Plasticizers are additives that are used to increase the flexibility of plastic during manufacturing. However, in injection molding processes, plasticizers cannot be generated with monomers because they can peel off from the plastics into the surrounding environment, water, or food, or become attached to skin. Among the various plasticizers that are used, 1,2-benzenedicarboxylic acid (phthalic acid) is a typical precursor to generate phthalates. In addition, phthalic acid is a metabolite of diethylhexyl phthalate (DEHP). According to Gene_Ontology gene/protein database, phthalates can cause genital diseases, cardiotoxicity, hepatotoxicity, nephrotoxicity, etc. In this study, a silanized linker (3-aminopropyl triethoxyslane, APTES) was deposited on silicon dioxides (SiO₂) particles and phthalate chemical probes were manufactured from phthalic acid and APTES–SiO₂. These probes could be used for detecting proteins that targeted phthalic acid and for protein-protein interactions. The phthalic acid chemical probes we produced were incubated with epithelioid cell lysates of normal rat kidney (NRK-52E cells) to detect the interactions between phthalic acid and NRK-52E extracted proteins. These chemical probes interacted with a number of chaperones such as protein disulfide-isomerase A6, heat shock proteins, and Serpin H1. Ingenuity Pathways Analysis (IPA) software showed that these chemical probes were a practical technique for protein-protein interaction analysis. Full article

Nodal status is one of the most important prognostic factors in breast cancer. Established tests such as touch imprint cytology and frozen sections currently used in the intra-operative setting show variations in sensitivity and specificity. This limitation has led to the development of molecular alternatives, such as GeneSearch, a commercial intra-operative real-time quantitative Polymerase Chain Reaction (RT-qPCR) assay that allows the surgeon to carry out axillary clearance as a one-step process. Since GeneSearch has been discontinued, we have developed the replacement Metasin assay, which targets the breast epithelial cell markers CK19 and mammaglobin mRNA and identifies metastatic disease in sentinel lymph nodes. The optimised assay can be completed within 32 min (6 min for RNA preparation and 26 min instrument run time), making its use feasible in the intraoperative setting. An analysis by Metasin of 154 archived lymph node homogenates previously analysed by both parallel histology and GeneSearch showed concordance for 148 cases. The sensitivity and specificity of Metasin compared with GeneSearch were 95% (CI 83%–99%) and 97% (CI 91%–99%) respectively; compared with histology they were 95% (CI 83%–99%) and 97% (CI 91%–99%), respectively. The sensitivity and specificity of GeneSearch compared with histology were 90% (CI 77%–96%) and 97% (CI 93%–99%) respectively. The positive predictive value of Metasin was 90% and negative predictive value was 98% for both histology and GeneSearch. The positive predictive value of GeneSearch was 92% and the negative predictive value was 97% compared to histology. The discordance rates of Metasin with both GeneSearch and histology were 3.89%. In comparison, the discordance rate of GeneSearch with histology was 4.5%. Metasin’s robustness was independently evaluated on 193 samples previously analysed by GeneSearch from the Jules Bordet Institute, where Metasin yielded comparable results. Full article

We report on optimized architectures containing layer-by-layer (LbL) films of natural rubber latex (NRL), carboxymethyl-chitosan (CMC) and magnetite (Fe₃O₄) nanoparticles (MNPs) deposited on flexible substrates, which could be easily bent by an external magnetic field. The mechanical response depended on the number of deposited layers and was explained semi-quantitatively with a fully atomistic model, where the LbL film was represented as superposing layers of hexagonal graphene-like atomic arrangements deposited on a stiffer substrate. The bending with no direct current or voltage being applied to a supramolecular structure containing biocompatible and antimicrobial materials represents a proof-of-principle experiment that is promising for tissue engineering applications in biomedicine. Full article

Galactomannan (GM) is widely used for detection of invasive aspergillosis in high-risk haemato-oncology patients. Recent publications have reported a lack of repeatability of GM detection. The objective of this retrospective study was to assess the repeatability of GM levels during storage of clinical samples. In a GM screening strategy, positive sera were repeat tested as per manufacturer’s recommendations. Short-term (ST) storage of samples was at +4 °C while long-term (LT) storage was at −80 °C. Bronchoalveolar (BAL) fluid was also repeating tested after ST storage and LT storage. Wilcoxon Signed Ranks Test was employed to assess the repeatability of GM levels. In a subset of 14 GM positive sera, repeat testing was performed on both the original serum and ethylenediaminetetraacetic acid (EDTA) pre-treated sample. There was a significant reduction in GM signals on repeat testing following ST storage (median GM index: 0.65 vs. 0.19; p < 0.001) and LT storage (median GM index: 0.56 vs. 0.10; p < 0.001) of serum samples. Of samples that were initially GM positive, an average GM index reduction of 50% was seen, with approximately two-thirds becoming GM negative on repeat testing of the same sample. In contrast, GM signal loss was not seen on repeat testing of BAL fluid following ST or LT storage. When GM positive serum samples were repeat tested using EDTA pre-treated serum from the first step of the testing protocol, all samples remained GM positive. In contrast, when the same samples were repeat tested from the original collected serum, 9 samples (64%) became GM negative. The significant reduction in GM signals during ST and LT storage of serum samples has implications for clinical management. Although the reasons for GM decline are unknown, they occur prior to the EDTA pre-treatment stage, indicating that the time from phlebotomy to testing should be minimized. BAL fluid GM index values remain stable. Full article

Achillea millefolium L. is a member of the Asteraceae family and has been used in folk medicine in many countries. In this study, 19 compounds in A. millefolium essential oil (AM-EO) have been identified; the major components are artemisia ketone (14.92%), camphor (11.64%), linalyl acetate (11.51%) and 1,8-cineole (10.15%). AM-EO can suppress the inflammatory responses of lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages, including decreased levels of cellular nitric oxide (NO) and superoxide anion production, lipid peroxidation and glutathione (GSH) concentration. This antioxidant activity is not a result of increased superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, but rather occurs as a result of the down-regulation of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and heme oxygenase-1 (HO-1) expression, thus reducing the inflammatory response. Therefore, AM-EO can be utilized in many applications, including the treatment of inflammatory diseases in the future. Full article

Plant mitochondrial NAD-malic enzyme (NAD-ME), which is composed of α- and β-subunits in many species, participates in many plant biosynthetic pathways and in plant respiratory metabolism. However, little is known about the properties of woody plant NAD-MEs. In this study, we analyzed four NAD-ME genes (PtNAD-ME1 through PtNAD-ME4) in the genome of Populus trichocarpa. PtNAD-ME1 and -2 encode putative α-subunits, while PtNAD-ME3 and -4 encode putative β-subunits. The Populus NAD-MEs were expressed in Escherichia coli cells as GST-tagged fusion proteins. Each recombinant GST-PtNAD-ME protein was purified to near homogeneity by glutathione-Sepharose 4B affinity chromatography. Milligram quantities of each native protein were obtained from 1 L bacterial cultures after cleavage of the GST tag. Analysis of the enzymatic properties of these proteins in vitro indicated that α-NAD-MEs are more active than β-NAD-MEs and that α- and β-NAD-MEs presented different kinetic properties (V_max, k_cat and k_cat/K_m). The effect of different amounts of metabolites on the activities of Populus α- and β-NAD-MEs was assessed in vitro. While none of the metabolites evaluated in our assays activated Populus NAD-ME, oxalacetate and citrate inhibited all α- and β-NAD-MEs and glucose-6-P and fructose inhibited only the α-NAD-MEs. Full article

Mitofusin-2 (Mfn2) is a mitochondrial outer membrane protein involved in mitochondrial fusion. Its mutation can cause Charcot-Marie-Tooth disease. Recent studies of Mfn2 in cancer research have not included gastric cancer. We confirmed that Mfn2 expression was lower in tumor tissue than in normal gastric mucosal tissue and that it was negatively correlated with tumor size, indicating an anti-tumor role for Mfn2. In vitro experiments showed that Mfn2 overexpression suppressed gastric cancer cell proliferation and colony formation, weakened the invasion and migratory ability of cancer cells by downregulating MMP-2 and MMP-9, halted the cell cycle and induced apoptosis. Western blotting indicated the likely involvement of P21 and PI3K/Akt signaling. Therefore, Mfn2 is a potential anti-tumor gene and a potential therapeutic target for treating gastric cancer. The progress of gastric cancer may be delayed by controlling Mfn2 expression. Full article

The aptitude of cyclodextrins (CDs) to form host-guest complexes has prompted an increase in the development of new drug formulations. In this study, the inclusion complexes of pipemidic acid (HPPA), a therapeutic agent for urinary tract infections, with native β-CD were prepared in solid state by kneading method and confirmed by FT-IR and ¹H NMR. The inclusion complex formation was also characterized in aqueous solution at different pH via UV-Vis titration and phase solubility studies obtaining the stability constant. The 1:1 stoichiometry was established by a Job plot and the inclusion mechanism was clarified using docking experiments. Finally, the antibacterial activity of HPPA and its inclusion complex was tested on P. aeruginosa, E. coli and S. aureus to determine the respective EC_50s and EC_90s. The results showed that the antibacterial activity of HPPA:β-CD against E. coli and S. aureus is higher than that of HPPA. Furthermore, HPPA and HPPA:β-CD, tested on human hepatoblastoma HepG2 and MCF-7 cell lines by MTT assay, exhibited, for the first time, antitumor activities, and the complex revealed a higher activity than that of HPPA. The use of β-CD allows an increase in the aqueous solubility of the drug, its bioavailability and then its bioactivity. Full article

The accumulation of weakly basic drugs into acidic organelles has recently been described as a contributor to resistance in childhood cancer rhabdomyosarcoma (RMS) cell lines with differential sensitivity to a novel topoisomerase II inhibitor, AS-DACA. The current study aims to explore the contribution of the endocytic pathway to AS-DACA sequestration in RMS cell lines. A 24-fold differential in AS-DACA cytotoxicity was detected between the RMS lines RD and Rh30. The effect of inhibitors of the endocytic pathway on AS-DACA sensitivity in RMS cell lines, coupled with the variations of endosomal marker expression, indicated the late endosomal/lysosomal compartment was implicated by confounding lines of evidence. Higher expression levels of Lysosomal-Associated Membrane Protein-1 (LAMP1) in the resistant RMS cell line, RD, provided correlations between the increased amount and activity of these compartments to AS-DACA resistance. The late endosomal inhibitor 3-methyladenine increased AS-DACA sensitivity solely in RD leading to the reduction of AS-DACA in membrane trafficking organelles. Acidification inhibitors did not produce an increase in AS-DACA sensitivity nor reduce its sequestration, indicating that the pH partitioning of weakly basic drugs into acidic compartments does not likely contribute to the AS-DACA sequestering resistance mechanism evident in RMS cells. Full article

Four new labdane-type diterpenoids: hedychicoronarin (1), peroxycoronarin D (2), 7β-hydroxycalcaratarin A (3), and (E)-7β-hydroxy-6-oxo-labda-8(17),12-diene-15,16-dial (4), have been isolated from the rhizomes of Hedychium coronarium, together with 13 known compounds (5–17). The structures of these new compounds were determined through spectroscopic and MS analyses. Compounds 3, 5, 6, and 10 exhibited inhibition (IC₅₀ values ≤4.52 μg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 3–6, 10, and 11 inhibited fMLP/CB-induced elastase release with IC₅₀ values ≤6.17 μg/mL. Full article

The aim of the study was to investigate the protective effects of sodium hydrosulfide (NaHS), a H₂S donor, against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs) and also to look into the possible mechanisms by which H₂S exerts this protective effect. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and scratch wound healing assay were chosen to measure the cell viability and migration-promoting effects. The fluorescent probe, DCFH-DA and 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) were applied to detect the reactive oxygen species (ROS) level and mitochondrial membrane potential (ΔΨ_m). Furthermore, western blots were used to measure the expressions of the apoptosis-related proteins. Under hypoxic conditions, 300 μM and 600 μM of H₂S could protect HUVECs against hypoxia-induced injury, as determined by MTT assay. Following the treatment of 60 µM NaHS for 18 h, scratch wound healing assays indicated that the scratch became much narrower than control group. After treatment with 60 µM, 120 µM, and 600 µM NaHS, and hypoxia for 30 min, flow cytometry demonstrated that the ROS concentrations decreased to 95.08% ± 5.52%, 73.14% ± 3.36%, and 73.51% ± 3.05%, respectively, compared with the control group. In addition, the JC-1 assay showed NaHS had a protective effect on mitochondria damage. Additionally, NaHS increased Bcl-2 expression and decreased the expression of Bax, Caspase-3 and Caspase-9 in a dose-dependent way. Our results suggest that H₂S can protect endothelial cells and promote migration under hypoxic condition in HUVECs. These effects are partially associated with the preservation of mitochondrial function mediated by regulating the mitochondrial-dependent apoptotic pathway. Full article

The aim of our study was to evaluate the diagnosis of desmoplastic reaction (DR) by immunostaining for α-smooth muscle actin (αSMA) and desmin, for predicting the depth of submucosal invasion in biopsy specimens of early colorectal carcinomas (CRCs). Thirty-eight cases of non-pedunculated early CRCs were included in this study. Positive for DR was defined as αSMA-positive and desmin-negative stroma in the CRC. The depth of submucosal invasion was measured in endoscopically or surgically resected specimens and the lesions were subsequently divided into two groups: Group A (carcinoma in situ/intramucosal carcinoma and submucosal invasive carcinoma with a depth <1000 μm) and Group B (submucosal invasion with a depth ≥1000 μm). Twenty-one cases were DR-positive and 17 were DR-negative. No statistical significance was found between the DR with regard to tumor size, location and histological type. All DR-positive cases belonged to Group B whereas 14 (82.4%) DR-negative lesions belonged to Group A (p < 0.001). The sensitivity, specificity, positive and negative predictive values and accuracy of DR positivity for diagnosis of Group B were 87.5%, 100%, 100%, 82.4% and 92.1%, respectively. Conclusively, detection of DR in biopsy specimens with ancillary immunohistochemistry (αSMA/desmin) would help in preoperative diagnosis for the depth of submucosal invasion of early CRC. Full article

