR was 1.18 (95% CI: 1.04, 1.32) on lag 0 day. There were similar effects on males and females. Our analyses provided preliminary evidence that smog constituted a significant risk factor of CHD in China. International Journal of Environmental Research and Public Health 2016-06-23 13 7 Article 10.3390/ijerph13070627 627 1660-4601 2016-06-23 doi: 10.3390/ijerph13070627 Fang Huang Renjie Chen Yuetian Shen Haidong Kan Xingya Kuang http://www.mdpi.com/2072-6643/8/7/386 Obesity is a leading cause of morbidity and mortality, yet is preventable. This study aimed to investigate associations between body mass index, body fat percentage and obesity-related eating behaviors. Women (n = 116; 18–44 years) were measured for height, weight and body fat using air displacement plethysmography (BodPod). Women completed the validated Three Factor Eating Questionnaire to assess their eating behaviors using Restraint, Disinhibition and Hunger eating factor categories and sub-categories. The eating behavior data were analyzed for associations with body mass index and body fat percentage, and comparisons across body mass index and body fat percentage categories (< vs. ≥25 kg/m2; < vs. ≥30%, respectively). Women had a mean (standard deviation) body mass index of 23.4 (3.5) kg/m2, and body fat percentage of 30.5 (7.6)%. Disinhibition was positively associated with both body mass index (p < 0.001) and body fat percentage (p < 0.001). Emotional Disinhibition was positively associated with body fat percentage (p < 0.028). Women with low Restraint and high Disinhibition had significantly higher body mass index and body fat percentage than women with high Restraint and low Disinhibition. Disinhibition seems likely to be an important contributor to obesity. Tailored intervention strategies focused on counteracting Disinhibition should be a key target area for managing weight/fat gain. Nutrients 2016-06-23 8 7 Article 10.3390/nu8070386 386 2072-6643 2016-06-23 doi: 10.3390/nu8070386 Rozanne Kruger Jacqui De Bray Kathryn Beck Cathryn Conlon Welma Stonehouse http://www.mdpi.com/2072-6643/8/7/390 (1) Objective: To compare the effects of isomaltulose (Palatinose™, PSE) vs. maltodextrin (MDX) ingestion on substrate utilization during endurance exercise and subsequent time trial performance; (2) Methods: 20 male athletes performed two experimental trials with ingestion of either 75 g PSE or MDX 45 min before the start of exercise. The exercise protocol consisted of 90 min cycling (60% VO2max) followed by a time trial; (3) Results: Time trial finishing time (−2.7%, 90% CI: ±3.0%, 89% likely beneficial; p = 0.147) and power output during the final 5 min (+4.6%, 90% CI: ±4.0%, 93% likely beneficial; p = 0.053) were improved with PSE compared with MDX. The blood glucose profile differed between trials (p = 0.013) with PSE resulting in lower glycemia during rest (95%–99% likelihood) and higher blood glucose concentrations during exercise (63%–86% likelihood). In comparison to MDX, fat oxidation was higher (88%–99% likelihood; p = 0.005) and carbohydrate oxidation was lower following PSE intake (85%–96% likelihood; p = 0.002). (4) Conclusion: PSE maintained a more stable blood glucose profile and higher fat oxidation during exercise which resulted in improved cycling performance compared with MDX. These results could be explained by the slower availability and the low-glycemic properties of Palatinose™ allowing a greater reliance on fat oxidation and sparing of glycogen during the initial endurance exercise. Nutrients 2016-06-23 8 7 Article 10.3390/nu8070390 390 2072-6643 2016-06-23 doi: 10.3390/nu8070390 Daniel König Denise Zdzieblik Anja Holz Stephan Theis Albert Gollhofer http://www.mdpi.com/2072-6643/8/7/385 Objective: To explore the relationship between the erythrocyte fatty acid profile and cognition in elderly Chinese adults. Methods: 60 mild cognitive impairment (MCI) subjects and 60 age- and gender-matched control adults (aged 55 years and above) were involved in this cross-sectional study. Cognitive function was measured by using the Montreal Cognitive Assessment (MoCA) test. Information regarding the demographic characteristics and lifestyle of the participants was collected with a questionnaire. A semi-quantified food frequency questionnaire (FFQ) method was used for dietary assessment. The erythrocytes fatty acid profile was measured. Results: The MCI subjects had a lower education level than the control subjects (p < 0.05). Compared with control subjects, MCI subjects had higher daily poultry intake and lower fish intake (p < 0.05). Erythrocyte fatty acid profile of the MCI subjects was characterized as lower erythrocyte proportions of 20:4 n-6, 20:5 n-3, and total n-3 fatty acids compared with control subjects (p < 0.05). An association of erythrocyte proportions of 18:0, 22:0, total SFA, 18:2 n-6, 24:4 n-6 fatty acids, docosahexaenoic acid (DHA), and total n-6 PUFAs with cognition in elderly Chinese adults was detected. Conclusion: The erythrocyte fatty acid profile was related to cognitionin the elderly. Lower erythrocyte unsaturated fatty acid and higher saturated fatty acid proportions might predict cognitive function decline in elderly Chinese adults. Nutrients 2016-06-23 8 7 Article 10.3390/nu8070385 385 2072-6643 2016-06-23 doi: 10.3390/nu8070385 Linhong Yuan Jie Zhen Weiwei Ma Can Cai Xiaochen Huang Rong Xiao http://www.mdpi.com/2072-6643/8/7/388 The aim of this study was to evaluate evidence for the efficacy of aloe vera on managing prediabetes and early non-treated diabetes mellitus. We performed a systematic search of PubMed, Embase, and Cochrane Central Register of Controlled Trials until 28 January 2016. A total of five randomized controlled trials (RCTs) involving 415 participants were included. Compared with the controls, aloe vera supplementation significantly reduced the concentrations of fasting blood glucose (FBG) (p = 0.02; weighed mean difference [WMD]: −30.05 mg/dL; 95% confidence interval [CI]: −54.87 to −5.23 mg/dL), glycosylated hemoglobin A1c (HbA1c) (p < 0.00001; WMD: −0.41%; 95% CI: −0.55% to −0.27%), triglyceride (p = 0.0001), total cholesterol (TC) (p < 0.00001), and low density lipoprotein-cholesterol (LDL-C) (p < 0.00001). Aloe vera was superior to placebo in increasing serum high density lipoprotein-cholesterol (HDL-C) levels (p = 0.04). Only one adverse event was reported. The evidence from RCTs showed that aloe vera might effectively reduce the levels of FBG, HbA1c, triglyceride, TC and LDL-C, and increase the levels of HDL-C on prediabetes and early non-treated diabetic patients. Limited evidence exists about the safety of aloe vera. Given the small number and poor quality of RCTs included in the meta-analysis, these results are inconclusive. A large-scale, well-designed RCT is needed to further