Int. J. Mol. Sci., Volume 13, Issue 9 (September 2012) – 92 articles , Pages 10660-12152

Among Gram-positive bacteria, CtsR (Class Three Stress gene Repressor) mainly regulates the expression of genes encoding the Clp ATPases and the ClpP protease. To gain a better understanding of the biological significance of the CtsR regulon in response to heat-shock conditions, we performed a global proteomic analysis of Lactobacillus plantarum WCFS1 and ∆ctsR mutant strains under optimal or heat stress temperatures. Total protein extracts from bacterial cells were analyzed by two-dimensional gel fractionation. By comparing maps from different culture conditions and different L. plantarum strains, image analysis revealed 23 spots with altered levels of expression. The proteomic analysis of L. plantarum WCFS1 and ctsR mutant strains confirms at the translational level the CtsR-mediated regulation of some members of the Clp family, as well as the heat induction of typical stress response genes. Heat activation of the putative CtsR regulon genes at transcriptional and translational levels, in the ∆ctsR mutant, suggests additional regulative mechanisms, as is the case of hsp1. Furthermore, isoforms of ClpE with different molecular mass were found, which might contribute to CtsR quality control. Our results could add new outlooks in order to determine the complex biological role of CtsR-mediated stress response in lactic acid bacteria. Full article

Polysaccharides play a key role in enhancing immune function and facilitating cellular communication. Here, we purified Nymphaea rubra Roxb. polysaccharides (NR-PS) by treating them with pullulanase. They were then cultured with immature dendritic cells (DCs) derived from rat bone marrow hematopoietic cells (BMHCs). After treatment with bioactive NR-PS with a degree of polymerization (DP) value of 359.8, we found that the DCs underwent morphological changes indicative of activation. CD80/86 (87.16% ± 8.49%) and MHC class II (52.01% ± 10.11%) expression levels were significantly up-regulated by this treatment compared to the controls (65.45% ± 0.97% and 34.87% ± 1.96%). In parallel, endocytosis was also reduced (167.94% ± 60.59%) after treatment with 25 μg/mL of NR-PS as measured by the medium fluorescence intensity compared to the control (261.67% ± 47.26%). Furthermore, the DCs after treatment with 25 μg/mL NR-PS showed increased IL-12 (102.09 ± 10.16 to 258.78 ± 25.26 pg/mL) and IFN-γ (11.76 ± 0.11 to 15.51 ± 1.66 pg/mL) secretion together with reduced IL-10 secretion (30.75 ± 3.35 to 15.37 ± 2.35 pg/mL), which indicates a T_H1 immune response. In conclusion, NR-PS exhibits stimulatory effects on rat DCs and promotes the secretion of T_H1 cytokines. Taken together, our studies are the first to show that NR-PS is an immunomodulator affecting the maturation and functioning of DCs. Full article

Treatment trends of retinoblastoma (RB) have gradually evolved from eye enucleation and external radiation to local treatment. Combined treatment with an oncolytic virus and chemotherapy is currently a new method in RB treatment. To investigate the therapeutic effect of oncolytic adenovirus SG600 in combination with vincristine (VCR) on retinoblastoma in vitro, the cell viability, cell cycle effects and apoptotic activity of HXO-RB₄₄ cells treated with SG600, VCR or SG600 plus VCR were measured using a cell counting kit-8-based procedure and flow cytometry. Western blot analysis for Akt, p-Akt, p-p53 and p-Rb protein was performed to investigate the underlying mechanisms of combined therapy. The combination therapy exerted a synergistic antitumor effect via a type of G₂/M and S phase arrest rather than the induction of apoptosis. The combination of VCR and SG600 further reduced Akt phosphorylation compared with cells treated with VCR alone, suggesting that SG600 could overcome chemoresistance, perhaps by down-regulating Akt in RB cells. An increase in the expression of p-p53 and decrease in p-Rb expression in HXO-RB₄₄ after co-treatment might be associated with cell cycle block. Western blot examination revealed that VCR might enhance SG600 replication. These results suggest that viro-chemo combination therapy is a feasible and potentially promising approach for the treatment of retinoblastoma. Full article

The small abalone, Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516 (2.26%; average length of 385 bp) reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity (mean heterozygosity = 0.81; mean allelic number = 15.5) was observed in both populations. A statistical analysis of the fixation index (F_ST) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations (F_ST = 0.002, p > 0.05). Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species. Full article

To evaluate the population genetics structure as a means of devising conservation strategies, we developed microsatellite primers for Sophora koreensis, a narrowly endemic and endangered species in Korea. Thirteen polymorphic microsatellite markers were developed in Korean populations of S. koreensis. Genetic diversity was analyzed in 40 individuals from two populations. The number of alleles per locus ranged from 4 to 14, with observed and expected heterozygosities ranging from 0.200 to 1.000 and from 0.189 to 0.864, respectively. The microsatellite markers described here are valuable tools for the population genetics research of S. koreensis. They can be used to obtain information for creating suitable management strategies to conserve this endemic and endangered species. Full article

Siamese mud carp (Henichorynchus siamensis) is a freshwater teleost of high economic importance in the Mekong River Basin. However, genetic data relevant for delineating wild stocks for management purposes currently are limited for this species. Here, we used 454 pyrosequencing to generate a partial genome survey sequence (GSS) dataset to develop simple sequence repeat (SSR) markers from H. siamensis genomic DNA. Data generated included a total of 65,954 sequence reads with average length of 264 nucleotides, of which 2.79% contain SSR motifs. Based on GSS-BLASTx results, 10.5% of contigs and 8.1% singletons possessed significant similarity (E value < 10^–5) with the majority matching well to reported fish sequences. KEGG analysis identified several metabolic pathways that provide insights into specific potential roles and functions of sequences involved in molecular processes in H. siamensis. Top protein domains detected included reverse transcriptase and the top putative functional transcript identified was an ORF2-encoded protein. One thousand eight hundred and thirty seven sequences containing SSR motifs were identified, of which 422 qualified for primer design and eight polymorphic loci have been tested with average observed and expected heterozygosity estimated at 0.75 and 0.83, respectively. Regardless of their relative levels of polymorphism and heterozygosity, microsatellite loci developed here are suitable for further population genetic studies in H. siamensis and may also be applicable to other related taxa. Full article