The innate immune response is the earliest cellular response to infectious agents and mediates the interactions between microbes and cells. Toll-like receptors (TLRs) play an important role in these interactions. We have already shown that TLRs are involved with the uptake of titanium dioxide nanoparticles (TiO₂ NPs) and promote inflammatory responses. In this paper, we compared role of cellular uptake and inflammatory response via TLR 4 to lipopolysaccharide (LPS) and TiO₂ NPs. In the case of LPS, LPS binds to LPS binding protein (LBP) and CD 14, and then this complex binds to TLR 4. In the case of TiO₂ NPs, the necessity of LBP and CD 14 to induce the inflammatory response and for uptake by cells was investigated using over-expression, antibody blocking, and siRNA knockdown experiments. Our results suggested that for cellular uptake of TiO₂ NPs, TLR 4 did not form a complex with LBP and CD 14. In the TiO₂ NP-mediated inflammatory response, TLR 4 acted as the signaling receptor without protein complex of LPS, LBP and CD 14. The results suggested that character of TiO₂ NPs might be similar to the complex of LPS, LBP and CD 14. These results are important for development of safer nanomaterials. Full article

The aim of the study was to assess the role of salicylic acid (SA) and abscisic acid (ABA) in osmotic stress tolerance of wheat seedlings. This was accomplished by determining the impact of the acids applied exogenously on seedlings grown under osmotic stress in hydroponics. The investigation was unique in its comprehensiveness, examining changes under osmotic stress and other conditions, and testing a number of parameters simultaneously. In both drought susceptible (SQ1) and drought resistant (CS) wheat cultivars, significant physiological and biochemical changes were observed upon the addition of SA (0.05 mM) or ABA (0.1 μM) to solutions containing half-strength Hoagland medium and PEG 6000 (−0.75 MPa). The most noticeable result of supplementing SA or ABA to the medium (PEG + SA and PEG + ABA) was a decrease in the length of leaves and roots in both cultivars. While PEG treatment reduced gas exchange parameters, chlorophyll content in CS, and osmotic potential, and conversely, increased lipid peroxidation, soluble carbohydrates in SQ1, proline content in both cultivars and total antioxidants activity in SQ1, PEG + SA or PEG + ABA did not change the values of these parameters. Furthermore, PEG caused a two-fold increase of endogenous ABA content in SQ1 and a four-fold increase in CS. PEG + ABA increased endogenous ABA only in SQ1, whereas PEG + SA caused a greater increase of ABA content in both cultivars compared to PEG. In PEG-treated plants growing until the harvest, a greater decrease of yield components was observed in SQ1 than in CS. PEG + SA, and particularly PEG + ABA, caused a greater increase of these yield parameters in CS compared to SQ1. In conclusion, SA and ABA ameliorate, particularly in the tolerant wheat cultivar, the harmful effects and after effects of osmotic stress induced by PEG in hydroponics through better osmotic adjustment achieved by an increase in proline and carbohydrate content as well as by an increase in antioxidant activity. Full article

It has been well established that in the oxidative folding of hen egg white lysozyme (HEL), which has four SS linkages in the native state (N), three des intermediates, i.e., des[76–94], des[64–80], and des [6–127], are populated at 20 °C and N is dominantly formed by the oxidation of des[64–80] and des[6–127]. To elucidate the temperature effects, the oxidative folding pathways of HEL were reinvestigated at 5–45 °C in the presence of 2 M urea at pH 8.0 by using a selenoxide reagent, DHS^ox. When reduced HEL was reacted with 1–4 equivalents of DHS^ox, 1S, 2S, 3S, and 4S intermediate ensembles with 1–4 SS linkages, respectively, were produced within 1 min. After the oxidation, 3S was slowly converted to the des intermediates with formation of the native structures through SS rearrangement. At 5 °C, des[76–94] was populated in the largest amount, but the oxidation to N was slower than that of des[64–80] and des[6–127]. At 35 °C, on the other hand, des[64–80] and des[6–127] were no longer stable, and only des[76–94] was populated. The results suggested that the major folding pathways of HEL can be switched from one to the other by temperature control. Full article

The cell type-, organ-, and species-specific expression of the Toll-like receptors (TLRs) are well described, but little is known about the respective expression profiles of their accessory molecules. We therefore determined the mRNA expression levels of LBP, MD2, CD36, CD14, granulin, HMGB1, LL37, GRP94, UNC93b1, TRIL, PRAT4A, AP3B1, AEP and the respective TLRs in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. In addition, the expression profiles in transient tissue inflammation upon renal ischemia-reperfusion injury, in spleens and kidneys from mice with lupus-like systemic autoimmunity, and in progressive tissue fibrosis upon unilateral ureteral obstruction were studied. Several TLR co-factors were specifically regulated during the different phases of these disease entities, suggesting a functional involvement in the disease process. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to TLR-mediated innate immunity, which seems to be involved in the tissue injury phase, in the phase of tissue regeneration, and in progressive tissue remodelling. Full article

During microRNA (miRNA) biogenesis, one strand of a 21–23 nucleotide RNA duplex is preferentially selected for entry into an RNA-induced silencing complex (RISC). The other strand, known as the miRNA* species, is typically thought to be degraded. Previous studies have provided miRNA* selection models, but it remains unclear how the dominance of one arm arises during the biogenesis of miRNA. Using miRNA sponge-like methods, we cloned four tandem target sequences (artificial target) of miR-7b* and then measured miR-7b* expression levels after transfection of the artificial target. miR-7b* levels were found to significantly increase after transfection of the artificial target. We postulate that the abundance of target transcripts drives miRNA arm selection. Full article

Membrane anchorage was tested as a strategy to accumulate recombinant proteins in transgenic plants. Transmembrane domains of different lengths and topology were fused to the cytosolic HIV antigen p24, to promote endoplasmic reticulum (ER) residence or traffic to distal compartments of the secretory pathway in transgenic tobacco. Fusions to a domain of the maize seed storage protein γ-zein were also expressed, as a reference strategy that leads to very high stability via the formation of large polymers in the ER lumen. Although all the membrane anchored constructs were less stable compared to the zein fusions, residence at the ER membrane either as a type I fusion (where the p24 sequence is luminal) or a tail-anchored fusion (where the p24 sequence is cytosolic) resulted in much higher stability than delivery to the plasma membrane or intermediate traffic compartments. Delivery to the tonoplast was never observed. The inclusion of a thrombin cleavage site allowed for the quantitative in vitro recovery of p24 from all constructs. These results point to the ER as suitable compartment for the accumulation of membrane-anchored recombinant proteins in plants. Full article

Recent studies have shown that cancer stem-like cells (CSCs) within a tumor have the capacity for self-renewal and differentiation, and are associated with an aggressive phenotype and therapeutic resistance. Studies have also associated tumor progression with alterations in the levels of intracellular reactive oxygen species (ROS). In this study, we cultured nasopharyngeal carcinoma (NPC) CSCs in conditions that allowed sphere formation. The resulting sphere cells displayed stemness properties, characteristics of the epithelial–mesenchymal transition (EMT), and increased expression of the CSC surface marker CD44. We further evaluated the association between CD44 expression and EMT marker expression, and any correlation with redox status, in these CSCs. We showed that the EMT in sphere cells is associated with the upregulation of CD44 expression and increased ROS generation, which might promote NPC aggressiveness. We also identified the coexpression of CD44 with the EMT marker N-cadherin in sphere cells, and downregulated CD44 expression after the addition of the antioxidant N-acetyl cysteine. Our results indicate that CD44 plays a role in the EMT phenotype of CSCs in NPC, and suggest its involvement in EMT-associated ROS production. These findings might facilitate the development of a novel therapy for the prevention of NPC recurrence and metastasis. Full article

Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to distant organs. Bradykinin (BK) is an inflammatory mediator and has recently been shown to mediate tumor growth and metastasis. The adhesion molecule intercellular adhesion molecule-1 (ICAM-1) plays a critical role during tumor metastasis. The aim of this study was to examine whether BK promotes prostate cancer cell migration via ICAM-1 expression. The motility of cancer cells was increased following BK treatment. Stimulation of prostate cancer cells with BK induced mRNA and protein expression of ICAM-1. Transfection of cells with ICAM-1 small interfering RNA reduced BK-increased cell migration. Pretreatment of prostate cancer cells with B2 receptor, phosphatidylinositol 3-kinase (PI3K), Akt, and activator protein 1 (AP-1) inhibitors or mutants abolished BK-promoted migration and ICAM-1 expression. In addition, treatment with a B2 receptor, PI3K, or Akt inhibitor also reduced BK-mediated AP-1 activation. Our results indicate that BK enhances the migration of prostate cancer cells by increasing ICAM-1 expression through a signal transduction pathway that involves the B2 receptor, PI3K, Akt, and AP-1. Thus, BK represents a promising new target for treating prostate cancer metastasis. Full article

Radish floral bud abortion (FBA) is an adverse biological phenomenon that occurs during reproduction. Although FBA is a frequent occurrence, its molecular mechanism remains unknown. A transcript-derived fragment (TDF72), which was obtained by cDNA amplified fragment length polymorphism (cDNA-AFLP), was up-regulated in the aborted buds and exhibited 89% sequence homology with the AtγVPE gene. In this study, TDF72 was used to clarify the role of VPE in FBA by isolation of the VPE gene RsVPE1 from radish flower buds. The full-length genomic DNA was 2346 bp including nine exons and eight introns. The full-length cDNA was 1825 bp, containing a complete open reading frame (ORF) of 1470 bp, which encoded a predicted protein containing 489 amino acid residues, with a calculated molecular mass of 53.735 kDa. Expression analysis demonstrated that RsVPE1 was expressed in all tested organs of radish at different levels. Highest expression was detected in aborted flower buds, suggesting that RsVPE1 has a role in FBA. In order to analyze the role of RsVPE1 in FBA, RsVPE1 was overexpressed in transgenic Arabidopsis thaliana plants. Aborted flower buds appeared in transgenic plants subjected to heat stress. In addition, RsVPE1 expression in the transgenic plants reached a maximum when subjected to heat stress for 24 h and increased by 2.1-fold to 2.8-fold in three homozygous transgenic lines. These results indicated that RsVPE1 led to FBA when its expression levels exceeded a particular threshold, and provided evidence for the involvement of RsVPE1 in promoting FBA under heat stress. Full article

We propose a kinetic model for the activation of the las regulon in the opportunistic pathogen Pseudomonas aeruginosa. The model is based on in vitro data and accounts for the LasR dimerization and consecutive activation by binding of two OdDHL signal molecules. Experimentally, the production of the active LasR quorum-sensing regulator was studied in an Escherichia coli background as a function of signal molecule concentration. The functional activity of the regulator was monitored via a GFP reporter fusion to lasB expressed from the native lasB promoter. The new data shows that the active form of the LasR dimer binds two signal molecules cooperatively and that the timescale for reaching saturation is independent of the signal molecule concentration. This favors a picture where the dimerized regulator is protected against proteases and remains protected as it is activated through binding of two successive signal molecules. In absence of signal molecules, the dimerized regulator can dissociate and degrade through proteolytic turnover of the monomer. This resolves the apparent contradiction between our data and recent reports that the fully protected dimer is able to “degrade” when the induction of LasR ceases. Full article

Adhesion molecules are known to play an important role in endothelial activation and angiogenesis. Here we determined the functional role of IQGAP1 in the regulation of endothelial adherens junctions. VE-cadherin is found to be associated with actin filaments and thus stable, but IQGAP1 at intercellular junctions is not bound to actin filaments and thus labile. Expression of GFP labeled VE-α-catenin is shown to increase the electrical resistance across HUVEC monolayers and diminishes endogenous labile IQGAP1 at the intercellular junctions. Knockdown of endogenous IQGAP1 enhances intercellular adhesion in HUVECs by increasing the association of VE-cadherin with P120 and β-catenin. IQGAP1 knockdown also decreases the interaction of N-cadherin with P120 and β-catenin. Together, these results suggest that a labile pool of IQGAP1 at intercellular junctions disassembles adherens junctions and thus impairs endothelial cell-cell adhesion. Full article

Magnetic particle mediated transport in combination with nanomaterial based drug carrier has a great potential for targeted cancer therapy. In this study, doxorubicin encapsulation into the apoferritin and its conjugation with magnetic particles was investigated by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The quantification of encapsulated doxorubicin was performed by fluorescence spectroscopy and compared to CE-LIF. Moreover, the significant enhancement of the doxorubicin signal was observed by addition of methanol into the sample solution. Full article