address this issue. Nutrients 2016-06-23 8 7 Article 10.3390/nu8070388 388 2072-6643 2016-06-23 doi: 10.3390/nu8070388 Yiyi Zhang Wen Liu Dan Liu Tieyun Zhao Haoming Tian http://www.mdpi.com/2072-6643/8/7/389 This study assessed the relationship between physical activity and beverage consumption among adolescents with a population based cross-sectional survey was carried out in the Balearic Islands, Spain (n = 1988; 12–17 years old). Body composition, educational and income level, physical activity (PA), and beverage consumption and energy intake were assessed. Sixty-two percent of adolescents engaged in >300 min/week of PA. Boys were more active than girls, younger adolescents were more active than older counterparts, low parental income was associated with physical inactivity, and time spent watching TV (including, TV, Internet or handheld cellular devices) was inversely associated with PA practice. The average beverage intake of the studied adolescents was 0.9 L/day, higher in boys than in girls. Beverage intake was positively associated with PA practice, and the highest amount of energy intake from beverages was observed in active boys and girls. Most of the studied adolescent population met the PA recommendations. Gender, age, parental income, and time spent watching TV were significant determinants of PA. Type and amount of beverages drunk varied according to gender and PA, and general daily total beverage intake was lower than recommended adequate fluid intake. PA behavior should be considered when analyzing beverage consumption in adolescents. Nutrients 2016-06-23 8 7 Article 10.3390/nu8070389 389 2072-6643 2016-06-23 doi: 10.3390/nu8070389 Maria Bibiloni Asli Özen Antoni Pons Marcela González-Gross Josep Tur http://www.mdpi.com/2072-6643/8/7/387 Anemia affects approximately 25% of school-aged children (SAC—aged 5.00–14.99 years) globally. We determined in three countries the prevalence and determinants of anemia in SAC. Data on sociodemographics, inflammation and nutrition status were obtained from the 2006 Mexican National Nutrition Survey, the 2003-6 US National Health and Nutrition Examination Surveys, and the 2010 Encuesta Nacional de Nutrición Situación Colombia. In the US, vitamin A and iron deficiency (ID) were available only for girls aged 12.00–14.99 years to which our analysis was limited. Associations were evaluated by country using multivariable logistic regression adjusting for confounders and complex survey design. The prevalence of anemia and ID were: Mexico 12% (ID 18%), n = 3660; US 4% (ID 10%), n = 733; and Colombia 4% (ID 9%), n = 8573. The percentage of anemia associated with ID was 22.4% in Mexico, 38.9% in the US and 16.7% in Colombia. In Mexico, anemia was associated with ID (adjusted OR: 1.5, p = 0.02) and overweight (aOR 0.4, p = 0.007). In the US, anemia was associated with black race/ethnicity (aOR: 14.1, p < 0.0001) and ID (aOR: 8.0, p < 0.0001). In Colombia, anemia was associated with black race/ethnicity (aOR: 1.6, p = 0.005), lowest socio-economic status quintile (aOR: 1.8, p = 0.0005), ID (aOR: 2.7, p < 0.0001), and being stunted (aOR: 1.6, p = 0.02). While anemia was uniformly associated with iron deficiency in Mexico, Columbia, and the United States, other measured factors showed inconsistent associations with anemia. Additional data on anemia determinants in SAC are needed to guide interventions. Nutrients 2016-06-23 8 7 Article 10.3390/nu8070387 387 2072-6643 2016-06-23 doi: 10.3390/nu8070387 Sana Syed O. Addo Vanessa De la Cruz-Góngora Fayrouz Ashour Thomas Ziegler Parminder Suchdev http://www.mdpi.com/2304-8158/5/3/45 Many consumers do not follow recommended food safety practices for cooking egg dishes, such as pies, quiches, and casseroles, potentially leading to foodborne illnesses such as Salmonellosis. The United States Department of Agriculture (USDA) recommends cooking egg mixtures until the center reaches 71 °C (160 °F). The objectives of this study were to determine what endpoint temperature information consumers receive from egg dish recipes, and if recipes would lead to safe temperatures when followed. Egg dish recipes (n = 226) from 65 websites, 50 cookbooks, and nine magazine titles (multiple issues of each) were analyzed. Time was the most frequently used indicator, given in 92% of the recipes, with 15% using only time. Other indicators included: set (89), browned (76), clean toothpick/knife (60), puffed (27), and jiggled (13). Only two recipes indicated final endpoint temperatures. Three recipes (a pie, a quiche, and an egg casserole) were chosen and prepared in triplicate to see if they would reach recommended temperatures. The pie and quiche were still liquid at 71 °C, and were well over the recommended temperature when cooked according to instructions, but the egg casserole was not consistently above 71 °C, when the recipe instructions indicated it was done and the center was light brown and "jiggled" This research indicates that consumers are not receiving information on endpoint temperatures in egg recipes, but the likelihood of foodborne illness is low since most dishes probably be cooked past the recommended temperature before the consumer considers them done unless there are many inclusions that may absorb liquid and reduce the appearance of liquid in the dish. Foods 2016-06-23 5 3 Article 10.3390/foods5030045 45 2304-8158 2016-06-23 doi: 10.3390/foods5030045 Sandria Godwin Curtis Maughan Edgar Chambers http://www.mdpi.com/2077-0383/5/7/61 Background: Ghrelin is a 28-amino acid peptide with an orexigenic property, which is predominantly produced by the stomach. Acylated ghrelin is the active form of this hormone. Obestatin is a 23-amino acid peptide which is produced by post-translational modification of a protein precursor that also produces ghrelin. Obestatin has the opposite effect of ghrelin on food intake. The aim of this study was to evaluate acylated ghrelin and obestatin levels and their ratio in obese and normal-weight children and adolescents, and their association with metabolic syndrome (MetS) parameters. Methods: Serum acyl-ghrelin, obestatin, leptin, insulin, fasting plasma glucose (FPG), lipid profile, and malondialdehyde (MDA) were evaluated in 73 children and adolescents (42 obese and 31 control). Insulin resistance was calculated by a homeostasis model assessment of insulin resistance (HOMA-IR). MetS was determined according to IDF criteria. Results: Acyl-ghrelin levels were significantly lower in obese subjects compared to the control group and lower in obese children with MetS compared to obese subjects without MetS. Obestatin was significantly higher in obese subjects compared to that of the control, but it did not differ significantly among those with or without MetS. Acyl-ghrelin to obestatin ratio was