Indoleamine 2,3-dioxygenase (IDO) has been implicated in preventing the fetus from undergoing maternal T cell-mediated immune responses, yet the mechanism underlying these kinds of IDO-mediated immune responses has not been fully elucidated. Since the CD4 molecule plays a central role in the onset and regulation of antigen-specific immune responses, and T cell is sensitive in the absence of tryptophan, we hypothesize that IDO may reduce cell surface CD4 expression. To test this hypothesis, an adenoviral vector-based construct IDO-EGFP was generated and the effect of IDO-EGFP on CD4 expression was determined on recombinant adenoviral infected C8166 and MT-2 cells, by flow cytometry and/or Western blot analysis. The results revealed a significant downregulation of cell membrane CD4 in pAd-IDOEGFP infected cells when compared to that of mock-infected cells or infection with empty vector pAd-EGFP. Further experiments disclosed that either an addition of tryptophan or IDO inhibitor could partly restore CD4 expression in pAd-IDOEGFP infected C8166 cells. Our findings suggest that downregulation of CD4 by IDO might be one of the mechanisms through which IDO regulates T cell-mediated immune responses. Full article

Azoles are generally fungistatic, and resistance to fluconazole is emerging in several fungal pathogens. In an attempt to find novel azole antifungal agents with improved activity, a series of tetrazole ring bearing acylhydrazone derivatives were synthesized and screened for their in vitro antifungal activity. The mechanism of their antifungal activity was assessed by studying their effect on the plasma membrane using flow cytometry and determination of the levels of ergosterol, a fungal-specific sterol. Propidium iodide rapidly penetrated a majority of yeast cells when they were treated with the synthesized compounds at concentrations just above MIC, implying that fungicidal activity resulted from extensive lesions of the plasma membrane. Target compounds also caused a considerable reduction in the amount of ergosterol. The results also showed that the presence and position of different substituents on the phenyl ring of the acylhydrazone pendant seem to play a role on the antifungal activity as well as in deciding the fungistatic and fungicidal nature of the compounds. Full article

Oxidative stress (OS) contributes to the cascade leading to the dysfunction or death of dopaminergic neurons during Parkinson’s disease (PD). A strategy to prevent the OS of dopaminergic neurons may be the use of phytochemicals as inducers of endogenous antioxidants and phase 2 enzymes. In this study, we demonstrated that treatment of the dopaminergic-like neuroblastoma SH-SY5Y cell line with isothiocyanate erucin (ER), a compound of cruciferous vegetables, resulted in significant increases of both total glutathione (GSH) levels and total antioxidant capacity at the cytosolic level. The increase of GSH levels was associated with an increase in the resistance of SH-SY5Y cells to neuronal death, in terms of apoptosis, induced by 6-hydroxydopamine (6-OHDA). The pretreatment of SH-SY5Y cells with ER was also shown to prevent the redox status impairment, in terms of intracellular ROS and O₂^•− formation, and loss of mitochondrial membrane potential, early events that are initiators of the apoptotic process, induced by 6-OHDA. Last, the antiapoptotic and antioxidant effects of ER were abolished by buthionine sulfoximine, supporting the main role of GSH in the neuroprotective effects recorded by ER. These results suggest that ER may prevent the oxidative damage induced by 6-OHDA. Full article

Graphene nanoplatelet (xGnP) was investigated as a novel reinforcement filler in mechanical properties for poly(lactic acid) (PLA)/epoxidized palm oil (EPO) blend. PLA/EPO/xGnP green nanocomposites were successfully prepared by melt blending method. PLA/EPO reinforced with xGnP resulted in an increase of up to 26.5% and 60.6% in the tensile strength and elongation at break of the nanocomposites respectively, compared to PLA/EPO blend. XRD pattern showed the presence of peak around 26.5° in PLA/EPO nanocomposites which corresponds to characteristic peak of graphene nanoplatelets. However, incorporation of xGnP has no effect on the flexural strength and modulus. Impact strength of PLA/5 wt% EPO improved by 73.6% with the presence of 0.5 wt% xGnP loading. Mechanical properties of PLA were greatly improved by the addition of a small amount of graphene nanoplatelets ( < 1 wt%). Full article

Peanut samples were irradiated (0.0, 5.2, 7.2 or 10.0 kGy), stored for a year (room temperature) and examined every three months. Mycotoxic fungi (MF) were detected in non-irradiated blanched peanuts. A dose of 5.2 kGy was found suitable to prevent MF growth in blanched samples. No MF was detected in in-shell peanuts, with or without irradiation. The colors of the control in-shell and blanched samples were, respectively, 44.72 and 60.21 (L *); 25.20 and 20.38 (Chroma); 53.05 and 86.46 (°Hue). The water activities (Aw) were 0.673 and 0.425. The corresponding fatty acids were 13.33% and 12.14% (C16:0), 44.94% and 44.92% (C18:1, ω9) and 37.10% and 37.63% (C18:2, ω6). The total phenolics (TP) were 4.62 and 2.52 mg GAE/g, with antioxidant activities (AA) of 16.97 and 10.36 μmol TEAC/g. Storage time negatively correlated with Aw (in-shell peanuts) or L *, linoleic acid, TP and AA (in-shell and blanched peanuts) but positively correlated with Aw (blanched peanuts), and with oleic acid (in-shell and blanched peanuts). Irradiation positively correlated with antioxidant activity (blanched peanuts). No correlation was found between irradiation and AA (in-shell samples) or fatty acids and TP (in-shell and blanched peanuts). Irradiation protected against MF and retained both the polyunsaturated fatty acids and polyphenols in the samples. Full article

NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyses the reduction of quinoid compounds to hydroquinones, preventing the generation of free radicals and reactive oxygen. A “C” to “T” transversion at position 609 of NQO1, leading to a nonsynonymous amino acid change (Pro187Ser, P187S), results in an altered enzyme activity. No NQO1 protein activity was detected in NQO1 ⁶⁰⁹TT genotype, and low to intermediate activity was detected in NQO1 ⁶⁰⁹CT genotype compared with ⁶⁰⁹CC genotype. Thus, this polymorphism may result in altered cancer predisposition. For prostate cancer, only sparse data are available. We therefore analyzed the distribution of the NQO1 P187S SNP (single nucleotide polymorphism) in prostate cancer patients and a healthy control group. Allelic variants were determined using RFLP analysis. Overall, 232 patients without any malignancy and 119 consecutive prostate cancer patients were investigated. The genotype distribution in our cohorts followed the Hardy–Weinberg equilibrium in cases and controls. The distribution of the NQO1 codon 187 SNP did not differ significantly between prostate cancer patients and the control group (p = 0.242). There was also no association between the allelic variants and stage or Gleason score of the tumors. The NQO1 P187S SNP was not significantly associated with an increased prostate cancer risk in our cohorts. The SNP has also no influence on histopathological characteristics of the tumors. A combined analysis of all available data from published European studies also showed no significant differences in the genotype distribution between controls and prostate cancer patients. Our data suggest a minor role of the NQO1 nucleotide 609 polymorphism in prostate carcinogenesis. Full article

Chitosan is a naturally occurring cationic polysaccharide and has attracted much attention in the past decade as an important ophthalmic biomaterial. We recently demonstrated that the genipin (GP) cross-linked chitosan is compatible with human retinal pigment epithelial cells. The present work aims to further investigate the in vivo biocompatibility of GP-treated chitosan (GP-chi group) by adopting the anterior chamber of a rabbit eye model. The glutaraldehyde (GTA) cross-linked samples (GTA-chi group) were used for comparison. The 7-mm-diameter membrane implants made from either non-cross-linked chitosan or chemically modified materials with a cross-linking degree of around 80% were inserted in the ocular anterior chamber for 24 weeks and characterized by slit-lamp and specular microscopic examinations, intraocular pressure measurements, and corneal thickness measurements. The interleukin-6 expressions at mRNA level were also detected by quantitative real-time reverse transcription polymerase chain reaction. Results of clinical observations showed that the overall ocular scores in the GTA-chi groups were relatively high. In contrast, the rabbits bearing GP-chi implants in the anterior chamber of the eye exhibited no signs of ocular inflammation. As compared to the non-cross-linked counterparts, the GP-chi samples improved the preservation of corneal endothelial cell density and possessed better anti-inflammatory activities, indicating the benefit action of the GP cross-linker. In summary, the intracameral tissue response to the chemically modified chitosan materials strongly depends on the selection of cross-linking agents. Full article

The interactions between chemosensors, 3-amino-5-(4,5,6,7-tetrahydro-1H-indol-2-yl)isoxazole-4-carboxamide (AIC) derivatives, and different anions (F⁻ Cl⁻, Br⁻, AcO⁻, and H₂PO₄⁻) have been theoretically investigated using DFT approaches. It turned out that the unique selectivity of AIC derivatives for F⁻ is ascribed to their ability of deprotonating the host sensors. Frontier molecular orbital (FMO) analyses have shown that the vertical electronic transitions of absorption and emission for the sensing signals are characterized as intramolecular charge transfer (ICT). The study of substituent effects suggests that all the substituted derivatives are expected to be promising candidates for fluoride chemosensors both in UV-vis and fluorescence spectra except for derivative with benzo[d]thieno[3,2-b]thiophene fragment that can serve as ratiometric fluorescent fluoride chemosensor only. Full article

Ionic liquid based, ultrasound-assisted extraction was successfully applied to the extraction of phenolcarboxylic acids, carnosic acid and rosmarinic acid, from Rosmarinus officinalis. Eight ionic liquids, with different cations and anions, were investigated in this work and [C₈mim]Br was selected as the optimal solvent. Ultrasound extraction parameters, including soaking time, solid–liquid ratio, ultrasound power and time, and the number of extraction cycles, were discussed by single factor experiments and the main influence factors were optimized by response surface methodology. The proposed approach was demonstrated as having higher efficiency, shorter extraction time and as a new alternative for the extraction of carnosic acid and rosmarinic acid from R. officinalis compared with traditional reference extraction methods. Ionic liquids are considered to be green solvents, in the ultrasound-assisted extraction of key chemicals from medicinal plants, and show great potential. Full article

In this study we aimed to confirm the emerging role of Chromatin Assembly Factor 1 (CAF-1 p60) as a new proliferation and prognostic marker for cancer and to test the usefulness of the tissue microarray technique (TMA) for CAF-1 p60 rapid screening in several human malignancies. CAF-1 is a histone chaperone, regulating chromatin dynamics during DNA replication and repair in eukaryotics. TMA is a powerful high-throughput methodology in the study of cancer, allowing simultaneous assessment of different biomarkers within large numbers of tissue specimens. We generated TMA taking 3 mm diameter-core biopsies from oral squamous cell carcinoma, prostate cancer, salivary gland tumours and skin melanoma specimens, which had been previously tested for CAF-1 p60 on routine tissue sections. We also analysed, for the first time, 30 larynx and 30 skin squamous cell carcinomas. CAF-1 p60 resulted over-expressed in both the tissue sections and the TMA specimens, with the highest levels of expression in tumours which were more aggressive and metastasizing. Notably, a high degree of agreement was found between the CAF-1 p60 assessment on TMAs and on routine tissue sections. Our findings confirm the prognostic role of CAF-1 p60 and indicate TMA as a really advantageous method for CAF-1 p60 immunohistochemical screening, allowing savings on both tissue quantity and operator-time. Full article

We present a method to modulate the fluorescence of non-polar single squaraine-derived rotaxanes molecules embedded in a polar poly(methyl methacrylate) (PMMA) matrix under an external electric field. The electron transfer between single molecules and the electron acceptors in a PMMA matrix contributes to the diverse responses of fluorescence intensities to the electric field. The observed instantaneous and non-instantaneous electric field dependence of single-molecule fluorescence reflects the redistribution of electron acceptors in PMMA induced by electronic polarization and orientation polarization of polar polymer chains in an electric field. Full article