Abscisic acid plays a pivotal role in the abiotic stress response in plants. Although great progress has been achieved explaining the complexity of the stress and ABA signaling cascade, there are still many questions to answer. Mutants are a valuable tool in the identification of new genes or new alleles of already known genes and in elucidating their role in signaling pathways. We applied a suppressor mutation approach in order to find new components of ABA and abiotic stress signaling in Arabidopsis. Using the abh1 (ABA hypersensitive 1) insertional mutant as a parental line for EMS mutagenesis, we selected several mutants with suppressed hypersensitivity to ABA during seed germination. Here, we present the response to ABA and a wide range of abiotic stresses during the seed germination and young seedling development of two suppressor mutants—soa2 (suppressor of abh1 hypersensitivity to ABA 2) and soa3 (suppressor of abh1 hypersensitivity to ABA 3). Generally, both mutants displayed a suppression of the hypersensitivity of abh1 to ABA, NaCl and mannitol during germination. Both mutants showed a higher level of tolerance than Columbia-0 (Col-0—the parental line of abh1) in high concentrations of glucose. Additionally, soa2 exhibited better root growth than Col-0 in the presence of high ABA concentrations. soa2 and soa3 were drought tolerant and both had about 50% fewer stomata per mm² than the wild-type but the same number as their parental line—abh1. Taking into account that suppressor mutants had the same genetic background as their parental line—abh1, it was necessary to backcross abh1 with Landsberg erecta four times for the map-based cloning approach. Mapping populations, derived from the cross of abh1 in the Landsberg erecta background with each suppressor mutant, were created. Map based cloning in order to identify the suppressor genes is in progress. Full article

c-Jun NH₂-terminal kinase (JNK) signaling is a highly conserved pathway that controls gene transcription in response to a wide variety of biological and environmental stresses. In this study, a JNK from the invasive Mediterranean (MED) species of the whitefly Bemisia tabaci complex was cloned and characterized. The full-length JNK cDNA of MED consists of 1565 bp, with an 1176 bp open reading frame encoding 392 amino acids. Comparison of JNK amino acid sequences among different species showed that the sequences of JNKs are highly conserved. To reveal its biological function, the gene expression and functional activation of JNK were analyzed during various stress conditions. Quantitative RT-PCR analysis showed that the relative expression level of JNK remained hardly unchanged when the insects were transferred from cotton (a suitable host plant) to tobacco (an unsuitable host plant), infected with bacteria and treated with high and low temperatures. However, the mRNA level of JNK significantly increased when treated with fungal pathogens. Furthermore, we found that the amount of phosphorylated JNK increased significantly after fungal infection, while there is no obvious change for phosphorylated p38 and ERK. Our results indicate that the whitefly JNK plays an important role in whitefly’s immune responses to fungal infection. Full article

Heparin has been conjugated to Fe₃O₄, Co₃O₄, and NiO nanoparticles (NPs) through electrostatic interactions, producing colloidal suspensions of hybrid metal oxide heparin NPs that are stable in water. Negative zeta potentials and retention of heparin’s ability to capture toluidine blue indicate that heparin’s negative charges are exposed on the surface of the coated NPs. IR results confirmed the formation of nanohybrids as did NMR experiments, which were also interpreted on the basis of toluidine blue tests. Transmission electron microscopy results revealed that the heparin coating does not modify the shape or dimension of the NPs. Dynamic light scattering and negative zeta potential measurements confirmed that heparin surface functionalisation is an effective strategy to prevent NP aggregation. Full article

Over the past few years, it has become evident that the distinctive pattern of miRNA expression seen in embryonic stem cells (ESCs) contributes to important signals in the choice of the cell fate. Thus, the identification of miRNAs and their targets, whose expression is linked to a specific step of differentiation, as well as the modulation of these miRNAs, may prove useful in the learning of how ESC potential is regulated. In this context, we have studied the expression profile of miRNAs during neural differentiation of ESCs. We have found that miR-125b is upregulated in the first steps of neural differentiation of ESCs. This miRNA targets the BMP4 co-receptor, Dies1, and, in turn, regulates the balance between BMP4 and Nodal/Activin signaling. The ectopic expression of miR-125b blocks ESC differentiation at the epiblast stage, and this arrest is rescued by restoring the expression of Dies1. Finally, opposite to miR-125a, whose expression is under the control of the BMP4, miR-125b is not directly regulated by Transforming Growth Factor beta (TGFβ) signals. These results highlight a new important role of miR-125b in the regulation of the transition from ESCs to the epiblast stage and add a new level of control on TGFβ signaling in ESCs. Full article

Electrochemical detection of quantum dots (QDs) has already been used in numerous applications. However, QDs have not been well characterized using voltammetry, with respect to their characterization and quantification. Therefore, the main aim was to characterize CdTe QDs using cyclic and differential pulse voltammetry. The obtained peaks were identified and the detection limit (3 S/N) was estimated down to 100 fg/mL. Based on the convincing results, a new method for how to study stability and quantify the dots was suggested. Thus, the approach was further utilized for the testing of QDs stability. Full article

Pokemon is an important proto-oncogene involved in various biological processes and cancer development, such as cell differentiation, tumorigenesis and metastasis. Pokemon is recognized as a transcription factor localized upstream of several oncogenes, regulating their expression. p38MAPKs act as key regulatory factors in cellular signaling pathways associated with inflammatory responses, cell proliferation, differentiation and survival. p38β, a member of p38MAPK family, is closely correlated with tumorigenesis, but the mechanism of activation remains unclear. In this study, we found overexpression of Pokemon promoted the growth, migration and invasion of HepG2 cells. However, a p38 inhibitor SB202190 efficiently attenuated the promoting effect of Pokemon in the HepG2 cells. Targeted expression or silencing of Pokemon changed cellular p38β protein level and phosphorylation of downstream ATF2 in the p38 signaling pathway. Both dual luciferase report assay and ChIP assay suggested that p38β is a novel regulatory target of the transcription factor Pokemon and positively regulated by Pokemon in hepatic cells. Full article

Epimedium sagittatum (Sieb. et Zucc) Maxim is a member of the Berberidaceae family of basal eudicot plants, widely distributed and used as a traditional medicinal plant in China for therapeutic effects on many diseases with a long history. Recent data shows that E. sagittatum has a relatively large genome, with a haploid genome size of ~4496 Mbp, divided into a small number of only 12 diploid chromosomes (2n = 2x = 12). However, little is known about Epimedium genome structure and composition. Here we present the analysis of 691 kb of high-quality genomic sequence derived from 672 randomly selected plasmid clones of E. sagittatum genomic DNA, representing ~0.0154% of the genome. The sampled sequences comprised at least 78.41% repetitive DNA elements and 2.51% confirmed annotated gene sequences, with a total GC% content of 39%. Retrotransposons represented the major class of transposable element (TE) repeats identified (65.37% of all TE repeats), particularly LTR (Long Terminal Repeat) retrotransposons (52.27% of all TE repeats). Chromosome analysis and Fluorescence in situ Hybridization of Gypsy-Ty3 retrotransposons were performed to survey the E. sagittatum genome at the cytological level. Our data provide the first insights into the composition and structure of the E. sagittatum genome, and will facilitate the functional genomic analysis of this valuable medicinal plant. Full article

Tumor metastasis is the main cause of lethality of prostate cancer, because conventional therapies like surgery and hormone treatment rarely work at this stage. Tumor cell migration, invasion and adhesion are necessary processes for metastasis. By providing nutrition and an escape route from the primary site, angiogenesis is also required for tumor metastasis. Phosphatidylinositol 3-kinases (PI3Ks) are well known to play important roles in tumorigenesis as well as metastasis. ZSTK474 is a specific PI3K inhibitor developed for solid tumor therapy. In the present report, antimetastatic activities of ZSTK474 were investigated in vitro by determining the effects on the main metastatic processes. ZSTK474 exhibited inhibitory effects on migration, invasion and adhesive ability of prostate cancer PC3 cells. Furthermore, ZSTK474 inhibited phosphorylation of Akt substrate-Girdin, and the secretion of matrix metalloproteinase (MMP), both of which were reported to be closely involved in migration and invasion. On the other hand, ZSTK474 inhibited the expression of HIF-1α and the secretion of vascular endothelial growth factor (VEGF), suggesting its potential antiangiogenic activity on PC3 cells. Moreover, we demonstrated the antiangiogenesis by determining the effect of ZSTK474-reduced VEGF on tube formation of human umbilical vein endothelial cells (HUVECs). In conclusion, ZSTK474 was demonstrated to have potential in vitro antimetastatic effects on PC3 cells via dual mechanisms: inhibition of metastatic processes including cell migration, invasion and adhesion, and antiangiogenesis via blockade of VEGF secretion. Full article

Polymers are currently widely used to replace a variety of natural materials with respect to their favourable physical and chemical properties, and due to their economic advantage. One of the most important branches of application of polymers is the production of different products for medical use. In this case, it is necessary to face a significant disadvantage of polymer products due to possible and very common colonization of the surface by various microorganisms that can pose a potential danger to the patient. One of the possible solutions is to prepare polymer with antibacterial/antimicrobial properties that is resistant to bacterial colonization. The aim of this study was to contribute to the development of antimicrobial polymeric material ideal for covering vascular implants with subsequent use in transplant surgery. Therefore, the complexes of polymeric substances (hyaluronic acid and chitosan) with silver nitrate or silver phosphate nanoparticles were created, and their effects on gram-positive bacterial culture of Staphylococcus aureus were monitored. Stages of formation of complexes of silver nitrate and silver phosphate nanoparticles with polymeric compounds were characterized using electrochemical and spectrophotometric methods. Furthermore, the antimicrobial activity of complexes was determined using the methods of determination of growth curves and zones of inhibition. The results of this study revealed that the complex of chitosan, with silver phosphate nanoparticles, was the most suitable in order to have an antibacterial effect on bacterial culture of Staphylococcus aureus. Formation of this complex was under way at low concentrations of chitosan. The results of electrochemical determination corresponded with the results of spectrophotometric methods and verified good interaction and formation of the complex. The complex has an outstanding antibacterial effect and this effect was of several orders higher compared to other investigated complexes. Full article

In this work, we studied the antimicrobial properties of a nanocomposite system based on a lactose-substituted chitosan and silver nanoparticles: Chitlac-nAg. Twofold serial dilutions of the colloidal Chitlac-nAg solution were both tested on Streptococcus mitis, Streptococcus mutans, and Streptococcus oralis planktonic phase and biofilm growth mode as well as on saliva samples. The minimum inhibitory and bactericidal concentrations of Chitlac-nAg were evaluated together with its effect on sessile cell viability, as well as both on biofilm formation and on preformed biofilm. In respect to the planktonic bacteria, Chitlac-nAg showed an inhibitory/bactericidal effect against all streptococcal strains at 0.1% (v/v), except for S. mitis ATCC 6249 that was inhibited at one step less. On preformed biofilm, Chitlac-nAg at a value of 0.2%, was able to inhibit the bacterial growth on the supernatant phase as well as on the mature biofilm. For S. mitis ATCC 6249, the biofilm inhibitory concentration of Chitlac-nAg was 0.1%. At sub-inhibitory concentrations, the Streptococcal strains adhesion capability on a polystyrene surface showed a general reduction following a concentration-dependent-way; a similar effect was obtained for the metabolic biofilm activity. From these results, Chitlac-nAg seems to be a promising antibacterial and antibiofilm agent able to hinder plaque formation. Full article

Phenylalanine ammonia-lyase (PAL), Cinnamic acid 4-hydroxylase (C4H) and 4-Coumarate: CoA ligase (4CL) catalyze the first three steps of the general phenylpropanoid pathway whereas chalcone synthase (CHS) catalyzes the first specific step towards flavonoids production. This class of specialized metabolites has a wide range of biological functions in plant development and defence and a broad spectrum of therapeutic activities for human health. In this study, we report the isolation of hemp PAL and 4CL cDNA and genomic clones. Through in silico analysis of their deduced amino acid sequences, more than an 80% identity with homologues genes of other plants was shown and phylogenetic relationships were highlighted. Quantitative expression analysis of the four above mentioned genes, PAL and 4CL enzymatic activities, lignin content and NMR metabolite fingerprinting in different Cannabis sativa tissues were evaluated. Furthermore, the use of different substrates to assay PAL and 4CL enzymatic activities indicated that different isoforms were active in different tissues. The diversity in secondary metabolites content observed in leaves (mainly flavonoids) and roots (mainly lignin) was discussed in relation to gene expression and enzymatic activities data. Full article

Auxin has pleiotropic effects on plant growth and development. AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins are short-lived transcriptional regulators that mediate auxin responses through interaction with an auxin receptor, the F-box protein transport inhibitor response 1 (TIR1). Most functions of Aux/IAA proteins have been identified in Arabidopsis by studying the gain-of-function mutants in domain II. In this study, we isolated and identified an Aux/IAA protein gene from rice, OsIAA4, whose protein contains a dominant mutation-type domain II. OsIAA4 has very low expression in the entire life cycle of rice. OsIAA4-overexpressing rice plants show dwarfism, increased tiller angles, reduced gravity response, and are less sensitive to synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Full article

Repair of parenchyma by stem/progenitor cells is seen as a possible alternative to cure acute and chronic renal failure in future. To learn about this therapeutic purpose, the formation of nephrons during organ growth is under focus of present research. This process is triggered by numerous morphogenetic interactions between epithelial and mesenchymal cells within the renal stem/progenitor cell niche. Recent data demonstrate that an astonishingly wide interstitial interface separates both types of stem/progenitor cells probably controlling coordinated cell-to-cell communication. Since conventional fixation by glutaraldehyde (GA) does not declare in transmission electron microscopy the spatial separation, improved contrasting procedures were applied. As a consequence, the embryonic cortex of neonatal rabbit kidneys was fixed in solutions containing glutaraldehyde in combination with cupromeronic blue, ruthenium red or tannic acid. To obtain a comparable view to the renal stem/progenitor cell niche, the specimens had to be orientated along the cortico-medullary axis of lining collecting ducts. Analysis of tissue samples fixed with GA, in combination with cupromeronic blue, demonstrates demasked extracellular matrix. Numerous braces of proteoglycans cover, as well, the basal lamina of epithelial stem/progenitor cells as projections of mesenchymal stem/progenitor cells crossing the interstitial interface. Fixation with GA containing ruthenium red or tannic acid illustrates strands of extracellular matrix that originate from the basal lamina of epithelial stem/progenitor cells and line through the interstitial interface. Thus, for the first time, improved contrasting techniques make it possible to analyze in detail a microheterogeneous composition of the interstitial interface within the renal stem/progenitor cell niche. Full article