significantly lower in obese subjects compared to that in normal subjects. Acyl-ghrelin showed significant negative and obestatin showed significant positive correlations with body mass index (BMI), BMI Z-score, leptin, insulin, and HOMA-IR. Acyl-ghrelin had a significant negative correlation with MDA as an index of oxidative stress. Conclusion: Ghrelin is decreased and obestatin is elevated in obesity. Both of these hormones are associated with insulin resistance, and ghrelin is associated with oxidative stress. The balance between ghrelin and obestatin seems to be disturbed in obesity. Journal of Clinical Medicine 2016-06-23 5 7 Article 10.3390/jcm5070061 61 2077-0383 2016-06-23 doi: 10.3390/jcm5070061 Maryam Razzaghy-Azar Mitra Nourbakhsh Abdolreza Pourmoteabed Mona Nourbakhsh Davod Ilbeigi Mohsen Khosravi http://www.mdpi.com/1660-4601/13/7/625 The purpose of this study was to investigate the effect of two-hour exposure to a forest environment on cytokine, anti-oxidant and stress levels among university students and to compare the results to those measured in urban environments. Forty-one subjects were recruited. For our crossover design, subjects were divided into two groups based on similar demographic characteristics. Group A remained in the urban environment and was asked to perform regular breathing for 2 h. Blood samples were collected and the serum levels of cytokines including interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and glutathione peroxidase (GPx) were examined. Subjects were moved to a small town in a rural area for an equal amount of time to exclude carryover effects, and then remained for another 2 h in a forest environment. The second set of blood samples was collected to assess the effect of exposure to the forest environment. Using the same method, Group B was first exposed to the forest environment, followed by exposure to the urban environment. Blood samples collected after the subjects were exposed to the forest environment showed significantly lower levels of IL-8 and TNF-α compared to those in samples collected after urban environment exposure (10.76 vs. 9.21, t = 4.559, p < 0.001, and 0.97 vs. 0.87, t = 4.130, p < 0.001). The GPx concentration increased significantly after exposure to the forest environment (LnGPx = 5.09 vs. LnGPx = 5.21, t = −2.039, p < 0.05). International Journal of Environmental Research and Public Health 2016-06-23 13 7 Article 10.3390/ijerph13070625 625 1660-4601 2016-06-23 doi: 10.3390/ijerph13070625 Su Im Han Choi Yo-Han Jeon Min-Kyu Song Won Kim Jong-Min Woo http://www.mdpi.com/2072-6651/8/7/193 Toxic weaponry in the form of venom and poison has evolved in most groups of animals, including all four major lineages of tetrapods. Moreover, the evolution of such traits has been linked to several key aspects of the biology of toxic animals including life-history and diversification. Despite this, attempts to investigate the macroevolutionary patterns underlying such weaponry are lacking. In this study we analyse patterns of venom and poison evolution across reptiles, amphibians, mammals, and birds using a suite of phylogenetic comparative methods. We find that each major lineage has a characteristic pattern of trait evolution, but mammals and reptiles evolve under a surprisingly similar regime, whilst that of amphibians appears to be particularly distinct and highly contrasting compared to other groups. Our results also suggest that the mechanism of toxin acquisition may be an important distinction in such evolutionary patterns; the evolution of biosynthesis is far less dynamic than that of sequestration of toxins from the diet. Finally, contrary to the situation in amphibians, other tetrapod groups show an association between the evolution of toxic weaponry and higher diversification rates. Taken together, our study provides the first broad-scale analysis of macroevolutionary patterns of venom and poison throughout tetrapods. Toxins 2016-06-23 8 7 Article 10.3390/toxins8070193 193 2072-6651 2016-06-23 doi: 10.3390/toxins8070193 Richard Harris Kevin Arbuckle http://www.mdpi.com/1660-4601/13/7/626 Malaria and iron-deficient anemia during pregnancy pose considerable risks for the mother and newborn. Intermittent Preventive Treatment during pregnancy with sulphadoxine-pyrimethamine (IPTp-SP) and iron supplement to prevent anemia to all pregnant women receiving antenatal care (ANC) services is highly recommended. However, their compliance remains low. This study aimed at identifying factors influencing non-compliance of medications among pregnant women. A descriptive cross-sectional study was conducted in Simiyu region in northwest Tanzania using a structured questionnaire to collect data from 430 women who were pregnant or gave birth 12 months prior to data collection. Data were analyzed using non-parametric statistical analysis with STATA 10. Overall, 284 (66%) and 195 (45%) of interviewed women received IPTp-SP and iron supplementation during their ANC visits, respectively. The majority (85%) of women whom received medications were aware if they had received IPTp-SP or iron supplementation. Of those received IPTp-SP, only 11% took all the three doses, while the remaining 89% took only two doses or one dose. For women who received iron supplementation, 29% reported that they did not take any dose at all. Reasons given for not complying with regiments included not liking the medications and disapproval from male partners. Our findings suggest that IPTp-SP and iron supplement compliance among pregnant women in Simiyu region is low. Intensification of community education, further qualitative research and administration of medication through directly-observed therapy (DOT) are recommended to address the problem. International Journal of Environmental Research and Public Health 2016-06-23 13 7 Article 10.3390/ijerph13070626 626 1660-4601 2016-06-23 doi: 10.3390/ijerph13070626 Benatus Sambili Ronald Kimambo Yun Peng Elison Ishunga Edna Matasha Godfrey Matumu Rita Noronha David Ngilangwa http://www.mdpi.com/2077-0383/5/7/60 Remote ischemic preconditioning (RIPC) is the set of ischemia episodes that protects against subsequent periods of prolonged ischemia through the cascade of adaptive responses; however, the mechanisms of RIPC are not entirely clear. Here, we aimed to study the impact of RIPC in patients with stable angina pectoris and compare it with healthy individuals with respect to arterial stiffness and heart rate variability. In the randomized, sham-controlled, crossover blind design study, a group of 30 coronary heart disease (CHD) patients (63.9 ± 1.6 years) with stable angina pectoris NYHA II-III and a control group of 20 healthy individuals (58.2 ± 2.49) were both randomly allocated for remote RIPC or sham RIPC. Arterial stiffness, pulse