Cephalotaxus oliveri is a scarce medicinal conifer endemic to the south central region of China and Vietnam. A small fragmented population presently exists due to anthropogenic disturbance. C. oliveri has been used for its alkaloids harringtonine and homoharringtonine, which are effective against leucocythemia and lymphadenosarcoma. Monoecious plants have been detected in nature, although they were understood to be dioecious. In order to study the mating system, population genetics and the genetic effects of habitat fragmentation on C. oliveri, 15 polymorphic and 12 monomorphic microsatellite loci were developed for C. oliveri by using the Fast Isolation by AFLP of Sequences Containing repeats (FIASCO) protocol. The polymorphisms were assessed in 96 individuals from three natural populations (32 individuals per population). The number of alleles per locus ranged from two to 33, the observed and expected heterozygosity per locus ranged from 0.000 to 1.000 and from 0.000 to 0.923, respectively. These loci would facilitate a comprehensive understanding of the genetic dynamics on C. oliveri, which will be useful for establishing effective conservation strategies for this species. Full article

Since the primordial of humanity, pollen has been considered a good source of nutrients and energy. Its promising healing properties have also been referred to. The present study aimed to characterize, for the first time, eight commercial pollens from Portugal and Spain available on the market studying the legislation on labeling, pollinic origin, physicochemical and microbiological analyses and identification of yeasts. Eleven botanical families were found amongst the samples. The most abundant family and the most dominant pollen was Cistaceae. The moisture content, ash, a_w, pH, reducing sugars, carbohydrates, proteins, lipids and energy were analyzed and the specific parameters were within the specifications required by some countries with legislation regarding these parameters. Microbiologically commercial pollen showed acceptable safety for the commercial quality and hygiene. All samples showed negative results for toxigenic species. The microorganisms studied were aerobic mesophiles, yeasts and moulds, coliforms, Escherichia coli, Staphylococcus aureus, Salmonella and sulfite-reducing Clostridium. During the work, six yeasts species were isolated from pollen, with Rhodotorula mucilaginosa being the most abundant, as it was present in four samples. Full article

In this study, 13 polymorphic microsatellite markers were isolated from the Phaseolus vulgaris L. (common bean) by using the Fast Isolation by AFLP of Sequence COntaining Repeats (FIASCO) protocol. These markers revealed two to seven alleles, with an average of 3.64 alleles per locus. The polymorphic information content (PIC) values ranged from 0.055 to 0.721 over 13 loci, with a mean value of 0.492, and 7 loci having PIC greater than 0.5. The expected heterozygosity (H_E) and observed heterozygosity (H_O) levels ranged from 0.057 to 0.814 and from 0.026 to 0.531, respectively. Cross-species amplification of the 13 prime pairs was performed in its related specie of Vigna unguiculata L. Seven out of all these markers showed cross-species transferability. These markers will be useful for future genetic diversity and population genetics studies for this agricultural specie and its related species. Full article

Four novel hydrogels based on chitosan were synthesized via a cross-linking reaction of chitosan with different concentrations of oxalyl bis 4-(2,5-dioxo-2H-pyrrol-1(5H)-yl)benzamide. Their structures were confirmed by fourier transform infrared X-ray (FTIR), scanning electron microscopy (SEM) and X-ray diffraction. The antimicrobial activities of the hydrogels against two crop-threatening pathogenic fungi namely: Aspergillus fumigatus (A. fumigatus, RCMBA 06002), and Aspergillus niger (A. niger, RCMBA 06106), and five bacterial species namely: Bacillis subtilis (B. subtilis, RCMBA 6005), Staphylococcus aureus (S. aureus, RCMBA 2004), Streptococcus pneumoniae (S. pneumonia, RCMB 000101) as Gram positive bacteria, and Salmonella typhimurium (S. typhimurium, RCMB 000104), and Escherichia coli (E. coli, RCMBA 5003) as Gram negative bacteria have been investigated. The prepared hydrogels showed much higher antimicrobial activities than that of the parent chitosan. The hydrogels were more potent in case of Gram-positive bacteria than Gram-negative bacteria. Increasing the degree of cross-linking in the hydrogels resulted in a weaker antimicrobial activity. Full article

Inducible Nitric Oxide Synthase (iNOS) has been involved in a variety of diseases, and thus it is interesting to discover and optimize new iNOS inhibitors. In previous studies, a series of benzimidazole-quinolinone derivatives with high inhibitory activity against human iNOS were discovered. In this work, three-dimensional quantitative structure-activity relationships (3D-QSAR), molecular docking and molecular dynamics (MD) simulation approaches were applied to investigate the functionalities of active molecular interaction between these active ligands and iNOS. A QSAR model with R² of 0.9356, Q² of 0.8373 and Pearson-R value of 0.9406 was constructed, which presents a good predictive ability in both internal and external validation. Furthermore, a combined analysis incorporating the obtained model and the MD results indicates: (1) compounds with the proper-size hydrophobic substituents at position 3 in ring-C (R₃ substituent), hydrophilic substituents near the X₆ of ring-D and hydrophilic or H-bond acceptor groups at position 2 in ring-B show enhanced biological activities; (2) Met368, Trp366, Gly365, Tyr367, Phe363, Pro344, Gln257, Val346, Asn364, Met349, Thr370, Glu371 and Tyr485 are key amino acids in the active pocket, and activities of iNOS inhibitors are consistent with their capability to alter the position of these important residues, especially Glu371 and Thr370. The results provide a set of useful guidelines for the rational design of novel iNOS inhibitors. Full article