Folate receptor alpha (FOLR1/FRA) is reported to be overexpressed in epithelial ovarian cancers (EOC), especially the serous histotype. Further, while dysregulation of the folate-dependent 1-carbon cycle has been implicated in tumorogenesis, little is known relative to the potential mechanism of action of FOLR1 expression in these processes. We therefore investigated the expression of FOLR1, other folate receptors, and genes within the 1-carbon cycle in samples of EOC, normal ovary and fallopian tube on a custom TaqMan Low Density Array. Also included on this array were known markers of EOC such as MSLN, MUC16 and HE4. While few differences were observed in the expression profiles of genes in the 1-carbon cycle, genes previously considered to be overexpressed in EOC (e.g., FOLR1, MSLN, MUC16 and HE4) showed significantly increased expression when comparing EOC to normal ovary. However, when the comparator was changed to normal fallopian tube, these differences were abolished, supporting the hypothesis that EOC derives from fallopian fimbriae and, further, that markers previously considered to be upregulated or overexpressed in EOC are most likely not of ovarian origin, but fallopian in derivation. Our findings therefore support the hypothesis that the cell of origin of EOC is tubal epithelium. Full article

A comparative investigation of the induction of double-strand DNA breaks (DSBs) in the Chinese hamster V79 cells by γ-radiation at dose rates of 1, 10 and 400 mGy/min (doses ranged from 0.36 to 4.32 Gy) was performed. The acute radiation exposure at a dose rate of 400 mGy/min resulted in the linear dose-dependent increase of the γ-H2AX foci formation. The dose-response curve for the acute exposure was well described by a linear function y = 1.22 + 19.7x, where “y” is an average number of γ-H2AX foci per a cell and “x” is the absorbed dose (Gy). The dose rate reduction down to 10 mGy/min lead to a decreased number of γ-H2AX foci, as well as to a change of the dose-response relationship. Thus, the foci number up to 1.44 Gy increased and reached the “plateau” area between 1.44 and 4.32 Gy. There was only a slight increase of the γ-H2AX foci number (up to 7) in cells after the protracted exposure (up to 72 h) to ionizing radiation at a dose rate of 1 mGy/min. Similar effects of the varying dose rates were obtained when DNA damage was assessed using the comet assay. In general, our results show that the reduction of the radiation dose rate resulted in a significant decrease of DSBs per cell per an absorbed dose. Full article

Members of the Burkholderia genus of Proteobacteria are capable of living freely in the environment and can also colonize human, animal and plant hosts. Certain members are considered to be clinically important from both medical and veterinary perspectives and furthermore may be important modulators of the rhizosphere. Quorum sensing via N-acyl homoserine lactone signals (AHL QS) is present in almost all Burkholderia species and is thought to play important roles in lifestyle changes such as colonization and niche invasion. Here we present a census of AHL QS genes retrieved from public databases and indicate that the local arrangement (topology) of QS genes, their location within chromosomes and their gene neighborhoods show characteristic patterns that differ between the known Burkholderia clades. In sequence phylogenies, AHL QS genes seem to cluster according to the local gene topology rather than according to the species, which suggests that the basic topology types were present prior to the appearance of current Burkholderia species. The data are available at http://net.icgeb.org/burkholderia/. Full article

In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2%) and 5 (7.4%) of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2%) out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications. Full article

Calorie restriction (CR) inhibits prostate cancer progression, partially through modulation of the IGF axis. IGF-1 receptor (IGF-1R) blockade reduces prostate cancer xenograft growth. We hypothesized that combining calorie restriction with IGF-1R blockade would have an additive effect on prostate cancer growth. Severe combined immunodeficient mice were subcutaneously injected with 22Rv1 cells and randomized to: (1) Ad libitum feeding/intraperitoneal saline (Ad-lib); (2) Ad-lib/20 mg/kg twice weekly, intraperitoneal ganitumab [anti-IGF-1R antibody (Ad-lib/Ab)]; (3) 40% calorie restriction/intraperitoneal saline (CR); (4) CR/ intraperitoneal ganitumab, (CR/Ab). CR and ganitumab treatment were initiated one week after tumor injection. Euthanasia occurred 19 days post treatment. Results showed that CR alone decreased final tumor weight, plasma insulin and IGF-1 levels, and increased apoptosis. Ganitumab therapy alone reduced tumor growth but had no effect on final tumor weight. The combination therapy (CR/Ab) further decreased final tumor weight and proliferation, increased apoptosis in comparison to the Ad-lib group, and lowered plasma insulin levels relative to the Ad-lib and Ad-lib/Ab groups. Tumor AKT activation directly correlated with plasma IGF-1 levels. In conclusion, whereas ganitumab therapy modestly affected 22Rv1 tumor growth, combining IGF-1R blockade with calorie restriction resulted in a significant decrease in final tumor weight and improved metabolic profile. Full article

Changes in flower morphology may influence the frequency and specificity of animal visitors. In Petunia (Solanaceae), adaptation to different pollinators is one of the factors leading to species diversification within the genus. This study provides evidence that differential expression patterns of MAWEWEST (MAW) homologs in different Petunia species may be associated with adaptive changes in floral morphology. The Petunia × hybrida MAW gene belongs to the WOX (WUSCHEL-related homeobox) transcription factor family and has been identified as a controller of petal fusion during corolla formation. We analyzed the expression patterns of P. inflata and P. axillaris MAW orthologs (PiMAW and PaMAW, respectively) by reverse transcriptase polymerase chain reaction (RT-PCR), reverse transcription–quantitative PCR (qRT-PCR) and in situ hybridization in different tissues and different developmental stages of flowers in both species. The spatial expression patterns of PiMAW and PaMAW were similar in P. inflata and P. axillaris. Nevertheless, PaMAW expression level in P. axillaris was higher during the late bud development stage as compared to PiMAW in P. inflata. This work represents an expansion of petunia developmental research to wild accessions. Full article

The reverse osmosis (RO) brine generated from the Al-Quds University wastewater treatment plant was treated using an epuvalisation system. The advanced integrated wastewater treatment plant included an activated sludge unit, two consecutive ultrafiltration (UF) membrane filters (20 kD and 100 kD cutoffs) followed by an activated carbon filter and a reverse osmosis membrane. The epuvalisation system consisted of salt tolerant plants grown in hydroponic channels under continuous water flowing in a closed loop system, and placed in a greenhouse at Al-Quds University. Sweet basil (Ocimum basilicum) plants were selected, and underwent two consecutive hydroponic flowing stages using different brine-concentrations: an adaptation stage, in which a 1:1 mixture of brine and fresh water was used; followed by a functioning stage, with 100% brine. A control treatment using fresh water was included as well. The experiment started in April and ended in June (2012). At the end of the experiment, analysis of the effluent brine showed a remarkable decrease of electroconductivity (EC), PO₄³⁻, chemical oxygen demand (COD) and K⁺ with a reduction of 60%, 74%, 70%, and 60%, respectively, as compared to the influent. The effluent of the control treatment showed 50%, 63%, 46%, and 90% reduction for the same parameters as compared to the influent. Plant growth parameters (plant height, fresh and dry weight) showed no significant difference between fresh water and brine treatments. Obtained results suggest that the epuvalisation system is a promising technique for inland brine treatment with added benefits. The increasing of channel number or closed loop time is estimated for enhancing the treatment process and increasing the nutrient uptake. Nevertheless, the epuvalisation technique is considered to be simple, efficient and low cost for inland RO brine treatment. Full article

Fatty acid-binding protein 3 (FABP3) is a member of the intracellular lipid-binding protein family, and is primarily expressed in cardiac muscle tissue. Previously, we found that FABP3 is highly expressed in patients with ventricular-septal defects and is often used as a plasma biomarker in idiopathic dilated cardiomyopathy, and may play a significant role in the development of these defects in humans. In the present study, we aimed to investigate the role of FABP3 in the embryonic development of the zebrafish heart, and specifically how morpholino (MO) mediated knockdown of FABP3 would affect heart development in this species. Our results revealed that knockdown of FABP3 caused significant impairment of cardiac development observed, including developmental delay, pericardial edema, a linear heart tube phenotype, incomplete cardiac loop formation, abnormal positioning of the ventricles and atria, downregulated expression of cardiac-specific markers and decreased heart rate. Mechanistically, our data showed that the retinoic acid (RA) catabolizing enzyme Cyp26a1 was upregulated in FABP3-MO zebrafish, as indicated by in situ hybridization and real-time PCR. On the other hand, the expression level of the RA synthesizing enzyme Raldh2 did not significantly change in FABP3-MO injected zebrafish. Collectively, our results indicated that FABP3 knockdown had significant effects on cardiac development, and that dysregulated RA signaling was one of the mechanisms underlying this effect. As a result, these studies identify FABP3 as a candidate gene underlying the etiology of congenital heart defects. Full article

The objective of these investigations was to determine the possible effects of the novel selective estrogen receptor modulator, ospemifene, on cytochrome P450 (CYP)-mediated drug metabolism. Ospemifene underwent testing for possible effects on CYP enzyme activity in human liver microsomes and in isolated human hepatocytes. Based on the results obtained in vitro, three Phase 1 crossover pharmacokinetic studies were conducted in healthy postmenopausal women to assess the in vivo effects of ospemifene on CYP-mediated drug metabolism. Ospemifene and its main metabolites 4-hydroxyospemifene and 4'-hydroxyospemifene weakly inhibited a number of CYPs (CYP2B6, CYP2C9, CYP2C19, CYP2C8, and CYP2D6) in vitro. However, only CYP2C9 activity was inhibited by 4-hydroxyospemifene at clinically relevant concentrations. Induction of CYPs by ospemifene in cultured human hepatocytes was 2.4-fold or less. The in vivo studies showed that ospemifene did not have significant effects on the areas under the plasma concentration-time curves of the tested CYP substrates warfarin (CYP2C9), bupropion (CYP2B6) and omeprazole (CYP2C19), demonstrating that pretreatment with ospemifene did not alter their metabolism. Therefore, the risk that ospemifene will affect the pharmacokinetics of drugs that are substrates for CYP enzymes is low. Full article

Leucine, a branched chain amino acid, is well known to stimulate protein synthesis in skeletal muscle. However, the role of leucine in myoblast proliferation remains unclear. In this study, we found that leucine could promote proliferation of C2C12 cells. Moreover, expressions of miR-27a and myostatin (a bona fide target of miR-27a) were upregulated and downregulated, respectively, following leucine treatment. We also found that miR-27a loss-of-function by transfection of a miR-27a inhibitor suppressed the promotion of myoblast proliferation caused by leucine. Our results suggest that miR-27a is induced by leucine and contributes to leucine-induced proliferation promotion of myoblast. Full article

Growing evidence has shown that proNGF-p75NTR-sortilin signaling might be a crucial factor in neurodegeneration, but it remains unclear if it may function in nigral neurons under aging and disease. The purpose of this study is to examine and quantify proNGF and sortilin expression in the substantia nigra and dynamic changes of aging in lactacystin and 6-hydroxydopamine (6-OHDA) rat models of Parkinson’s disease using immunofluorescence, electronic microscopy, western blot and FLIVO staining methods. The expression of proNGF and sortilin was abundantly and selectively identified in tyrosine hydroxylase (TH)-containing dopamine neurons in the substantia nigra. These proNGF/TH, sortilin/TH-positive neurons were densely distributed in the ventral tier, while they were less distributed in the dorsal tier, where calbindin-D28K-containing neurons were numerously located. A correlated decrease of proNGF, sortilin and TH was also detected during animal aging process. While increase of proNGF, sortilin and cleaved (active) caspase-3 expression was found in the lactacystin model, dynamic proNGF and sortilin changes along with dopamine neuronal loss were demonstrated in the substantia nigra of both the lactacystin and 6-OHDA models. This study has thus revealed the presence of the proNGF-sortilin signaling complex in nigral dopamine neurons and its response to aging, lactacystin and 6-OHDA insults, suggesting that it might contribute to neuronal apoptosis or neurodegeneration during pathogenesis and disease progression of Parkinson’s disease; the underlying mechanism and key signaling pathways involved warrant further investigation. Full article

IL-1β, a pro-inflammatory cytokine, has been shown to contribute to radiation injury. Sirt1, an NAD⁺-dependent class III protein deacetylase, plays an important role in the regulation of the proinflammatory cytokines involved in inflammation-associated diseases. The relationship between Sirt1 and IL-1β, however, has remained elusive. The present study was designed to explore the potential effect of Sirt1 on IL-1β expression induced by radiation and to provide a new target for the development of radiation protection drugs. Our results showed that radiation significantly increased IL-1β mRNA and protein expression and that pretreatment with resveratrol, a Sirt1 activator, inhibited the radiation-induced IL-1β expression in a concentration-dependent manner, whereas the knockdown or inhibition of Sirt1 by nicotinamide significantly enhanced radiation-induced IL-1β expression. This effect can likely be attributed to Sirt1-mediated inhibition of NLRP-3 inflammasome activation because Sirt1 inhibits the transactivation potential of NF-κb by deacetylation, which then suppresses NLRP3 transcription. Taken together, the results demonstrate that Sirt1 exerts anti-inflammatory effects by regulating NLRP3 expression partially through the NF-κb pathway in mesenchymal stem cells. More importantly, our findings suggest that resveratrol is an effective agent in protecting against radiation injury, and we provide a theoretical basis for developing a drug to protect against radiation injury by targeting Sirt1. Full article