wave velocity (Spygmacor, Australia), and heart rate variability (HRV) were recorded before and after the procedure followed by the crossover examination. In the group of healthy individuals, RIPC showed virtually no impact on the cardiovascular parameters, while, in the CHD group, the systolic and central systolic blood pressure, central pulse pressure, and augmentation decreased, and total power of HRV improved. We conclude that ischemic preconditioning reduces not only systolic blood pressure, but also reduces central systolic blood pressure and improves arterial compliance and heart rate modulation reserve, which may be associated with the antianginal effect of preconditioning. Journal of Clinical Medicine 2016-06-23 5 7 Article 10.3390/jcm5070060 60 2077-0383 2016-06-23 doi: 10.3390/jcm5070060 Naufal Zagidullin Elena Scherbakova Yuliana Safina Rustem Zulkarneev Shamil Zagidullin http://www.mdpi.com/1999-4915/8/6/177 Badnaviruses (Family: Caulimoviridae; Genus: Badnavirus) are non-enveloped bacilliform DNA viruses with a monopartite genome containing about 7.2 to 9.2 kb of dsDNA with three to seven open reading frames. They are transmitted by mealybugs and a few species by aphids in a semi-persistent manner. They are one of the most important plant virus groups and have emerged as serious pathogens affecting the cultivation of several horticultural crops in the tropics, especially banana, black pepper, cocoa, citrus, sugarcane, taro, and yam. Some badnaviruses are also known as endogenous viruses integrated into their host genomes and a few such endogenous viruses can be awakened, e.g., through abiotic stress, giving rise to infective episomal forms. The presence of endogenous badnaviruses poses a new challenge for the fool-proof diagnosis, taxonomy, and management of the diseases. The present review aims to highlight emerging disease problems, virus characteristics, transmission, and diagnosis of badnaviruses. Viruses 2016-06-22 8 6 Review 10.3390/v8060177 177 1999-4915 2016-06-22 doi: 10.3390/v8060177 Alangar Bhat Thomas Hohn Ramasamy Selvarajan http://www.mdpi.com/1422-0067/17/6/837 TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is an endogenous ligand, which plays role in immune surveillance and anti-tumor immunity. It has ability to selectively kill tumor cells showing no toxicity to normal cells. We tested the apoptotic and cytotoxic activities of xanthohumol, a prenylated chalcone found in Humulus lupulus on androgen-sensitive human prostate adenocarcinoma cells (LNCaP) in combination with TRAIL. Cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium reduction assay (MTT) and lactate dehydrogenase assay (LDH). The expression of death receptors (DR4/TRAIL-R1 and DR5/TRAIL-R2) and apoptosis were detected using flow cytometry. We examined mitochondrial membrane potential (ΔΨm) by DePsipher reagent using fluorescence microscopy. The intracellular expression of proteins was evaluated by Western blotting. Our study showed that xanthohumol enhanced cytotoxic and apoptotic effects of TRAIL. The tested compounds activated caspases-3, -8, -9, Bid, and increased the expression of Bax. They also decreased expression of Bcl-xL and decreased mitochondrial membrane potential, while the expression of death receptors was not changed. The findings suggest that xanthohumol is a compound of potential use in chemoprevention of prostate cancer due to its sensitization of cancer cells to TRAIL-mediated apoptosis. International Journal of Molecular Sciences 2016-06-22 17 6 Article 10.3390/ijms17060837 837 1422-0067 2016-06-22 doi: 10.3390/ijms17060837 Małgorzata Kłósek Anna Mertas Wojciech Król Dagmara Jaworska Jan Szymszal Ewelina Szliszka http://www.mdpi.com/1422-0067/17/6/992 Staphylococcus epidermidis, especially methicillin-resistant strains, may be the source of surgical site infections and may be a reservoir of staphylococcal cassette chromosome mec (SCCmec) for S. aureus. The aim of this study was to investigate the prevalence of methicillin-resistant S. epidermidis (MRSE) on the abdominal skin of females before laparotomy and determine the molecular characteristics and antimicrobial susceptibility patterns of these isolates. MRSE was found in 54 of 157 isolates based on mecA gene detection, and there was no difference in icaA gene carriage rate between MRSE and methicillin-susceptible S. epidermidis (MSSE) isolates. Antimicrobial susceptibility profiles were determined by broth microdilution antimicrobial susceptibility testing according to the latest CLSI manuals. All MRSE isolates had unfavorable antimicrobial susceptibility patterns. Twenty-three MRSE strains (42.6%) were multi-drug resistant. SCCmec typing and pulsed field gel electrophoresis (PFGE) typing was performed. Thirty-nine (72.2%) had a single SCCmec type, whereas 1.9% had two types. Fourteen strains (25.9%) were non-typeable (NT). The most frequent MRSE genotype was SCCmec type IVa. High diversity with PFGE patterns was obtained for MRSE, and there were no isolates exhibiting identical pulsotype. The results confirm that methicillin-resistant strains are frequently present among S. epidermidis on the abdominal skin of females before laparotomy. Moreover, resistance profiles seem to have no association with the SCCmec types or PFGE types for most common antibiotics. International Journal of Molecular Sciences 2016-06-22 17 6 Article 10.3390/ijms17060992 992 1422-0067 2016-06-22 doi: 10.3390/ijms17060992 Pin-Jia Wang Cheng-Bin Xie Feng-Hui Sun Li-Juan Guo Min Dai Xi Cheng Yong-Xin Ma http://www.mdpi.com/1422-0067/17/6/990 Pulmonary hypertension (PH) is defined by a mean pulmonary arterial pressure over 25 mmHg at rest and is diagnosed by right heart catheterization. Among the different groups of PH, pulmonary arterial hypertension (PAH) is characterized by a progressive obstruction of distal pulmonary arteries, related to endothelial cell dysfunction and vascular cell proliferation, which leads to an increased pulmonary vascular resistance, right ventricular hypertrophy, and right heart failure. Although the primary trigger of PAH remains unknown, oxidative stress and inflammation have been shown to play a key role in the development and progression of vascular remodeling. These factors are known to increase DNA damage that might favor the emergence of the proliferative and apoptosis-resistant phenotype observed in PAH vascular cells. High levels of DNA damage were reported to occur in PAH lungs and remodeled arteries as well as in animal models of PH. Moreover, recent studies have demonstrated that impaired DNA-response mechanisms may lead to an increased mutagen sensitivity in PAH patients. Finally, PAH was linked with decreased breast cancer 1 protein (BRCA1) and DNA topoisomerase 2-binding protein 1 (TopBP1) expression, both