We previously reported a gradual increase of relative mitochondrial DNA (mtDNA) copy number during the progression of esophageal squamous cell carcinoma (ESCC). Because mitochondria are the intracellular organelles responsible for ATP production, we investigated the associations among mtDNA copy number, mitochondrial bioenergetic function, tumor invasion and the expression levels of epithelial mesenchymal transition (EMT) markers in a series of seven ESCC cell lines, including 48T, 81T, 146T, TE1, TE2, TE6 and TE9. Among them, TE1 had the highest relative mtDNA copy number of 240.7%. The mRNA of mtDNA-encoded ND1 gene (2.80), succinate-supported oxygen consumption rate (11.21 nmol/min/10⁶ cells), ATP content (10.7 fmol/cell), and the protein level of mitochondrial transcription factor A (TFAM) were the highest and the lactate concentration in the culture medium (3.34 mM) was the lowest in TE1. These findings indicate that TE1 exhibited the highest bioenergetic function of mitochondria. Furthermore, TE1 showed the highest trans-well migration activity of 223.0 cells/field, the highest vimentin but the lowest E-cadherin protein expression levels, which suggest that TE1 had the highest invasion capability. We then conducted a knockdown study using pLKO.1-based lentiviral particles to infect TE1 cells to suppress the expression of TFAM. Molecular analyses of the parental TE1, control TE1-NT and TFAM knockdown TE1-sh-TFAM(97) cells were performed. Interestingly, as compared to the control TE1-NT, TE1-sh-TFAM(97) exhibited lower levels of the relative mtDNA copy number (p = 0.001), mRNA of mtDNA-encoded ND1 gene (p = 0.050), succinate-supported oxygen consumption rate (p = 0.065), and ATP content (p = 0.007), but had a higher lactate concentration in the culture medium (p = 0.010) and higher protein level of lactate dehydrogenase. A decline in mitochondrial bioenergetic function was observed in TE1-sh-TFAM(97). Significantly, compared to the control TE1-NT, TE1-sh-TFAM(97) had a lower trans-well migration activity (p < 0.001), a higher E-cadherin level but a lower vimentin protein level, which indicates a decrease of invasiveness. Taken together, we suggest that high relative mtDNA copy number and bioenergetic function of mitochondria may confer an advantage for tumor invasion of ESCC. Full article

The dried roots of Euphorbia kansui (kansui) have been used for centuries in China as a herbal medicine for edema, ascites, and asthma. The 95% ethanol extract showed a significant inhibition of cell proliferation against human normal cell lines L-O2 and GES-1. Bioassay-guided separation of the 95% ethanol extract from the roots of E. kansui led to the isolation of 12 diverse terpenoids whose structures were identified by ¹H, ¹³C NMR spectroscopy and ESI-MS as kansuinine A (1), kansuinine B (2), kansuinine C (3), kansuiphorin C (4), 3-O-(2'E,4'Z-decadienoyl)-20-O-acetylingenol (5), 3-O-(2'E,4'E-decadienoyl)-20-O-acetylingenol (6), 3-O-(2'E,4'Z-decadienoyl)-20-deoxyingenol (7), 3-O-benzoyl-20-deoxyingenol (8), 5-O-benzoyl-20-deoxyingenol (9), kansenone (10), epi-kansenone (11), euphol (12). All these 12 terpernoids were evaluated in vitro for cytotoxicity on L-O2 and GES-1 cell lines. Most ingenane-type diterpenoids and 8-ene-7-one triterpenoids (5–11) exhibited a relatively lower IC₅₀ value; therefore, these compounds had stronger cytotoxicity against human normal cell lines L-O2 and GES-1 with dose-dependent relationships. These results will be significantly helpful to reveal the mechanism of toxicity of kansui and to effectively guide safer clinical application of this herb. Full article

Mangiferin is a natural xanthonoid with various biological activities. Quantification of mangiferin in fruit peel, pulp, and seed kernel was carried out in 11 Chinese mango (Mangifera indica L.) cultivars. The highest mangiferin content was found in the peel of Lvpimang (LPM) fruit (7.49 mg/g DW). Efficient purification of mangiferin from mango fruit peel was then established for the first time by combination of macroporous HPD100 resin chromatography with optimized high-speed counter-current chromatography (HSCCC). Purified mangiferin was identified by both HPLC and LC-MS, and it showed higher DPPH^• free-radical scavenging capacities and ferric reducing ability of plasma (FRAP) than by l-ascorbic acid (Vc) or Trolox. In addition, it showed significant protective effects on human umbilical vein endothelial cells (HUVEC) under H₂O₂-induced stress. Cells treated with mangiferin resulted in significant enhanced cell survival under of H₂O₂ stress. Therefore, mangiferin from mango fruit provides a promising perspective for the prevention of oxidative stress-associated diseases. Full article

Ustiloxins are cyclopeptide mycotoxins produced by the pathogenic fungus Villosiclava virens of rice false smut. Ustiloxins A and B as two main mycotoxins were determined conveniently by LC-ESI-MS in the water extract from rice false smut balls which were mostly composed of the chlamydospores and mycelia of the pathogen. Both ustiloxins A and B in the water extract were also quantitatively analyzed by HPLC. This is the first report on the determination and analysis of ustiloxins A and B simultaneously by LC-ESI-MS and HPLC in false smut balls of rice. Full article

Fish oil was extracted from the viscera of African Catfish using supercritical carbon dioxide (SC-CO₂). A Central Composite Design of Response Surface methodology (RSM) was employed to optimize the SC-CO₂ extraction parameters. The oil yield (Y) as response variable was executed against the four independent variables, namely pressure, temperature, flow rate and soaking time. The oil yield varied with the linear, quadratic and interaction of pressure, temperature, flow rate and soaking time. Optimum points were observed within the variables of temperature from 35 °C to 80 °C, pressure from 10 MPa to 40 MPa, flow rate from 1 mL/min to 3 mL/min and soaking time from 1 h to 4 h. However, the extraction parameters were found to be optimized at temperature 57.5 °C, pressure 40 MPa, flow rate 2.0 mL/min and soaking time 2.5 h. At this optimized condition, the highest oil yields were found to be 67.0% (g oil/100 g sample on dry basis) in the viscera of catfish which was reasonable to the yields of 78.0% extracted using the Soxhlet method. Full article

We recently identified a novel dataset of snRNA-like trascriptional units in the human genome. The investigation of a subset of these elements showed that they play relevant roles in physiology and/or pathology. In this work we expand our collection of small RNAs taking advantage of a newly developed algorithm able to identify genome sequence stretches with RNA polymerase (pol) III type 3 promoter features thus constituting putative pol III binding sites. The bioinformatic analysis of a subset of these elements that map in introns of protein-coding genes in antisense configuration suggest their association with alternative splicing, similarly to other recently characterized small RNAs. Interestingly, the analysis of the transcriptional activity of these novel promoters shows that they are active in a cell-type specific manner, in accordance with the emerging body of evidence of a tissue/cell-specific activity of pol III. Full article