There is a paucity of large animal models to study both the extent and the health risk of ionizing radiation exposure in humans. One promising candidate for such a model is the minipig. Here, we evaluate the minipig for its potential in γ-H2AX-based biodosimetry after exposure to ionizing radiation using both Cs137 and Co60 sources. γ-H2AX foci were enumerated in blood lymphocytes and normal fibroblasts of human and porcine origin after ex vivo g-ray irradiation. DNA double-strand break repair kinetics in minipig blood lymphocytes and fibroblasts, based on the γ-H2AX assay, were similar to those observed in their human counterparts. To substantiate the similarity observed between the human and minipig we show that minipig fibroblast radiosensitivity was similar to that observed with human fibroblasts. Finally, a strong γ-H2AX induction was observed in blood lymphocytes following minipig total body irradiation. Significant responses were detected 3 days after 1.8 Gy and 1 week after 3.8 and 5 Gy with residual γ-H2AX foci proportional to the initial radiation doses. These findings show that the Gottingen minipig provides a useful in vivo model for validation of γ-H2AX biodosimetry for dose assessment in humans. Full article

The discovery of microRNAs (miRNAs) provides a new and powerful tool for studying the mechanism, diagnosis and treatment of human cancers. Currently, the methylation epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is emerging as a common hallmark of different tumors. Here we showed that miR-433 and miR-127 were significantly down-regulated in gastric cancer (GC) tissues compared with the adjacent normal regions in 86 paired samples. Moreover, the lower level of miR-433 and miR-127 was associated with pM or pTNM stage in clinical gastric cancer patients. The restored expression of miR-433 and miR-127 in GC cells upon 5-Aza-CdR and TSA treatment suggested the loss of miR-433 and miR-127 was at least partly regulated by epigenetic modification in GC. Furthermore, the ectopic expression of miR-433 and miR-127 in gastric cancer cell lines HGC-27 inhibits cell proliferation, cell cycle progression, cell migration and invasion by directly interacting with the mRNA encoding oncogenic factors KRAS and MAPK4 respectively. Taken together, our results showed that miR-433 and miR-127 might act as tumor suppressors in GC, and it may provide novel diagnostic and therapeutic options for human GC clinical operation in the near future. Full article

This study investigated the interaction between eupatorin and bovine serum albumin (BSA) using ultraviolet-visible (UV-vis) absorption, fluorescence, synchronous fluorescence, circular dichroism (CD) spectroscopies, and molecular modeling at pH 7.4. Results of UV-vis and fluorescence spectroscopies illustrated that BSA fluorescence was quenched by eupatorin via a static quenching mechanism. Thermodynamic parameters revealed that hydrophobic and electrostatic interactions played major roles in the interaction. Moreover, the efficiency of energy transfer, and the distance between BSA and acceptor eupatorin, were calculated. The effects of eupatorin on the BSA conformation were analyzed using UV-vis, CD, and synchronous fluorescence. Finally, the binding of eupatorin to BSA was modeled using the molecular docking method. Full article

The FeCo/Fe₃O₄ nanocomposite was synthesized using the hydrothermal approach, in which the FeCo alloy and Fe₃O₄ are formed by one step. The structure of the FeCo/Fe₃O₄ nanocomposite was characterized by means of Scanning electron microscopy (SEM), X-ray diffraction (XRD) and X-ray energy-dispersive spectrometer spectroscopy (EDX). They show that the mass ratio of FeCo/Fe₃O₄ strongly depends on the reaction temperature. Such various architectures follow a stepwise growth mechanism of the composites prepared in various reaction temperatures were also discussed. It indicates that this strategy is facile, effective and controllable for the synthesis of FeCo/Fe₃O₄ by the one-step method. Furthermore, the magnetic and wave-absorbing properties of the nanocomposites with various structures were investigated in detail. The results show that the FeCo/Fe₃O₄ with higher mass ratio has higher magnetic properties. Moreover, the FeCo/Fe₃O₄ nanocomposite shows high wave-absorbing properties (e.g., –37.9 dB), which are expected to apply in microwave absorbing materials. Full article

This research investigated the effect of enzymatically digested low molecular weight (MW) chitosan oligosaccharide on type 2 diabetes prevention. Three different chitosan oligosaccharide samples with varying MW were evaluated in vitro for inhibition of rat small intestinal α-glucosidase and porcine pancreatic α-amylase (GO2KA1; <1000 Da, GO2KA2; 1000–10,000 Da, GO2KA3; MW > 10,000 Da). The in vitro results showed that all tested samples had similar rat α-glucosidase inhibitory and porcine α-amylase inhibitory activity. Based on these observations, we decided to further investigate the effect of all three samples at a dose of 0.1 g/kg, on reducing postprandial blood glucose levels in Sprague-Dawley (SD) rat model after sucrose loading test. In the animal trial, all tested samples had postprandial blood glucose reduction effect, when compared to control, however GO2KA1 supplementation had the strongest effect. The glucose peak (C_max) for GO2KA1 and control was 152 mg/dL and 193 mg/dL, respectively. The area under the blood glucose-time curve (AUC) for GO2KA1 and control was 262 h mg/dL and 305 h mg/dL, respectively. Furthermore, the time of peak plasma concentration of blood glucose (T_max) for GO2KA1 was significantly delayed (0.9 h) compared to control (0.5 h). These results suggest that GO2KA1 could have a beneficial effect for blood glucose management relevant to diabetes prevention in normal and pre-diabetic individuals. The suggested mechanism of action is via inhibition of the carbohydrate hydrolysis enzyme α-glucosidase and since GO2KA1 (MW < 1000 Da) had higher in vivo effect, we hypothesize that it is more readily absorbed and might exert further biological effect once it is absorbed in the blood stream, relevant to blood glucose management. Full article

Methione tRNA synthetase (MetRS) is an essential enzyme involved in protein biosynthesis in all living organisms and is a potential antibacterial target. In the current study, the structure-based pharmacophore (SBP)-guided method has been suggested to generate a comprehensive pharmacophore of MetRS based on fourteen crystal structures of MetRS-inhibitor complexes. In this investigation, a hybrid protocol of a virtual screening method, comprised of pharmacophore model-based virtual screening (PBVS), rigid and flexible docking-based virtual screenings (DBVS), is used for retrieving new MetRS inhibitors from commercially available chemical databases. This hybrid virtual screening approach was then applied to screen the Specs (202,408 compounds) database, a structurally diverse chemical database. Fifteen hit compounds were selected from the final hits and shifted to experimental studies. These results may provide important information for further research of novel MetRS inhibitors as antibacterial agents. Full article

Brassinosteroids (BRs) are endogenous plant hormones and are essential for normal plant growth and development. MicroRNAs (miRNAs) of Arabidopsis thaliana are involved in mediating cell proliferation in leaves, stress tolerance, and root development. The specifics of BR mechanisms involving miRNAs are unknown. Using customized miRNA array analysis, we identified miRNAs from A. thaliana ecotype Columbia (Col-0) regulated by 24-epibrassinolide (EBR, a highly active BR). We found that miR395a was significantly up-regulated by EBR treatment and validated its expression under these conditions. miR395a was over expressed in leaf veins and root tissues in EBR-treated miR395a promoter::GUS plants. We integrated bioinformatics methods and publicly available DNA microarray data to predict potential targets of miR395a. GUN5—a multifunctional protein involved in plant metabolic functions such as chlorophyll synthesis and the abscisic acid (ABA) pathway—was identified as a possible target. ABI4 and ABI5, both genes positively regulated by ABA, were down-regulated by EBR treatment. In summary, our results suggest that EBR regulates seedling development and root growth of A. thaliana through miR395a by suppressing GUN5 expression and its downstream signal transduction. Full article

Despite the high prevalence of histological Benign Prostatic Hypeplasia (BPH) in elderly men, little is known regarding the molecular mechanisms and networks underlying the development and progression of the disease. Here, we explored the effects of a phytotherapeutic agent, Lipidosterolic extract of the dwarf palm plant Serenoa repens (LSESr), on the mRNA gene expression profiles of two representative models of BPH, BPH1 cell line and primary stromal cells derived from BPH. Treatment of these cells with LSESr significantly altered gene expression patterns as assessed by comparative gene expression profiling on gene chip arrays. The expression changes were manifested three hours following in vitro administration of LSESr, suggesting a rapid action for this compound. Among the genes most consistently affected by LSESr treatment, we found numerous genes that were categorized as part of proliferative, apoptotic, and inflammatory pathways. Validation studies using quantitative real-time PCR confirmed the deregulation of genes known to exhibit key roles in these biological processes including IL1B, IL1A, CXCL6, IL1R1, PTGS2, ALOX5, GAS1, PHLDA1, IL6, IL8, NFkBIZ, NFKB1, TFRC, JUN, CDKN1B, and ERBB3. Subsequent analyses also indicated that LSESr treatment can impede the stimulatory effects of certain proinflammatory cytokines such as IL6, IL17, and IL15 in these cells. These results suggest that LSESr may be useful to treat BPH that manifest inflammation characteristics. This also supports a role for inflammation in BPH presumably by mediating the balance between apoptosis and proliferation. Full article

Thrombospondin-1 (TSP-1), a matricellular protein widely acclaimed to be involved in the inhibition of angiogenesis and tumorigenesis, is synthesized and secreted by many cell types, including osteoblast and cancer cells. TSP-1 is highly upregulated during early stage of osteogenesis, whereas it inhibits terminal osteoblast differentiation. Expression of TSP-1 is downregulated in cancer cells, and its ectopic expression has been shown to restrain tumor growth. Transcriptional regulation of TSP-1 in osteogenesis and cancer is poorly understood; this prompted us to study its regulation by the two key regulators of the aforementioned processes: Runx2 and Runx3. Through a PCR-based cDNA subtraction technique, we identified and cloned a cDNA fragment for mouse TSP-1, whose expression was dramatically upregulated in response to Runx2 expression in mesenchymal stem cells. Moreover, TSP-1 expression was considerably reduced in the lung of Runx2 knockout mouse. On the other hand, TSP-1 gene expression drastically increased at both the transcriptional and translational levels in response to Runx3 expression in B16-F10 melanoma cells. In line with this, Runx2 and Runx3 bound to the TSP-1 promoter and stimulated its activity. Hence, these results provide first line of evidence that TSP-1 is a transcriptional target gene of Runx2 and Runx3. Full article

Our recent in vitro experiments suggest that fluvastatin may influence tyrosinase (key enzyme of melanogenesis) synthesis. The aim of the present study was to verify those findings in experiments, in vitro, in melanoma cell line, and in vivo, in mice. The expression of tyrosinase in B16F10 melanoma cell line, after induction of melanogenesis by UVB irradiation, was examined by Western blot analysis. Afterwards, the effect of fluvastatin on melanin synthesis in hair follicles of C57Bl/6 mice was investigated. The expression of tyrosinase was reduced in the presence of fluvastatin. In mice after anagen induction over the dorsal skin, gel containing fluvastatin in various concentrations was injected subcutaneously, while in part of control groups of mice, gel with placebo was injected. In addition, gel with fluvastatin was injected to four week-old mice (mice in first postnatal anagen) without anagen induction. In extension, injections of gel with fluvastatin or placebo were performed in mice without anagen induction (but after first postnatal anagen). In part of study group of mice (mice after anagen induction and injection of fluvastatin) regrowth of depigmented hair was observed, while in all control groups (mice after injection of placebo), such hair depigmentation over the skin area was not found. In conclusion, this study, for the first time, shows that fluvastatin might affect melanin synthesis in vivo. Full article

Anabaena sp. was used to examine the toxicity of exposure to a nano-TiO₂ suspension, Zn²⁺ solution, and mixtures of nano-TiO₂ and Zn²⁺ suspensions. Typical chlorophyll fluorescence parameters, including effective quantum yield, photosynthetic efficiency and maximal electron transport rate, were measured by a pulse-amplitude modulated fluorometer. Nano-TiO₂ particles exhibited no significant toxicity at concentrations lower than 10.0 mg/L. The 96 h concentration for the 50% maximal effect (EC₅₀) of Zn²⁺ alone to Anabaena sp. was 0.38 ± 0.004 mg/L. The presence of nano-TiO₂ at low concentrations (<1.0 mg/L) significantly enhanced the toxicity of Zn²⁺ and consequently reduced the EC₅₀ value to 0.29 ± 0.003 mg/L. However, the toxicity of the Zn²⁺/TiO₂ system decreased with increasing nano-TiO₂ concentration because of the substantial adsorption of Zn²⁺ by nano-TiO₂. The toxicity curve of the Zn²⁺/TiO₂ system as a function of incremental nano-TiO₂ concentrations was parabolic. The toxicity significantly increased at the initial stage, reached its maximum, and then decreased with increasing nano-TiO₂ concentration. Hydrodynamic sizes, concentration of nano-TiO₂ and Zn²⁺ loaded nano-TiO₂ were the main parameters for synergistic toxicity. Full article