involved in maintaining genome integrity. This review aims to provide an overview of recent evidence of DNA damage and DNA repair deficiency and their implication in PAH pathogenesis. International Journal of Molecular Sciences 2016-06-22 17 6 Review 10.3390/ijms17060990 990 1422-0067 2016-06-22 doi: 10.3390/ijms17060990 Benoît Ranchoux Jolyane Meloche Roxane Paulin Olivier Boucherat Steeve Provencher Sébastien Bonnet http://www.mdpi.com/1422-0067/17/6/991 Substance P (SP), a member of tachykinin family, is involved in the inflammation of the central nervous system and in the appearance of cerebral edema. Higher serum levels of SP have been found in 18 patients with cerebral ischemia compared with healthy controls. The aim of our multi-center study was to analyze the possible association between serum levels of SP and mortality in ischemic stroke patients. We included patients with malignant middle cerebral artery infarction (MMCAI) and a Glasgow Coma Scale (GCS) lower than 9. Non-surviving patients at 30 days (n = 31) had higher serum concentrations of SP levels at diagnosis of severe MMCAI than survivors (n = 30) (p < 0.001). We found in multiple regression an association between serum concentrations of SP higher than 362 pg/mL and mortality at 30 days (Odds Ratio = 5.33; 95% confidence interval = 1.541–18.470; p = 0.008) after controlling for age and GCS. Thus, the major novel finding of our study was the association between serum levels of SP and mortality in patients suffering from severe acute ischemic stroke. International Journal of Molecular Sciences 2016-06-22 17 6 Communication 10.3390/ijms17060991 991 1422-0067 2016-06-22 doi: 10.3390/ijms17060991 Leonardo Lorente María Martín Teresa Almeida Antonia Pérez-Cejas Luis Ramos Mónica Argueso Marta Riaño-Ruiz Jordi Solé-Violán Mariano Hernández http://www.mdpi.com/1422-0067/17/6/989 Breast cancer develops over a timeframe of 2–3 decades prior to clinical detection. Given this prolonged latency, it is somewhat unexpected from a biological perspective that obesity has no effect or reduces the risk for breast cancer in premenopausal women yet increases the risk for breast cancer in postmenopausal women. This conundrum is particularly striking in light of the generally negative effects of obesity on breast cancer outcomes, including larger tumor size at diagnosis and poorer prognosis in both pre- and postmenopausal women. This review and analysis identifies factors that may contribute to this apparent conundrum, issues that merit further investigation, and characteristics of preclinical models for breast cancer and obesity that should be considered if animal models are used to deconstruct the conundrum. International Journal of Molecular Sciences 2016-06-22 17 6 Review 10.3390/ijms17060989 989 1422-0067 2016-06-22 doi: 10.3390/ijms17060989 Shawna Matthews Henry Thompson http://www.mdpi.com/1422-0067/17/6/986 Polysaccharides from Morchella esculenta have been proven to be functional and helpful for humans. The purpose of this study was to investigate the chemical structure and anti-proliferating and antitumor activities of a Morchella esculenta polysaccharide (MEP) extracted by pulsed electric field (PEF) in submerged fermentation. The endo-polysaccharide was separated and purified by column chromatography and Gel permeation chromatography, and analyzed by gas chromatography. The MEP with an average molecular weight of 81,835 Da consisted of xylose, glucose, mannose, rhamnose and galactose at the ratio of 5.4:5.0:6.5:7.8:72.3. Structure of MEP was further analyzed by Fourier-transform infrared spectroscopy and 1H and 13C liquid-state nuclear magnetic resonance spectroscopy. Apoptosis tests proved that MEP could inhibit the proliferation and growth of human colon cancer HT-29 cells in a time- and dose-dependent manner within 48 h. This study provides more information on chemical structure of anti-proliferating polysaccharides isolated from Morchella esculenta. International Journal of Molecular Sciences 2016-06-22 17 6 Article 10.3390/ijms17060986 986 1422-0067 2016-06-22 doi: 10.3390/ijms17060986 Chao Liu Yonghai Sun Qian Mao Xiaolei Guo Peng Li Yang Liu Na Xu http://www.mdpi.com/1660-4601/13/6/623 Certain attitudes toward suicide may be a risk factor for suicide among the bereaved. To explore this possibility, we examined the relationship between attitudes toward suicide and family history of suicide. We focused on two specific attitudes indicating resignation in a survey: #1 "When a person chooses to die by suicide, the suicide is inevitable" (i.e., inevitability belief); and #2 "A suicide cannot be stopped by any person, because suicide is unpreventable" (i.e., unpreventable belief). The data of 5117 fully completed questionnaires were analyzed. Logistic regression analysis revealed that the two attitudes of resignation were significantly associated with a family history of suicide. The adjusted odds ratio for #1 was 1.39 (95% CI, 1.07–1.79) for individuals having experienced suicide by a family member or relative, while that for #2 was 1.57 (95% CI, 1.27–1.95) for experiencing a suicide by a family member or relative and 1.25 (95% CI, 1.05–1.49) for experiencing a suicide by a friend, business associate, partner or other. These two attitudes of resignation toward suicide were significantly associated with a family history of suicide. These attitudes might increase suicide risk among the bereaved. International Journal of Environmental Research and Public Health 2016-06-22 13 6 Article 10.3390/ijerph13060623 623 1660-4601 2016-06-22 doi: 10.3390/ijerph13060623 Teruomi Tsukahara Hiroaki Arai Tomoko Kamijo Yoshikiyo Kobayashi Shinsuke Washizuka Heihachiro Arito Tetsuo Nomiyama http://www.mdpi.com/1422-0067/17/6/985 MicroRNAs act by post-transcriptionally regulating the gene expression of 30%–60% of mammalian genomes. MicroRNAs are key regulators in all cellular processes, though the mechanism by which the cell activates or represses microRNA-mediated translational regulation is poorly understood. In this review, we discuss the RNA binding protein Fragile X Mental Retardation Protein (FMRP) and its role in microRNA-mediated translational regulation. Historically, FMRP is known to function as a translational suppressor. However, emerging data suggests that FMRP has both an agonistic and antagonistic role in regulating microRNA-mediated translational suppression. This bi-functional role is dependent on FMRP's interaction with the RNA helicase Moloney leukemia virus 10 (MOV10), which modifies the structural landscape of bound mRNA, therefore facilitating or inhibiting its association with the RNA-Induced Silencing Complex. International Journal of Molecular Sciences 2016-06-22 17 6 Review 10.3390/ijms17060985 985 1422-0067 2016-06-22 doi: 