The industrial use of sucrose phosphorylase (SP), an interesting biocatalyst for the selective transfer of α-glucosyl residues to various acceptor molecules, has been hampered by a lack of long-term stability and low activity towards alternative substrates. We have recently shown that the stability of the SP from Bifidobacterium adolescentis can be significantly improved by the formation of a cross-linked enzyme aggregate (CLEA). In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate. To obtain proof of concept, SP was imprinted with α-glucosyl glycerol and subsequently cross-linked with glutaraldehyde. As a consequence, the enzyme’s specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C. This procedure can be performed in an aqueous environment and gives rise to a new enzyme formulation called iCLEA. Full article

Passiflora contracta Vitta (Passifloraceae) is an endemic species of the Atlantic Rainforest, one of the most species-rich ecoregions in the world, although extremely endangered. We have developed an enriched microsatellite library in order to fine-scale studies of the genetic structure of P. contracta. Twelve pairs of microsatellite primers were designed, and seven loci were successfully amplified and characterized by genotyping two wild populations of P. contracta. All seven loci were polymorphic, with an average number of alleles found being 4.8 and 5 per population. The cross-species transferability was tested using sister species Passiflora ovalis Vell. Ex Roemer. The development of these markers will contribute to the studies of population genetics in P. contracta as well as future studies concerning diversity patterns in the Atlantic Rainforest, and may also help to establish strategies for the conservation of this species. Full article

Halimodendron halodendron has been used as forage in northwestern China for a long time. Its young leaves and flowers are edible and favored by indigenous people. In this study, eleven phenolic compounds were bioassay-guided and isolated from the aerial parts of H. halodendron for the first time. They were identified by means of physicochemical and spectrometric analysis as quercetin (1), 3,5,7,8,4'-pentahydroxy-3'-methoxy flavone (2), 3-O-methylquercetin (3), 3,3'-di-O-methylquercetin (4), 3,3'-di-O-methylquercetin-7-O-β-D-glucopyranoside (5), isorhamentin-3-O-β-D-rutinoside (6), 8-O-methylretusin (7), 8-O-methylretusin-7-O-β-D-glucopyranoside (8), salicylic acid (9), p-hydroxybenzoic acid (ferulic acid) (10), and 4-hydroxy-3-methoxy cinnamic acid (11). They were sorted as flavonols (1–6), soflavones (7 and 8), and phenolic acids (9–11). Among the compounds, flanools 1–4 revealed a strong antibacterial activity with minimum inhibitory concentration (MIC) values of 50–150 μg/mL, and median inhibitory concentration (IC₅₀) values of 26.8–125.1 μg/mL. The two isoflavones (7 and 8) showed moderate inhibitory activity on the test bacteria. Three phenolic acids (9, 10 and 11) showed strong antibacterial activity with IC₅₀ values of 28.1–149.7 μg/mL. Antifungal activities of the compounds were similar to their antibacterial activities. All these phenolic compounds showed significant antimicrobial activity with a broad spectrum as well as antioxidant activity based on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and β-carotene-linoleic acid bleaching assays. In general, the flavonol aglycones with relatively low polarity exhibited stronger activities than the glycosides. The results suggest the potential of this plant as a source of functional food ingredients and provide support data for its utilization as forage as well. Full article

Response surface methodology (central composite design of experiments) was employed to simultaneously optimize enzyme production and productivities of two ligninolytic enzymes produced by Ceriporiopsis subvermispora. Concentrations of glucose, ammonium tartrate and Polysorbate 80 were varied to establish the optimal composition of liquid media (OLM), where the highest experimentally obtained activities and productivities were 41 U L⁻¹ and 16 U L⁻¹ day⁻¹ for laccase (Lac), and 193 U L⁻¹ and 80 U L⁻¹ day⁻¹ for manganese peroxidase (MnP). Considering culture growth in OLM on various types of immobilization support, the best results were obtained with 1 cm beech wood cubes (BWCM). Enzyme activities in culture filtrate were 152 U L⁻¹ for Lac and 58 U L⁻¹ for MnP, since the chemical composition of this immobilization material induced higher Lac activity. Lower enzyme activities were obtained with polyurethane foam. Culture filtrates of OLM and BWCM were applied for dye decolorization. Remazol Brilliant Blue R (RBBR) was decolorized faster and more efficiently than Copper(II)phthalocyanine (CuP) with BWCM (80% and 60%), since Lac played a crucial role. Decolorization of CuP was initially faster than that of RBBR, due to higher MnP activities in OLM. The extent of decolorization after 14 h was 60% for both dyes. Full article

The FLOWERING LOCUS T (FT) gene plays crucial roles in regulating the transition from the vegetative to reproductive phase. To understand the molecular mechanism of reproduction, three homologous FT genes were isolated and characterized from Cymbidium sinense “Qi Jian Bai Mo”, Cymbidium goeringii and Cymbidium ensifolium “Jin Si Ma Wei”. The three genes contained 618-bp nucleotides with a 531-bp open reading frame (ORF) of encoding 176 amino acids (AAs). Alignment of the AA sequences revealed that CsFT, CgFT and CeFT contain a conserved domain, which is characteristic of the PEBP-RKIP superfamily, and which share high identity with FT of other plants in GenBank: 94% with OnFT from Oncidium Gower Ramsey, 79% with Hd3a from Oryza sativa, and 74% with FT from Arabidopsis thaliana. qRT-PCR analysis showed a diurnal expression pattern of CsFT, CgFT and CeFT following both long day (LD, 16-h light/8-h dark) and short day (SD, 8-h light/16-h dark) treatment. While the transcripts of both CsFT and CeFT under LD were significantly higher than under SD, those of CgFT were higher under SD. Ectopic expression of CgFT in transgenic Arabidopsis plants resulted in early flowering compared to wild-type plants and significant up-regulation of APETALA1 (AP1) expression. Our data indicates that CgFT is a putative phosphatidylethanolamine-binding protein gene in Cymbidium that may regulate the vegetative to reproductive transition in flowers, similar to its Arabidopsis ortholog. Full article