To explore the athermal effect of an external electrostatic field on the stabilities of protein conformations and the binding affinities of protein-protein/ligand interactions, the dependences of the polar and hydrophobic interactions on the external electrostatic field, −E^ext, were studied using molecular dynamics (MD) simulations. By decomposing E^ext into, along, and perpendicular to the direction formed by the two solutes, the effect of E^ext on the interactions between these two solutes can be estimated based on the effects from these two components. E^ext was applied along the direction of the electric dipole formed by two solutes with opposite charges. The attractive interaction free energy between these two solutes decreased for solutes treated as point charges. In contrast, the attractive interaction free energy between these two solutes increased, as observed by MD simulations, for E^ext = 40 or 60 MV/cm. E^ext was applied perpendicular to the direction of the electric dipole formed by these two solutes. The attractive interaction free energy was increased for E^ext = 100 MV/cm as a result of dielectric saturation. The force on the solutes along the direction of E^ext computed from MD simulations was greater than that estimated from a continuum solvent in which the solutes were treated as point charges. To explore the hydrophobic interactions, E^ext was applied to a water cluster containing two neutral solutes. The repulsive force between these solutes was decreased/increased for E^ext along/perpendicular to the direction of the electric dipole formed by these two solutes. Full article

The measurement of autoantibodies to thyroid-stimulating hormone receptor (TSHR) is important for the diagnosis of autoimmune thyroid disease such as Graves’ disease (GD). Although TSHR from porcine thyroid membrane is commonly used for the measurement of TSHR autoantibodies (TRAb), recombinant human TSHR (hTSHR) remains ideal in terms of stable supply and species identity. Here we set out to express recombinant hTSHR on the lipid-bilayer surface of magnetic nanoparticles from a magnetotactic bacterium, Magnetospirillum magneticum AMB-1. Using a tetracycline-inducible expression system, we successfully overexpressed functional hTSHR on bacterial magnetic particles (BacMPs) in AMB-1 via an anchor protein specific for BacMPs. The overexpressed hTSHR was membrane integrated and possessed both ligand and autoantibody binding activity. Our data suggest that hTSHR-displayed BacMPs have potential as novel tools for ligand-receptor interaction analysis or for TRAb immunoassay in GD patients. Full article

Cholesteatoma is a destructive and expanding growth of keratinizing squamous epithelium in the middle ear or petrous apex. The molecular and cellular processes of the pathogenesis of acquired middle ear cholesteatoma have not been fully understood. In this study, comparative proteomic analysis was conducted to investigate the roles of specific proteins in the pathways regarding keratinocyte proliferation in cholesteatoma. The differential proteins were detected by comparing the two-dimension electrophoresis (2-DE) maps of the epithelial tissues of 12 attic cholesteatomas with those of retroauricular skins. There were 14 upregulated proteins in the epithelial tissues of cholesteatoma in comparison with retroauricular skin. The modulation of five crucial proteins, HSP27, PRDX2, GRP75, GRP78 and GRP94, was further determined by RT-PCR, Western blot and immunohistochemistry. Phosphorylation of HSP27 at Ser-82 was identified by mass spectroscopy. The results of this study suggested that phosphorylated HSP27 is the end expression of two potential signal-transduction pathways, and together with PRDX2, they are very likely involved in the proliferation of keratinocytes in cholesteatoma. Upregulations of GRP75, GRP78 and GRP94 in keratinocytes may be able to counter endoplasmic reticulum stress, to inhibit cell apoptosis, to prevent protein unfolding and to promote cholesteatoma growth. Full article

New skin substitutes for burn medicine or reconstructive surgery pose an important issue in plastic surgery. Matriderm^® is a clinically approved three-dimensional bovine collagen-elastin matrix which is already used as a dermal substitute of full thickness burn wounds. The drawback of an avital matrix is the limited integration in full thickness skin defects, depending on the defect size. To further optimize this process, Matriderm^® has also been studied as a matrix for tissue engineering of skin albeit long-term cultivation of the matrix with cells has been difficult. Cells have generally been seeded onto the matrix with high cell loss and minimal time-consuming migration. Here we developed a cell seeded skin equivalent after microtransfer of cells directly into the matrix. First, cells were cultured, and microinjected into Matriderm^®. Then, cell viability in the matrix was determined by histology in vitro. As a next step, the skin substitute was applied in vivo into a full thickness rodent wound model. The wound coverage and healing was observed over a period of two weeks followed by histological examination assessing cell viability, proliferation and integration into the host. Viable and proliferating cells could be found throughout the entire matrix. The presented skin substitute resembles healthy skin in morphology and integrity. Based on this study, future investigations are planned to examine behaviour of epidermal stem cells injected into a collagen-elastin matrix under the aspects of establishment of stem cell niches and differentiation. Full article

Embryo implantation is a highly synchronized bioprocess between an activated blastocyst and a receptive uterus. In mice, successful implantation relies on the dynamic interplay of estrogen and progesterone; however, the key mediators downstream of these hormones that act on blastocyst competency and endometrium receptivity acquisition are largely unknown. In this study, we showed that the expression of osteopontin (OPN) in mouse blastocysts is regulated by ovarian estrogen and uterine micro-environment. OPN mRNA is up-regulated in mouse blastocyst on day 4 of pregnancy, which is associated with ovarian estrogen secretion peak. Hormone treatment in vivo demonstrated that OPN expression in a blastocyst is regulated by estrogen through an estrogen receptor (ER). Our results of the delayed and activated implantation model showed that OPN expression is induced after estrogen injection. While estrogen treatment during embryo culture in vitro showed less effect on OPN expression, the tubal ligation model on day 3 of pregnancy confirmed that the regulation of estrogen on OPN expression in blastocyst might, through some specific cytokines, have existed in a uterine micro-environment. Collectively, our study presents that estrogen regulates OPN expression and it may play an important role during embryo implantation by activating blastocyst competence and facilitating the endometrium acceptable for active blastocyst. Full article

Recent experiments in function mechanism study reported that a pH low-insertion peptide (pHLIP) can insert into a zwitterionic palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer at acidic pH while binding to the bilayer surface at basic pH. However, the atomic details of the pH-dependent interaction of pHLIP with a POPC bilayer are not well understood. In this study, we investigate the detailed interactions of pHLIP with a POPC bilayer at acidic and basic pH conditions as those used in function mechanism study, using all-atom molecular dynamics (MD) simulations. Simulations have been performed by employing the initial configurations, where pHLIP is placed in aqueous solution, parallel to bilayer surface (system S), partially-inserted (system P), or fully-inserted (system F) in POPC bilayers. On the basis of multiple 200-ns MD simulations, we found (1) pHLIP in system S can spontaneously insert into a POPC bilayer at acidic pH, while binding to the membrane surface at basic pH; (2) pHLIP in system P can insert deep into a POPC bilayer at acidic pH, while it has a tendency to exit, and stays at bilayer surface at basic pH; (3) pHLIP in system F keeps in an α-helical structure at acidic pH while partially unfolding at basic pH. This study provides at atomic-level the pH-induced insertion of pHLIP into POPC bilayer. Full article

Despite the large efforts to prepare super paramagnetic iron oxide nanoparticles (MNPs) for biomedical applications, the number of FDA or EMA approved formulations is few. It is not known commonly that the approved formulations in many instances have already been withdrawn or discontinued by the producers; at present, hardly any approved formulations are produced and marketed. Literature survey reveals that there is a lack for a commonly accepted physicochemical practice in designing and qualifying formulations before they enter in vitro and in vivo biological testing. Such a standard procedure would exclude inadequate formulations from clinical trials thus improving their outcome. Here we present a straightforward route to assess eligibility of carboxylated MNPs for biomedical tests applied for a series of our core-shell products, i.e., citric acid, gallic acid, poly(acrylic acid) and poly(acrylic acid-co-maleic acid) coated MNPs. The discussion is based on physicochemical studies (carboxylate adsorption/desorption, FTIR-ATR, iron dissolution, zeta potential, particle size, coagulation kinetics and magnetization measurements) and involves in vitro and in vivo tests. Our procedure can serve as an example to construct adequate physico-chemical selection strategies for preparation of other types of core-shell nanoparticles as well. Full article

A common feature of biomineralization proteins is their self-assembly to produce a surface consistent in size with the inorganic crystals that they produce. Mms6, a small protein of 60 amino acids from Magnetospirillum magneticum strain AMB-1 that promotes the in vitro growth of superparamagnetic magnetite nanocrystals, assembles in aqueous solution to form spherical micelles that could be visualized by TEM and AFM. The results reported here are consistent with the view that the N and C-terminal domains interact with each other within one polypeptide chain and across protein units in the assembly. From studies to determine the amino acid residues important for self-assembly, we identified the unique GL repeat in the N-terminal domain with additional contributions from amino acids in other positions, throughout the molecule. Analysis by CD spectroscopy identified a structural change in the iron-binding C-terminal domain in the presence of Fe³⁺. A change in the intrinsic fluorescence of tryptophan in the N-terminal domain showed that this structural change is transmitted through the protein. Thus, self-assembly of Mms6 involves an interlaced structure of intra- and inter-molecular interactions that results in a coordinated structural change in the protein assembly with iron binding. Full article

Many Gram-negative plant pathogenic bacteria employ a N-acylhomoserine lactone (AHL)-based quorum sensing (QS) system to regulate their virulence traits. A sustainable biocontrol strategy has been developed using quorum quenching (QQ) bacteria to interfere with QS and protect plants from pathogens. Here, the prevalence and the diversity of QQ strains inhabiting tobacco leaf surfaces were explored. A total of 1177 leaf-associated isolates were screened for their ability to disrupt AHL-mediated QS, using the biosensor Chromobacterium violaceum CV026. One hundred and sixty-eight strains (14%) are capable of interfering with AHL activity. Among these, 106 strains (63%) of the culturable quenchers can enzymatically degrade AHL molecules, while the remaining strains might use other QS inhibitors to interrupt the chemical communication. Moreover, almost 79% of the QQ strains capable of inactivating AHLs enzymatically have lactonase activity. Further phylogenetic analysis based on 16S rDNA revealed that the leaf-associated QQ bacteria can be classified as Bacillus sp., Acinetobacter sp., Lysinibacillus sp., Serratia sp., Pseudomonas sp., and Myroides sp. The naturally occurring diversity of bacterial quenchers might provide opportunities to use them as effective biocontrol reagents for suppressing plant pathogen in situ. Full article

Members of the microRNA-29 (miR-29) family directly target the DNA methyltransferases, DNMT3A and DNMT3B. Disturbances in the expression levels of miR-29 have been linked to tumorigenesis and tumor aggressiveness. Members of the miR-29 family are currently thought to repress DNA methylation and suppress tumorigenesis by protecting against de novo methylation. Here, we report that members of the miR-29 family repress the activities of DNA methyltransferases and DNA demethylases, which have opposing roles in control of DNA methylation status. Members of the miR-29 family directly inhibited DNA methyltransferases and two major factors involved in DNA demethylation, namely tet methylcytosine dioxygenase 1 (TET1) and thymine DNA glycosylase (TDG). Overexpression of miR-29 upregulated the global DNA methylation level in some cancer cells and downregulated DNA methylation in other cancer cells, suggesting that miR-29 suppresses tumorigenesis by protecting against changes in the existing DNA methylation status rather than by preventing de novo methylation of DNA. Full article

Triple-negative breast cancer (TNBC) is characterized by the lack of immunohistochemical staining for estrogen receptors (ER), progesterone receptors (PR), and lack of overexpression or amplification of human epidermal growth factor receptor 2 (HER2). Our aim was to investigate the expression of transmembrane protease, serine 4 (TMPRSS4) in TNBC patients and its possible relationship to the outcome of the disease. A total of 72 TNBC patients and 109 non-TNBC patients who were diagnosed between 2003 and 2008 were enrolled in this study. Immunohistochemistry was used to compare the expression pattern of TMPRSS4 in TNBC and non-TNBC groups, and the prognostic significance was assessed by Kaplan-Meier analysis and Cox proportional hazards regression in TNBC patients. The rate of high expression of TMPRSS4 was significantly higher in TNBC group than that in non-TNBC group. High expression of TMPRSS4 was significantly correlated with lymph node metastasis, histological grade, and tumor size. TNBC patients with high TMPRSS4 expression showed the poorer overall survival (OS) and disease-free survival (DFS) than those patients with low TMPRSS4 expression. In multivariate analysis, only lymph node metastasis and TMPRSS4 expression were the independent prognostic factors for OS and DFS in TNBC. Our study provides evidence that TMPRSS4 expression is associated with lymph node metastasis, tumor size, and histological grade in TNBC patients, and also is an independent prognostic factor for TNBC. Full article

In this study, we present the applicability of imaging epidermal growth factor (EGF) receptor levels in preclinical models of COLO205 carcinoma cells in vitro, mice with orthotopic tumors and ex vivo colorectal tumor biopsies, using EGF-labeled with IRDye800CW (EGF-NIR). The near infrared (NIR) bio-imaging of COLO205 cultures indicated specific and selective binding, reflecting EGF receptors levels. In vivo imaging of tumors in mice showed that the highest signal/background ratio between tumor and adjacent tissue was achieved 48 hours post-injection. Dissected colorectal cancer tissues from different patients demonstrated ex vivo specific imaging using the NIR bio-imaging platform of the heterogeneous distributed EGF receptors. Moreover, in the adjacent gastrointestinal tissue of the same patients, which by Western blotting was demonstrated as EGF receptor negative, no labeling with EGF-NIR probe was detected. Present results support the concept of tumor imaging by measuring EGF receptor levels using EGF-NIR probe. This platform is advantageous for EGF receptor bio-imaging of the NCI-60 recommended panel of tumor cell lines including 6–9 colorectal cell lines, since it avoids radioactive probes and is appropriate for use in the clinical setting using NIR technologies in a real-time manner. Full article