10.3390/ijms17060985 Phillip Kenny Stephanie Ceman http://www.mdpi.com/1660-4601/13/6/622 In this paper, we assume that a professional pollutant treatment enterprise treats all of the pollutants emitted by multiple small and medium-sized enterprises (SMEs). In order to determine the treatment price, SMEs can bargain with the pollutant treatment enterprise individually, or through forming alliances. We propose a bargaining game model of centralized pollutant treatment to study how the pollutant treatment price is determined through negotiation. Then, we consider that there is a moral hazard from SMEs in centralized pollutant treatment; in other words, they may break their agreement concerning their quantities of production and pollutant emissions with the pollutant treatment enterprise. We study how the pollutant treatment enterprise can prevent this by pricing mechanism design. It is found that the pollutant treatment enterprise can prevent SMEs' moral hazard through tiered pricing. If the marginal treatment cost of the pollutant treatment enterprise is a constant, SMEs could bargain with the pollutant treatment enterprise individually, otherwise, they should form a grand alliance to bargain with it as a whole. International Journal of Environmental Research and Public Health 2016-06-22 13 6 Article 10.3390/ijerph13060622 622 1660-4601 2016-06-22 doi: 10.3390/ijerph13060622 Yuyu Li Bo Huang Fengming Tao http://www.mdpi.com/1422-0067/17/6/966 Stipa purpurea (S. purpurea) is the dominant plant species in the alpine steppe of the Qinghai-Tibet Plateau, China. It is highly resistant to cold and drought conditions. However, the underlying mechanisms regulating the stress tolerance are unknown. In this study, a CIPK gene from S. purpurea (SpCIPK26) was isolated. The SpCIPK26 coding region consisted of 1392 bp that encoded 464 amino acids. The protein has a highly conserved catalytic structure and regulatory domain. The expression of SpCIPK26 was induced by drought and salt stress. SpCIPK26 overexpression in Arabidopsis thaliana (A. thaliana) plants provided increased tolerance to drought and salt stress in an abscisic acid (ABA)-dependent manner. Compared with wild-type A. thaliana plants, SpCIPK26-overexpressing plants had higher survival rates, water potentials, and photosynthetic efficiency (Fv/Fm), as well as lower levels of reactive oxygen species (ROS) following exposure to drought and salt stress. Gene expression analyses indicated stress-inducible genes (RD29A, RD29B, and ABF2) and a ROS-scavenger gene (CAT1) were upregulated in SpCIPK26-overexpressing plants after stress treatments. All of these marker genes are associated with ABA-responsive cis-acting elements. Additionally, the similarities in the gene expression patterns following ABA, mannitol, and NaCl treatments suggest SpCIPK26 has an important role during plant responses to drought and salt stress and in regulating ABA signaling. International Journal of Molecular Sciences 2016-06-22 17 6 Article 10.3390/ijms17060966 966 1422-0067 2016-06-22 doi: 10.3390/ijms17060966 Yanli Zhou Xudong Sun Yunqiang Yang Xiong Li Ying Cheng Yongping Yang http://www.mdpi.com/2079-6382/5/2/23 Bacterial biofilm is an emerging clinical problem recognized in the treatment of infectious diseases within the last two decades. The appearance of microbial biofilm in clinical settings is steadily increasing due to several reasons including the increased use of quality of life-improving artificial devices. In contrast to infections caused by planktonic bacteria that respond relatively well to standard antibiotic therapy, biofilm-forming bacteria tend to cause chronic infections whereby infections persist despite seemingly adequate antibiotic therapy. This review briefly describes the responses of biofilm matrix components and biofilm-associated bacteria towards sub-lethal concentrations of antimicrobial agents, which may include the generation of genetic and phenotypic variabilities. Clinical implications of bacterial biofilms in relation to antibiotic treatments are also discussed. Antibiotics 2016-06-22 5 2 Review 10.3390/antibiotics5020023 23 2079-6382 2016-06-22 doi: 10.3390/antibiotics5020023 Tianyan Song Marylise Duperthuy Sun Wai http://www.mdpi.com/1422-0067/17/6/987 About one-third of patients with advanced renal cell carcinoma (RCC) have bone metastasis that are often osteolytic and cause substantial morbidity, such as pain, pathologic fracture, spinal cord compression and hypercalcemia. The presence of bone metastasis in RCC is also associated with poor prognosis. Bone-targeted treatment using bisphosphonate and denosumab can reduce skeletal complications in RCC, but does not cure the disease or improve survival. Elucidating the molecular mechanisms of tumor-induced changes in the bone microenvironment is needed to develop effective treatment. The "vicious cycle" hypothesis has been used to describe how tumor cells interact with the bone microenvironment to drive bone destruction and tumor growth. Tumor cells secrete factors like parathyroid hormone-related peptide, transforming growth factor-β and vascular endothelial growth factor, which stimulate osteoblasts and increase the production of the receptor activator of nuclear factor κB ligand (RANKL). In turn, the overexpression of RANKL leads to increased osteoclast formation, activation and survival, thereby enhancing bone resorption. This review presents a general survey on bone metastasis in RCC by natural history, interaction among the immune system, bone and tumor, molecular mechanisms, bone turnover markers, therapies and healthcare burden. International Journal of Molecular Sciences 2016-06-22 17 6 Review 10.3390/ijms17060987 987 1422-0067 2016-06-22 doi: 10.3390/ijms17060987 Szu-Chia Chen Po-Lin Kuo http://www.mdpi.com/1422-0067/17/6/968 Quantitative real-time PCR (qRT-PCR) is the most classical technique in the field of gene expression study. This method requires an appropriate reference gene to normalize mRNA levels. In this study, the expression stability of four frequently-used reference genes in epididymal white adipose tissue (eWAT), inguinal beige adipose tissue (iBeAT) and brown adipose tissue (BAT) from obese and lean rats were evaluated by geNorm, NormFinder and BestKeeper. Based on the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, the two most stable reference genes were recommended in each type of adipose tissue. Two target genes were applied to test the stability of the reference genes. The geNorm and NormFinder results revealed that GAPDH and 36B4 exhibited the highest expression stabilities in eWAT, while 36B4 and β-actin had the highest expression stabilities in iBeAT and BAT. According to the results of the BestKeeper analysis, 36B4 was the most stable gene in eWAT, iBeAT and