Diamondoids in crude oil are useful for assessing the maturity of oil in high maturation. However, they are very difficult to separate and accurately quantify by conventional geochemical methods due to their low abundance in oil. In this paper, we use comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) to study the compounds in condensates from the Junggar Basin of northwest China and address their geological and geochemical applications. GC×GC-TOFMS improves the resolution and separation efficiency of the compounds. It not only separates the compounds that coelute in conventional GC-MS (e.g., 4, 8-dimethyl-diamantane and trimethyl-diamantane) but also allows the identification of compounds that were not previously detected (e.g., trimethyl-diamantane (15A)). A reversed-phase column system improves the separation capabilities over the normal phase column system. The diamondoid indexes indicate that a representative condensate from Well DX 10 is highly mature with equivalent Ro being approximately 1.5%. Full article

Berkleasmium sp. Dzf12, an endophytic fungus from Dioscorea zingiberensis, is a high producer of spirobisnaphthalenes with various bioactivities. The exopolysaccharide (EPS) produced by this fungus also shows excellent antioxidant activity. In this study, the experimental designs based on statistics were employed to evaluate and optimize the medium for EPS production in liquid culture of Berkleasmium sp. Dzf12. For increasing EPS yield, the concentrations of glucose, peptone, KH₂PO₄, MgSO₄∙7H₂O and FeSO₄∙7H₂O in medium were optimized using response surface methodology (RSM). Both the fractional factorial design (FFD) and central composite design (CCD) were applied to optimize the main factors which significantly affected EPS production. The concentrations of glucose, peptone and MgSO₄∙7H₂O were found to be the main effective factors for EPS production by FFD experimental analysis. Based on the further CCD optimization and RSM analysis, a quadratic polynomial regression equation was derived from the EPS yield and three variables. Statistical analysis showed the polynomial regression model was in good agreement with the experimental results with the determination coefficient (adj-R²) as 0.9434. By solving the quadratic regression equation, the optimal concentrations of glucose, peptone and MgSO₄∙7H₂O for EPS production were determined as 63.80, 20.76 and 2.74 g/L, respectively. Under the optimum conditions, the predicted EPS yield reached the maximum (13.22 g/L). Verification experiment confirmed the validity with the actual EPS yield as 13.97 g/L, which was 6.29-fold in comparison with that (2.22 g/L) in the original basal medium. The results provide the support data for EPS production in large scale and also speed up the application of Berkleasmium sp. Dzf12. Full article

Cannabinoids and opioids systems share numerous pharmacological properties and antinociception is one of them. Previous findings have shown that mitragynine (MG), a major indole alkaloid found in Mitragyna speciosa (MS) can exert its antinociceptive effects through the opioids system. In the present study, the action of MG was investigated as the antinociceptive agent acting on Cannabinoid receptor type 1 (CB1) and effects on the opioids receptor. The latency time was recorded until the mice showed pain responses such as shaking, licking or jumping and the duration of latency was measured for 2 h at every 15 min interval by hot plate analysis. To investigate the beneficial effects of MG as antinociceptive agent, it was administered intraperitoneally 15 min prior to pain induction with a single dosage (3, 10, 15, 30, and 35 mg/kg b.wt). In this investigation, 35 mg/kg of MG showed significant increase in the latency time and this dosage was used in the antagonist receptor study. The treated groups were administered with AM251 (cannabinoid receptor-1 antagonist), naloxone (non-selective opioid antagonist), naltrindole (δ-opioid antagonist) naloxonazine (µ₁-receptor antagonist) and norbinaltorpimine (κ-opioid antagonist) respectively, prior to administration of MG (35 mg/kg). The results showed that the antinociceptive effect of MG was not antagonized by AM251; naloxone and naltrindole were effectively blocked; and norbinaltorpimine partially blocked the antinociceptive effect of MG. Naloxonazine did inhibit the effect of MG, but it was not statistically significant. These results demonstrate that CB1 does not directly have a role in the antinociceptive action of MG where the effect was observed with the activation of opioid receptor. Full article

Aminopropyl-functionalized SBA-15 mesoporous silica was used as a support to adsorb myoglobin. Then, in order to avoid the leakage of adsorbed myoglobin, lysozyme was covalently tethered to the internal and external surface of the mesoporous silica with glutaraldehyde as the coupling agent. The property of amino-functionalized mesoporous silica was characterized by N₂ adsorption-desorption and thermogravimetric (TG) analysis. The feature of the silica-based matrix before and after myoglobin adsorption was identified by fourier transform infrared (FTIR) and UV/VIS measurement. With o-dianisidine and H₂O₂ as the substrate, the peroxidase activity of adsorbed myoglobin was determined. With Micrococus lysodeilicus as the substrate, the antibacterial activity of covalently tethered lysozyme was measured. Results demonstrated that the final product not only presented peroxidase activity of the myoglobin but yielded antibacterial activity of the lysozyme. Full article

The Jinchuan yak is a new yak population identified in Sichuan, China. This population has a special anatomical characteristic: an additional pair of ribs compared with other yak breeds. The genetic structure of this population is unknown. In the present study, we investigated the maternal phylogeny of this special yak population using the mitochondrial DNA variation. A total of 23 Jinchuan yaks were sequenced for a 823-bp fragment of D-loop control region and three individuals were sequenced for the whole mtDNA genome with a length of 16,371-bp. To compare with the data from other yaks, we extracted sequence data from Genebank, including D-loop of 398 yaks (from 12 breeds) and 55 wild yaks, and whole mitochondrial genomes of 53 yaks (from 12 breeds) and 21 wild yaks. A total of 127 haplotypes were defined, based on the D-loop data. Thirteen haplotypes were defined from 23 mtDNA D-loop sequences of Jinchuan yaks, six of which were shared only by Jinchuan, and one was shared by Jinchuan and wild yaks. The Jinquan yaks were found to carry clades A and B from lineage I and clade C of lineage II, respectively. It was also suggested that the Jinchuan population has no distinct different phylogenetic relationship in maternal inheritance with other breeds of yak. The highly haplotype diversity of the Pali breed, Jinchuan population, Maiwa breed and Jiulong breed suggested that the yak was first domesticated from wild yaks in the middle Himalayan region and the northern Hengduan Mountains. The special anatomic characteristic that we found in the Jinchuan population needs further studies based on nuclear data. Full article