The production of small RNAs (sRNAs) from phased positions set by microRNA-directed cleavage of trans-acting-siRNA-producing locus (TAS) transcript has been characterized extensively; however, the production of sRNAs from non-phased positions remains unknown. We report three cis-cleavages that occurred in TAS3 transcripts in Vitis vinifera, by combining high-throughput sRNA deep sequencing information with evolutional conservation and genome-wide RNA degradome analysis. The three cis-cleavages can be deciphered to generate an orderly cleavage cascade, and can also produce distinct phasing patterns. Each of the patterns, either upstream or downstream of the cis-cleaved position, had a set of sRNAs arranged in 21-nucleotide increments. Part of the cascading cis-cleavages was also conserved in Arabidopsis thaliana. Our results will enhance the understanding of the production of sRNAs from non-phased positions that are not set by microRNA-directed cleavage. Full article

Metabolic syndrome is associated with an increased risk of colorectal cancer. This study investigated the impact of hypertension, a component of metabolic syndrome, on azoxymethane (AOM)-induced colorectal carcinogenesis using SHRSP/Izm (SHRSP) non-diabetic/hypertensive rats and SHRSP.Z-Lepr^fa/IzmDmcr (SHRSP-ZF) diabetic/hypertensive rats. Male 6-week-old SHRSP, SHRSP-ZF, and control non-diabetic/normotensive Wister Kyoto/Izm (WKY) rats were given 2 weekly intraperitoneal injections of AOM (20 mg/kg body weight). Two weeks after the last injection of AOM, the SHRSP and SHRSP-ZF rats became hypertensive compared to the control WKY rats. Serum levels of angiotensin-II, the active product of the renin-angiotensin system, were elevated in both SHRSP and SHRSP-ZF rats, but only the SHRSP-ZF rats developed insulin resistance, dyslipidemia, and hyperleptinemia and exhibited an increase in adipose tissue. The development of AOM-induced colonic preneoplastic lesions and aberrant crypts foci, was significantly accelerated in both SHRSP and SHRSP-ZF hypertensive rats, compared to WKY normotensive rats. Furthermore, induction of oxidative stress and exacerbation of inflammation were observed in the colonic mucosa and systemically in SHRSP and SHRSP-ZF rats. Our findings suggest that hypertension plays a role in the early stage of colorectal carcinogenesis by inducing oxidative stress and chronic inflammation, which might be associated with activation of the renin-angiotensin system. Full article

T-20 and T-1249 fusion inhibitor peptides were shown to interact with 1-palmitoyl-2-oleyl-phosphatidylcholine (POPC) (liquid disordered, ld) and POPC/cholesterol (1:1) (POPC/Chol) (liquid ordered, lo) bilayers, and they do so to different extents. Although they both possess a tryptophan-rich domain (TRD), T-20 lacks a pocket binding domain (PBD), which is present in T-1249. It has been postulated that the PBD domain enhances FI interaction with HIV gp41 protein and with model membranes. Interaction of these fusion inhibitor peptides with both the cell membrane and the viral envelope membrane is important for function, i.e., inhibition of the fusion process. We address this problem with a molecular dynamics approach focusing on lipid properties, trying to ascertain the consequences and the differences in the interaction of T-20 and T-1249 with ld and lo model membranes. T-20 and T-1249 interactions with model membranes are shown to have measurable and different effects on bilayer structural and dynamical parameters. T-1249’s adsorption to the membrane surface has generally a stronger influence in the measured parameters. The presence of both binding domains in T-1249 appears to be paramount to its stronger interaction, and is shown to have a definite importance in membrane properties upon peptide adsorption. Full article

Thuja sutchuenensis Franch. is a critically endangered plant endemic to the North-East Chongqing, China. Genetic variation was studied to assess the distribution of genetic diversity within and among seven populations from the single remnant locations, using inter-simple sequence repeat (ISSR) markers. A total of 15 primers generated 310 well defined bands, with an average of 20.7 bands per primer. The seven populations revealed a relatively high level of genetic diversity in the species. The percentage of polymorphic bands, Nei’s gene diversity and Shannon’s information index at the population and species level were 76.1%, 0.155, 0.252 and 100%, 0.165, 0.295, respectively. A low level of genetic differentiation among populations (G_ST = 0.102), in line with the results of Analyses of Molecular Variance (AMOVA), and a high level of gene flow (N_m = 4.407) were observed. Both the Unweighted Pair Group Method with Arithmatic Mean (UPGMA) cluster analysis and Principal Coordinates Analysis (PCoA) supported the grouping of all seven populations into two groups. In addition, Mantel test revealed no significant correlation between genetic and geographical distances (r = 0.329, p = 0.100). The low genetic differentiation among populations implies that the conservation efforts should aim to preserve all the extant populations of this endangered species. Full article

YABBY gene family plays an important role in the polarity development of lateral organs. We isolated the BraYAB1-702 gene, a member of the YABBY gene family, from young leaves of Chinese cabbage line 06J45. The full-length gene has a 937 bp CDNA sequence and contains an open reading frame (ORF) of 702 bp. The subcellular localization analysis showed that the expression product of the gene was localized in the nucleus. Ectopic expression of BraYAB1-702 in Arabidopsis thaliana caused leaf curling from the adaxial epidermises to abaxial epidermises; the partial abaxialization of the adaxial epidermises of leaves; leaf trichomes and stomata numbers being significantly increased; the plants being severely stunted; the flowering stage being remarkably delayed and inhibiting the development of shoot apical meristem (SAM) with the down-regulation of the expression of SHOOT MERISTEMLESS (STM), Brevipedicellus (BP) and KNAT2 which were related to the development of shoot apical meristem. These results from the present research help to reveal the molecular mechanism of BraYAB1-702 gene in the establishment of adaxial–abaxial polarity of the lateral organs in Chinese cabbage. Full article

Protein structure information is essential to understand protein function. Computational methods to accurately predict protein structure from the sequence have primarily been evaluated on protein sequences representing full-length native proteins. Here, we demonstrate that top-performing structure prediction methods can accurately predict the partial structures of proteins encoded by sequences that contain approximately 50% or more of the full-length protein sequence. We hypothesize that structure prediction may be useful for predicting functions of proteins whose corresponding genes are mapped expressed sequence tags (ESTs) that encode partial-length amino acid sequences. Additionally, we identify a confidence score representing the quality of a predicted structure as a useful means of predicting the likelihood that an arbitrary polypeptide sequence represents a portion of a foldable protein sequence (“foldability”). This work has ramifications for the prediction of protein structure with limited or noisy sequence information, as well as genome annotation. Full article

rRNA post transcriptional modifications play a role in cancer development by affecting ribosomal function. In particular, the snoRNA U50, mediating the methylation of C2848 in 28S rRNA, has been suggested as a potential tumor suppressor-like gene playing a role in breast and prostate cancers and B-cell lymphoma. Indeed, we observed the downregulation of U50 in colon cancer cell lines as well as tumors. We then investigated the relationship between U50 and proliferation in lymphocytes stimulated by phytohemagglutinin (PHA) and observed a strong decrease in U50 levels associated with a reduced C2848 methylation. This reduction was due to an alteration of U50 stability and to an increase of its consumption. Indeed, the blockade of ribosome biogenesis induced only an early decrease in U50 followed by a stabilization of U50 levels when ribosome biogenesis was almost completely blocked. Similar results were found with other snoRNAs. Lastly, we observed that U50 modulation affects ribosome efficiency in IRES-mediated translation, demonstrating that changes in the methylation levels of a single specific site on 28S rRNA may alter ribosome function. In conclusion, our results link U50 to the cellular proliferation rate and ribosome biogenesis and these findings may explain why its levels are often greatly reduced in cancers. Full article

Olfaction is crucial in many insects for critical behaviors, including those regulating survival and reproduction. Insect odorant-binding proteins (OBPs) function in the first step of the olfactory system and play an essential role in the perception of odorants, such as pheromones and host chemicals. The oriental fruit fly, Bactrocera dorsalis, is a destructive fruit-eating pest, due to its wide host range of up to 250 different types of fruits and vegetables, and this fly causes severe economic damage to the fruit and vegetable industry. However, OBP genes have not been largely identified in B. dorsalis. Based on our previously constructed B. dorsalis cDNA library, ten OBP genes were identified in B. dorsalis for the first time. A phylogenetic tree was generated to show the relationships among the 10 OBPs of B. dorsalis to OBP sequences of two other Dipteran species, including Drosophila melanogaster and the mosquito Anopheles gambiae. The expression profiles of the ten OBPs in different tissues (heads, thoraxes, abdomens, legs, wings, male antennae and female antenna) of the mated adults were analyzed by real-time PCR. The results showed that nine of them are highly expressed in the antenna of both sexes, except BdorOBP7. Four OBPs (BdorOBP1, BdorOBP4, BdorOBP8, and BdorOBP10) are also enriched in the abdomen, and BdorOBP7 is specifically expressed in leg, indicating that it may function in other biological processes. This work will provide insight into the roles of OBPs in chemoreception and help develop new pest-control strategies. Full article

Pak choi plants (Brassica rapa ssp. chinensis) were treated with different signaling molecules methyl jasmonate, jasmonic acid, linolenic acid, and methyl salicylate and were analyzed for specific changes in their glucosinolate profile. Glucosinolate levels were quantified using HPLC-DAD-UV, with focus on induction of indole glucosinolates and special emphasis on 1-methoxy-indol-3-ylmethyl glucosinolate. Furthermore, the effects of the different signaling molecules on indole glucosinolate accumulation were analyzed on the level of gene expression using semi-quantitative realtime RT-PCR of selected genes. The treatments with signaling molecules were performed on sprouts and mature leaves to determine ontogenetic differences in glucosinolate accumulation and related gene expression. The highest increase of indole glucosinolate levels, with considerable enhancement of the 1-methoxy-indol-3-ylmethyl glucosinolate content, was achieved with treatments of sprouts and mature leaves with methyl jasmonate and jasmonic acid. This increase was accompanied by increased expression of genes putatively involved in the indole glucosinolate biosynthetic pathway. The high levels of indole glucosinolates enabled the plant to preferentially produce the respective breakdown products after tissue damage. Thus, pak choi plants treated with methyl jasmonate or jasmonic acid, are a valuable tool to analyze the specific protection functions of 1-methoxy-indole-3-carbinole in the plants defense strategy in the future. Full article

The results of studies on the biological influence of magnetic fields are controversial and do not provide clear answers regarding their impact on cell functioning. Fluoride compounds are substances that influence free radical processes, which occur when the reactive forms of oxygen are present. It is not known whether static magnetic fields (SMF) cause any changes in fluoride assimilation or activity. Therefore, the aim of this work was to determine the potential relationship between magnetic field exposure to, and the antioxidant system of, fibroblasts cultured with fluoride ions. Three chambers with static magnetic fields of different intensities (0.4, 0.6, and 0.7 T) were used in this work. Fluoride ions were added at a concentration of 0.12 mM, which did not cause the precipitation of calcium or magnesium. The results of this study show that static magnetic fields reduce the oxidative stress caused by fluoride ions and normalize the activities of antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT). Static magnetic fields modify the energy state of fibroblasts, causing an increase in the ATP concentration and a decrease in the MDA concentration. These results suggest that exposure to fluoride and an SMF improves the tolerance of cells to the oxidative stress induced by fluoride ions. Full article

Uveal melanoma (UM) is the most common primary intraocular malignancy and the leading potentially fatal primary intraocular disease in adults. Melanoma antigen recognized by T-cells (MART-1) has been studied extensively as a clinically important diagnostic marker for melanoma, however, its biological function remains unclear. In the present study, the UM cell line SP6.5, which showed a high level of MART-1 expression, was subjected to small interfering RNA-mediated silencing of MART-1. Silencing of MART-1 expression increased the migration ability of SP6.5 cells and down-regulated the expression of the metastasis suppressor NM23. Our results suggest that MART-1 is a candidate target for the development of therapeutic strategies for UM and in particular for the suppression of metastasis associated with this malignancy. Full article

Mutations in BEST1 gene, encoding the bestrophin-1 (Best1) protein are associated with macular dystrophies. Best1 is predominantly expressed in the retinal pigment epithelium (RPE), and is inserted in its basolateral membrane. We investigated the cellular localization in polarized MDCKII cells of disease-associated Best1 mutant proteins to study specific sorting motifs of Best1. Real-time PCR and western blots for endogenous expression of BEST1 in MDCK cells were performed. Best1 mutant constructs were generated using site-directed mutagenesis and transfected in MDCK cells. For protein sorting, confocal microscopy studies, biotinylation assays and statistical methods for quantification of mislocalization were used. Analysis of endogenous expression of BEST1 in MDCK cells revealed the presence of BEST1 transcript but no protein. Confocal microscopy and quantitative analyses indicate that transfected normal human Best1 displays a basolateral localization in MDCK cells, while cell sorting of several Best1 mutants (Y85H, Q96R, L100R, Y227N, Y227E) was altered. In contrast to constitutively active Y227E, constitutively inactive Y227F Best1 mutant localized basolaterally similar to the normal Best1 protein. Our data suggest that at least three basolateral sorting motifs might be implicated in proper Best1 basolateral localization. In addition, non-phosphorylated tyrosine 227 could play a role for basolateral delivery. Full article

Biotechnological approaches using genetic modifications such as homologous gene overexpression can be used to decode gene functions under well-defined circumstances. However, only the recording of the resulting phenotypes allows inferences about the impact of the modification on the organisms’ evolutionary, ecological or economic performance. We here compare a potato wild-type cell line with two genetically engineered cell cultures homologously overexpressing Pathogenesis Related Protein 10a (pr-10a). A detailed analysis of the relative gene-expression patterns of pr-10a and its regulators sebf and pti4 over time provides insights into the molecular response of heterotrophic cells to distinct osmotic and salt-stress conditions. Furthermore, this system serves as an exemplar for the tracing of respiration kinetics as a faster and more sensitive alternative to the laborious and time-consuming recording of growth curves. The utility and characteristics of the resulting data type and the requirements for its appropriate analysis are figured out. It is demonstrated how this novel type of phenotypic information together with the gene-expression-data provides valuable insights into the effect of genetic modifications on the behaviour of cells on both the molecular and the macroscopic level. Full article