BAT, in terms of the coefficient of variance. In terms of the coefficient of correlation, GAPDH, 36B4 and β-actin were the most stable genes in eWAT, iBeAT and BAT, respectively. Additionally, expected results and statistical significance were obtained using a combination of two suitable reference genes for data normalization. In conclusion, 36B4 and GAPDH, in combination, are the best reference genes for eWAT, while 36B4 and β-actin are two most suitable reference genes for both iBeAT and BAT. We recommend using these reference genes accordingly. International Journal of Molecular Sciences 2016-06-22 17 6 Article 10.3390/ijms17060968 968 1422-0067 2016-06-22 doi: 10.3390/ijms17060968 Wan-Xia Zhang Jie Fan Jing Ma Yi-Song Rao Li Zhang You-E Yan http://www.mdpi.com/1420-3049/21/6/807 Natural products have always been exploited to promote health and served as a valuable source for the discovery of new drugs. In this review, the great potential of natural compounds and medicinal plants for the treatment or prevention of cardiovascular and metabolic disorders, global health problems with rising prevalence, is addressed. Special emphasis is laid on natural products for which efficacy and safety have already been proven and which are in clinical trials, as well as on plants used in traditional medicine. Potential benefits from certain dietary habits and dietary constituents, as well as common molecular targets of natural products, are also briefly discussed. A glimpse at the history of statins and biguanides, two prominent representatives of natural products (or their derivatives) in the fight against metabolic disease, is also included. The present review aims to serve as an "opening" of this special issue of Molecules, presenting key historical developments, recent advances, and future perspectives outlining the potential of natural products for prevention or therapy of cardiovascular and metabolic disease. Molecules 2016-06-22 21 6 Review 10.3390/molecules21060807 807 1420-3049 2016-06-22 doi: 10.3390/molecules21060807 Birgit Waltenberger Andrei Mocan Karel Šmejkal Elke Heiss Atanas Atanasov http://www.mdpi.com/1420-3049/21/6/819 Three new diterpenes, uprolide N (1), uprolide O (2), uprolide P (3) and a known one, dolabellane (4), were isolated from the CH2Cl2-MeOH extract of the gorgonian octocoral Eunicea succinea, collected from Bocas del Toro, on the Caribbean coast of Panama. Their structures were determined using spectroscopic analyses, including 1D and 2D NMR and high-resolution mass spectrometry (HRMS) together with molecular modeling studies. Compounds 1–3 displayed anti-inflammatory properties by inhibiting production of Tumor Necrosis Factor (TNF) and Interleukin (IL)-6 induced by lipopolysaccharide (LPS) in murine macrophages. Molecules 2016-06-22 21 6 Article 10.3390/molecules21060819 819 1420-3049 2016-06-22 doi: 10.3390/molecules21060819 Daniel Torres-Mendoza Yisett González José Gómez-Reyes Héctor Guzmán José López-Perez William Gerwick Patricia Fernandez Marcelino Gutiérrez http://www.mdpi.com/1422-0067/17/6/984 Reactive oxygen species (ROS) represent reactive products belonging to the partial reduction of oxygen. It has been reported that ROS are involved in different signaling pathways to control cellular stability. Under normal conditions, the correct function of redox systems leads to the prevention of cell oxidative damage. When ROS exceed the antioxidant defense system, cellular stress occurs. The cellular redox impairment is strictly related to tumorigenesis. Tumor cells, through the generation of hydrogen peroxide, tend to the alteration of cell cycle phases and, finally to cancer progression. In adults, the most common form of primary malignant brain tumors is represented by gliomas. The gliomagenesis is characterized by numerous molecular processes all characterized by an altered production of growth factor receptors. The difficulty to treat brain cancer depends on several biological mechanisms such as failure of drug delivery through the blood-brain barrier, tumor response to chemotherapy, and intrinsic resistance of tumor cells. Understanding the mechanisms of ROS action could allow the formulation of new therapeutic protocols to treat brain gliomas. International Journal of Molecular Sciences 2016-06-22 17 6 Review 10.3390/ijms17060984 984 1422-0067 2016-06-22 doi: 10.3390/ijms17060984 Mariagrazia Rinaldi Maria Caffo Letteria Minutoli Herbert Marini Rosaria Abbritti Francesco Squadrito Vincenzo Trichilo Andrea Valenti Valeria Barresi Domenica Altavilla Marcello Passalacqua Gerardo Caruso http://www.mdpi.com/1420-3049/21/6/808 A series of novel 5-substituted 2-(arylmethylthio)-4-chloro-N-(5-aryl-1,2,4-triazin-3-yl) benzenesulfonamide derivatives 27–60 have been synthesized by the reaction of aminoguanidines with an appropriate phenylglyoxal hydrate in glacial acetic acid. A majority of the compounds showed cytotoxic activity toward the human cancer cell lines HCT-116, HeLa and MCF-7, with IC50 values below 100 μM. It was found that for the analogues 36–38 the naphthyl moiety contributed significantly to the anticancer activity. Cytometric analysis of translocation of phosphatidylserine as well as mitochondrial membrane potential and cell cycle revealed that the most active compounds 37 (HCT-116 and HeLa) and 46 (MCF-7) inhibited the proliferation of cells by increasing the number of apoptotic cells. Apoptotic-like, dose dependent changes in morphology of cell lines were also noticed after treatment with 37 and 46. Moreover, triazines 37 and 46 induced caspase activity in the HCT-116, HeLa and MCF-7 cell lines. Selected compounds were tested for metabolic stability in the presence of pooled human liver microsomes and NADPH, both R2 and Ar = 4-CF3-C6H4 moiety in 2-(R2-methylthio)-N-(5-aryl-1,2,4-triazin-3-yl)benzenesulfonamides simultaneously increased metabolic stability. The results pointed to 37 as a hit compound with a good cytotoxicity against HCT-116 (IC50 = 36 μM), HeLa (IC50 = 34 μM) cell lines, apoptosis-inducing activity and moderate metabolic stability. Molecules 2016-06-22 21 6 Article 10.3390/molecules21060808 808 1420-3049 2016-06-22 doi: 10.3390/molecules21060808 Beata Żołnowska Jarosław Sławiński Aneta Pogorzelska Krzysztof Szafrański Anna Kawiak Grzegorz Stasiłojć Mariusz Belka Szymon Ulenberg Tomasz Bączek Jarosław Chojnacki http://www.mdpi.com/1420-3049/21/6/815 Aza-Michael reactions between primary aliphatic and aromatic amines and various Michael acceptors have been performed under environmentally-friendly solventless conditions using acidic alumina as a heterogeneous catalyst to selectively obtain the corresponding mono-adducts in high yields. Ethyl acrylate was the main acceptor used, although others such as acrylonitrile, methyl acrylate and acrylamide were also utilized successfully. Bi-functional amines also gave the mono-adducts in good to excellent yields. Such compounds can serve as intermediates for the synthesis of anti-cancer and antibiotic drugs. Molecules 2016-06-22 21 6 Article 10.3390/molecules21060815 815 1420-3049 2016-06-22 doi: 10.3390/molecules21060815 Giovanna Bosica Roderick Abdilla http://www.mdpi.com/1660-4601/13/6/624 Infant and neonatal mortality indicators are known to vary geographically, possibly as a result of socioeconomic and environmental inequalities. To better understand how these factors contribute to spatial and temporal patterns, we conducted a French ecological study comparing two time periods between 2002 and 2009 for three (purposefully distinct) Metropolitan Areas (MAs) and the city of Paris, using the French census block of parental residence as the geographic unit of analysis. We identified areas of excess risk and assessed the role of neighborhood deprivation and average nitrogen dioxide concentrations using generalized additive models to generate maps smoothed on longitude and latitude. Comparison of the two time periods indicated that statistically significant areas of elevated infant and neonatal mortality shifted northwards for the city of Paris, are present only in the earlier time period for Lille MA, only in the later time period for Lyon MA, and decrease over time for Marseille MA. These city-specific geographic patterns in neonatal and infant mortality are largely explained by socioeconomic and environmental inequalities. Spatial analysis can be a useful tool for understanding how risk factors contribute to disparities in health outcomes ranging from infant mortality to infectious disease—a leading cause of infant mortality. International Journal of Environmental Research and Public Health 2016-06-22 13 6 Article 10.3390/ijerph13060624 624 1660-4601 2016-06-22 doi: 10.3390/ijerph13060624 Cindy Padilla Wahida Kihal-Talantikit Verónica Vieira Séverine Deguen http://www.mdpi.com/1420-3049/21/6/818 Melandrii Herba (MH) is a traditional Asian medicinal herb used to treat breast cancer, anuria, and diseases of lactation. However, its biological properties and molecular mechanisms have not been fully elucidated. The purpose of this study was to investigate the anti-inflammatory activity and underlying molecular mechanism of MH ethanol extract (MHE) on the lipopolysaccharide (LPS)-mediated inflammatory response in macrophages. MHE cytotoxicity was determined using a cell counting kit (CCK) assay. The effects of MHE on the production of NO, inflammatory cytokines, and related proteins and mRNAs were determined using the Griess test, ELISA, Western blotting, and real-time RT-PCR, respectively. In addition, intracellular signaling pathways, such as NF-κB, MAPK, and HO-1, were analyzed using Western blotting. Our results revealed that MHE treatment significantly inhibited the secretion of NO and inflammatory cytokines, including TNF-α, IL-6, and IL-1β in macrophages, at sub-cytotoxic concentrations. Furthermore, MHE treatment inhibited iNOS expression and induced HO-1 expression. Finally, the transcriptional activities of NF-κB and MAPK activation were significantly suppressed by MHE in LPS-stimulated macrophages. The results indicate that MHE exerts anti-inflammatory effects by suppressing inflammatory mediator production via NF-κB and MAPK signaling pathways inhibition and induction of HO-1 expression in macrophages. Therefore, our results suggest the potential value of MHE as an inflammatory therapeutic agent developed from a natural substance. Molecules 2016-06-22 21 6 Article 10.3390/molecules21060818 818 1420-3049 2016-06-22 doi: 10.3390/molecules21060818 Yun Jeong You-Chang Oh Won-Kyung Cho Bohyoung Lee Jin Ma http://www.mdpi.com/1420-3049/21/6/817 The folate receptor (FR) is overexpressed in a wide variety of human tumors. In our study, the multimeric concept was used to synthesize a dimeric folate derivative via a click reaction. The novel folate derivative (HYNIC-D1-FA2) was radiolabeled with 99mTc using tricine and trisodium triphenylphosphine-3,3′,3″-trisulfonate (TPPTS) as coligands (99mTc-HYNIC-D1-FA2) and its in vitro physicochemical properties, ex vivo biodistribution and in vivo micro-SPECT/CT imaging as a potential FR targeted agent were evaluated. It is a hydrophilic compound (log P = −2.52 ± 0.13) with high binding affinity (IC50 = 19.06 nM). Biodistribution in KB tumor-bearing mice showed that 99mTc-HYNIC-D1-FA2 had high uptake in FR overexpressed tumor and kidney at all time-points, and both of them could obviously be inhibited when blocking with free FA in the blocking studies. From the in vivo micro-SPECT/CT imaging results, good tumor uptake of 99mTc-HYNIC-D1-FA2 was observed in KB tumor-bearing mice and it could be blocked obviously. Based on the results, this new radiolabeled dimeric FA tracer might be a promising candidate for FR-targeting imaging with high affinity and selectivity. Molecules 2016-06-22 21 6 Article 10.3390/molecules21060817 817 1420-3049 2016-06-22 doi: 10.3390/molecules21060817 Zhide Guo Mengna Gao Manli Song Changrong Shi Pu Zhang Duo Xu Linyi You Rongqiang Zhuang Xinhui Su Ting Liu Jin Du Xianzhong Zhang http://www.mdpi.com/1420-3049/21/6/816 Ultrahigh pressure (UHP) treatments are non-thermal processing methods that have customarily been employed to enhance the quality and productivity of plant consumables. We aimed to evaluate the effects of UHP treatments on ginseng samples (white ginseng: WG; UHP-treated WG: UWG; red ginseng: RG; UHP-treated RG: URG; ginseng berries: GB; and UHP-treated GB: UGB) using metabolite profiling based on ultrahigh performance liquid chromatography-linear trap quadrupole-ion trap-tandem mass spectrometry (UHPLC-LTQ-IT-MS/MS) and gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). Multivariate data analyses revealed a clear demarcation among the GB and UGB samples, and the phenotypic evaluations correlated the highest antioxidant activities and the total phenolic and flavonoid compositions with the UGB samples. Overall, eight amino acids, seven organic acids, seven sugars and sugar derivatives, two fatty acids, three notoginsenosides, three malonylginsenosides, and three ginsenosides, were identified as significantly discriminant metabolites between the GB and UGB samples, with relatively higher proportions in the latter. Ideally, these metabolites can be used as quality biomarkers for the assessment of ginseng products and our results indicate that UHP treatment likely led to an elevation in the proportions of total extractable metabolites in ginseng samples. Molecules 2016-06-22 21 6 Article 10.3390/molecules21060816 816 1420-3049 2016-06-22 doi: 10.3390/molecules21060816 Mee Lee Digar Singh Sung Kim Sang Lee Choong Lee