This study investigated the 1-year longitudinal effect of professional training in adolescent runners on redox balance during intense endurance exercise. Changes in selected serum oxidant and antioxidant status in response to a 21-km running time trial in 10 runners (15.5 ± 1.3 years) undergoing professional training were evaluated twice in 12 months (pre- and post-evaluation). Venous blood samples were collected immediately before and 4-h following the 21-km run for analysis of serum concentrations of thiobarbituric acid-reactive substances (TBARS), xanthine oxidase (XO), catalase (CAT), reduced glutathione (GSH), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC). In pre-evaluation trial, serum TBARS and SOD decreased after the 21-km run (p < 0.05) while XO, GSH, CAT and TAOC were unchanged. In post-evaluation trial, serum TBARS and SOD decreased, whereas XO and CAT increased post-exercise (p < 0.05). Furthermore, pre-exercise serum T-AOC, post-exercise serum XO, CAT, T-AOC (p < 0.05), and GSH (p = 0.057) appeared to be higher than the corresponding pre-evaluation values. The current findings suggest that a professional training regime in adolescent runners is not likely to jeopardize the development of their antioxidant defense. However, uncertainties in the maintenance of redox balance in runners facing increased exercise-induced oxidative stress as a consequence of training-induced enhancement of exercise capacity await further elucidation. Full article

Clathrin, a three-legged triskelion composed of three clathrin heavy chains (CHCs) and three light chains (CLCs), plays a critical role in clathrin-mediated endocytosis (CME) in eukaryotic cells. In this study, the genes ZmCHC1 and ZmCHC2 encoding clathrin heavy chain in maize were cloned and characterized for the first time in monocots. ZmCHC1 encodes a 1693-amino acid-protein including 29 exons and 28 introns, and ZmCHC2 encodes a 1746-amino acid-protein including 28 exons and 27 introns. The high similarities of gene structure, protein sequences and 3D models among ZmCHC1, and Arabidopsis AtCHC1 and AtCHC2 suggest their similar functions in CME. ZmCHC1 gene is predominantly expressed in maize roots instead of ubiquitous expression of ZmCHC2. Consistent with a typical predicted salicylic acid (SA)-responsive element and four predicted ABA-responsive elements (ABREs) in the promoter sequence of ZmCHC1, the expression of ZmCHC1 instead of ZmCHC2 in maize roots is significantly up-regulated by SA or ABA, suggesting that ZmCHC1 gene may be involved in the SA signaling pathway in maize defense responses. The expressions of ZmCHC1 and ZmCHC2 genes in maize are down-regulated by azide or cold treatment, further revealing the energy requirement of CME and suggesting that CME in plants is sensitive to low temperatures. Full article

MicroRNAs are small, noncoding RNA molecules that regulate a considerable amount of human genes on the post-transcriptional level, and participate in many key biological processes. MicroRNA deregulation has been found associated with major kidney diseases. Here, we summarize current knowledge on the role of microRNAs in renal glomerular and tubular pathologies, with emphasis on the mesangial cell and podocyte dysfunction in diabetic nephropathy, the proximal tubular cell survival in acute kidney injury, the transport function of the thick ascending limb in Ca⁺⁺ imbalance diseases, and the regulation of salt, K⁺ and blood pressure in the distal tubules. Identification of microRNAs and their target genes provides novel therapeutic candidates for treating these diseases. Manipulation of microRNA function with its sense or antisense oligonucleotide enables coordinated regulation of the entire downstream gene network, which has effectively ameliorated several renal disease phenotypes. The therapeutic potentials of microRNA based treatments, though promising, are confounded by major safety issues related to its target specificity, which remain to be fully elucidated. Full article

Hormesis describes the drug action of low dose stimulation and high dose inhibition. The hormesis phenomenon has been observed in a wide range of biological systems. Although known in its descriptive context, the underlying mode-of-action of hormesis is largely unexplored. Recently, the hormesis concept has been receiving increasing attention in the field of aging research. It has been proposed that within a certain concentration window, reactive oxygen species (ROS) or reactive nitrogen species (RNS) could act as major mediators of anti-aging and neuroprotective processes. Such hormetic phenomena could have potential therapeutic applications, if properly employed. Here, we review the current theories of hormetic phenomena in regard to aging and neurodegeneration, with the focus on its underlying mechanism. Facilitated by a simple mathematical model, we show for the first time that ROS-mediated hormesis can be explained by the addition of different biomolecular reactions including oxidative damage, MAPK signaling and autophagy stimulation. Due to their divergent scales, the optimal hormetic window is sensitive to each kinetic parameter, which may vary between individuals. Therefore, therapeutic utilization of hormesis requires quantitative characterizations in order to access the optimal hormetic window for each individual. This calls for a personalized medicine approach for a longer human healthspan. Full article

Spore photoproduct lyase (SPL) repairs a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct, or SP, in germinating endospores. SP is the exclusive DNA photo-damaging product found in endospores; its generation and swift repair by SPL are responsible for the spores’ extremely high UV resistance. Early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair SP in the absence of light. Recently, it has been established that SPL belongs to the radical S-adenosylmethionine (SAM) superfamily. The enzymes in this superfamily utilize a tri-cysteine CXXXCXXC motif to bind a [4Fe-4S] cluster. The cluster provides an electron to the S-adenosylmethionine (SAM) to reductively cleave its C5'-S bond, generating a reactive 5'-deoxyadenosyl (5'-dA) radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. The H atom donor is suggested to be a conserved cysteine141 in B. subtilis SPL; the resulting thiyl radical likely interacts with a neighboring tyrosine99 before oxidizing the 5'-dA to 5'-dA radical and, subsequently, regenerating SAM. These findings suggest SPL to be the first enzyme in the large radical SAM superfamily (>44,000 members) to utilize a radical transfer pathway for catalysis; its study should shed light on the mechanistic understanding of the SAM regeneration process in other members of the superfamily. Full article

The nonstructural protein 3 (NS3) from the hepatitis C virus (HCV) is responsible for processing the non-structural region of the viral precursor polyprotein in infected hepatic cells. NS3 protease activity, located at the N-terminal domain, is a zinc-dependent serine protease. A zinc ion, required for the hydrolytic activity, has been considered as a structural metal ion essential for the structural integrity of the protein. In addition, NS3 interacts with another cofactor, NS4A, an accessory viral protein that induces a conformational change enhancing the hydrolytic activity. Biophysical studies on the isolated protease domain, whose behavior is similar to that of the full-length protein (e.g., catalytic activity, allosteric mechanism and susceptibility to inhibitors), suggest that a considerable global conformational change in the protein is coupled to zinc binding. Zinc binding to NS3 protease can be considered as a folding event, an extreme case of induced-fit binding. Therefore, NS3 protease is an intrinsically (partially) disordered protein with a complex conformational landscape due to its inherent plasticity and to the interaction with its different effectors. Here we summarize the results from a detailed biophysical characterization of this enzyme and present new experimental data. Full article

Growing evidence shows a close association of transposable elements (TE) with non-coding RNAs (ncRNA), and a significant number of small ncRNAs originate from TEs. Further, ncRNAs linked with TE sequences participate in a wide-range of regulatory functions. Alu elements in particular are critical players in gene regulation and molecular pathways. Alu sequences embedded in both long non-coding RNAs (lncRNA) and mRNAs form the basis of targeted mRNA decay via short imperfect base-pairing. Imperfect pairing is prominent in most ncRNA/target RNA interactions and found throughout all biological kingdoms. The piRNA-Piwi complex is multifunctional, but plays a major role in protection against invasion by transposons. This is an RNA-based genetic immune system similar to the one found in prokaryotes, the CRISPR system. Thousands of long intergenic non-coding RNAs (lincRNAs) are associated with endogenous retrovirus LTR transposable elements in human cells. These TEs can provide regulatory signals for lincRNA genes. A surprisingly large number of long circular ncRNAs have been discovered in human fibroblasts. These serve as “sponges” for miRNAs. Alu sequences, encoded in introns that flank exons are proposed to participate in RNA circularization via Alu/Alu base-pairing. Diseases are increasingly found to have a TE/ncRNA etiology. A single point mutation in a SINE/Alu sequence in a human long non-coding RNA leads to brainstem atrophy and death. On the other hand, genomic clusters of repeat sequences as well as lncRNAs function in epigenetic regulation. Some clusters are unstable, which can lead to formation of diseases such as facioscapulohumeral muscular dystrophy. The future may hold more surprises regarding diseases associated with ncRNAs andTEs. Full article

Arginine-Glycine-Aspartic (RGD), is the specific recognition site of integrins with theirs ligands, and regulates cell-cell and cell-extracellular matrix interactions. The RGD motif can be combined with integrins overexpressed on the tumor neovasculature and tumor cells with a certain affinity, becoming the new target for imaging agents, and drugs, and gene delivery for tumor treatment. Further, RGD as a biomimetic peptide can also promote cell adherence to the matrix, prevent cell apoptosis and accelerate new tissue regeneration. Functionalizing material surfaces with RGD can improve cell/biomaterial interactions, which facilitates the generation of tissue-engineered constructs. This paper reviews the main functions and advantages of RGD, describes the applications of RGD in imaging agents, drugs, gene delivery for tumor therapy, and highlights the role of RGD in promoting the development of tissue engineering (bone regeneration, cornea repair, artificial neovascularization) in recent years. Full article

The current models for colorectal cancer (CRC) are essentially linear in nature with a sequential progression from adenoma through to carcinoma. However, these views of CRC development do not explain the full body of published knowledge and tend to discount environmental influences. This paper proposes that CRC is a cellular response to prolonged exposure to cytotoxic agents (e.g., free ammonia) as key events within a sustained high-risk colonic luminal environment. This environment is low in substrate for the colonocytes (short chain fatty acids, SCFA) and consequently of higher pH with higher levels of free ammonia and decreased mucosal oxygen supply as a result of lower visceral blood flow. All of these lead to greater and prolonged exposure of the colonic epithelium to a cytotoxic agent with diminished aerobic energy availability. Normal colonocytes faced with this unfavourable environment can transform into CRC cells for survival through epigenetic reprogramming to express genes which increase mobility to allow migration and proliferation. Recent data with high protein diets confirm that genetic damage can be increased, consistent with greater CRC risk. However, this damage can be reversed by increasing SCFA supply by feeding fermentable fibre as resistant starch or arabinoxylan. High protein, low carbohydrate diets have been shown to alter the colonic environment with lower butyrate levels and apparently greater mucosal exposure to ammonia, consistent with our hypothesis. Evidence is drawn from in vivo and in vitro genomic and biochemical studies to frame experiments to test this proposition. Full article

Applications of laser therapy, including low-level laser therapy (LLLT), phototherapy and photodynamic therapy (PDT), have been proven to be beneficial and relatively less invasive therapeutic modalities for numerous diseases and disease conditions. Using specific types of laser irradiation, specific cellular activities can be induced. Because multiple cellular signaling cascades are simultaneously activated in cells exposed to lasers, understanding the molecular responses within cells will aid in the development of laser therapies. In order to understand in detail the molecular mechanisms of LLLT and PDT-related responses, it will be useful to characterize the specific expression of miRNAs and proteins. Such analyses will provide an important source for new applications of laser therapy, as well as for the development of individualized treatments. Although several miRNAs should be up- or down-regulated upon stimulation by LLLT, phototherapy and PDT, very few published studies address the effect of laser therapy on miRNA expression. In this review, we focus on LLLT, phototherapy and PDT as representative laser therapies and discuss the effects of these therapies on miRNA expression. Full article

Epidermal stem cells sustain the adult skin for a lifetime through self-renewal and the production of committed progenitors. These stem cells generate progeny that will undergo terminal differentiation leading to the development of a protective epidermal barrier. Whereas the molecular mechanisms that govern epidermal barrier repair and renewal have been extensively studied, pathways controlling stem cell differentiation remain poorly understood. Asymmetric cell divisions, small non-coding RNAs (microRNAs), chromatin remodeling complexes, and multiple differentiation factors tightly control the balance of stem and progenitor cell proliferation and differentiation, and disruption of this balance leads to skin diseases. In this review, we summarize and discuss current advances in our understanding of the mechanisms regulating epidermal stem and progenitor cell differentiation, and explore new relationships for maintenance of skin barrier function. Full article

Malaria is one of the most severe tropical infectious diseases. More than 220 million people around the world have a clinical malaria infection and about one million die because of Plasmodium annually. This parasitic pathogen replicates efficiently in its human host making it difficult to eradicate. It is transmitted by mosquito vectors and so far mosquito control programs have not effectively eliminated this transmission. Because of malaria’s enormous health and economic impact and the need to develop new control and eventual elimination strategies, a big research effort has been made to better understand the biology of this parasite and its interactions with its vertebrate host. Determination of the genome sequence and organization, the elucidation of the role of key proteins, and cell signaling studies have helped to develop an understanding of the molecular mechanisms that provide the parasite’s versatility. The parasite can sense its environment and adapt to benefit its survival, indeed this is essential for it to complete its life cycle. For many years we have studied how the Plasmodium parasite is able to sense melatonin. In this review we discuss the melatonin signaling pathway and its role in the control of Plasmodium replication and development. Full article