Powdery mildew (PM), caused by fungus Erysiphe necator, is one of the most devastating diseases of grapevine. To better understand grapevine-PM interaction and provide candidate resources for grapevine breeding, a suppression subtractive hybridization (SSH) cDNA library was constructed from E. necator-infected leaves of a resistant Chinese wild Vitis quinquangularis clone “Shang-24”. A total of 492 high quality expressed sequence tags (ESTs) were obtained and assembled into 266 unigenes. Gene ontology (GO) analysis indicated that 188 unigenes could be assigned with at least one GO term in the biological process category, and 176 in the molecular function category. Sequence analysis showed that a large number of these genes were homologous to those involved in defense responses. Genes involved in metabolism, photosynthesis, transport and signal transduction were also enriched in the library. Expression analysis of 13 selected genes by qRT-PCR revealed that most were induced more quickly and intensely in the resistant material “Shang-24” than in the sensitive V. pseudoreticulata clone “Hunan-1” by E. necator infection. The ESTs reported here provide new clues to understand the disease-resistance mechanism in Chinese wild grapevine species and may enable us to investigate E. necator-responsive genes involved in PM resistance in grapevine germplasm. Full article

Two new 14-membered cyclopeptide alkaloids, Oxyphylline B (4) and Oxyphylline C (5), along with three known 13-membered cyclopeptide alkaloids, were isolated from stem and roots of Zizyphus oxyphylla Edgew. The compounds were tested for antibacterial activity. Oxyphylline B (4) showed comparatively better antibacterial activities against Escherichia coli (MIC, 5 µg/mL) than other compounds. This compound also exhibited weak antimicrobial activities against Staphylococcus aureus (MIC, 25 µg/mL), Pseudomonas aeruginosa (MIC, 50 µg/mL) and Salmonella typhi (MIC, 50 µg/mL). Full article

Recent evidence suggests that the development of castration resistant prostate cancer (CRPCa) is commonly associated with an aberrant, ligand-independent activation of the androgen receptor (AR). A putative mechanism allowing prostate cancer (PCa) cells to grow under low levels of androgens, is the expression of constitutively active, C-terminally truncated AR lacking the AR-ligand binding domain (LBD). Due to the absence of a LBD, these receptors, termed ARΔLBD, are unable to respond to any form of anti-hormonal therapies. In this study we demonstrate that the multikinase inhibitor sorafenib inhibits AR as well as ARΔLBD-signalling in CRPCa cells. This inhibition was paralleled by proteasomal degradation of the AR- and ARΔLBD-molecules. In line with these observations, maximal antiproliferative effects of sorafenib were achieved in AR and ARΔLBD-positive PCa cells. The present findings warrant further investigations on sorafenib as an option for the treatment of advanced AR-positive PCa. Full article

The aim of this work was to compare protective and anti-apoptotic effects of α-tocopherol at nanomolar and micromolar concentrations against 0.2 mM H₂O₂-induced toxicity in the PC12 neuronal cell line and to reveal protein kinases that contribute to α-tocopherol protective action. The protection by 100 nM α-tocopherol against H₂O₂-induced PC12 cell death was pronounced if the time of pre-incubation with α-tocopherol was 3–18 h. For the first time, the protective effect of α-tocopherol was shown to depend on its concentration in the nanomolar range (1 nM < 10 nM < 100 nM), if the pre-incubation time was 18 h. Nanomolar and micromolar α-tocopherol decreased the number of PC12 cells in late apoptosis induced by H₂O₂ to the same extent if pre-incubation time was 18 h. Immunoblotting data showed that α-tocopherol markedly diminished the time of maximal activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt)-induced in PC12 cells by H₂O₂. Inhibitors of MEK 1/2, PI 3-kinase and protein kinase C (PKC) diminished the protective effect of α-tocopherol against H₂O₂-initiated toxicity if the pre-incubation time was long. The modulation of ERK 1/2, Akt and PKC activities appears to participate in the protection by α-tocopherol against H₂O₂-induced death of PC12 cells. The data obtained suggest that inhibition by α-tocopherol in late stage ERK 1/2 and Akt activation induced by H₂O₂ in PC12 cells makes contribution to its protective effect, while total inhibition of these enzymes is not protective. Full article

Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated. Incubation of rat brain mitochondria with biotinylated ubiquitin followed by affinity chromatography on avidin-agarose, intensive washing, tryptic digestion of proteins bound to the affinity sorbent and their mass spectrometry analysis resulted in reliable identification of 50 proteins belonging to mitochondrial and extramitochondrial compartments. Since all these proteins were bound to avidin-agarose only after preincubation of the mitochondrial fraction with biotinylated ubiquitin, they could therefore be referred to as specifically bound proteins. A search for specific ubiquitination signature masses revealed several extramitochondrial and intramitochondrial ubiquitinated proteins representing about 20% of total number of proteins bound to avidin-agarose. The interactome analysis suggests that the identified non-ubiquitinated proteins obviously form tight complexes either with ubiquitinated proteins or with their partners and/or mitochondrial membrane components. Results of the present study demonstrate that the use of biotinylated ubiquitin may be considered as the method of choice for in vitro evaluation of endogenous ubiquitin-conjugating machinery in particular subcellular organelles and changes in ubiquitin/organelle associated interactomes. This may be useful for evaluation of changes in interactomes induced by protein ubiquitination under norm and various brain pathologies. Full article

The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of (R,S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide (DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications. Full article

Doxorubicin (DOX) is currently used in cancer chemotherapy to treat many tumors and shows improved delivery, reduced toxicity and higher treatment efficacy when being part of nanoscale delivery systems. However, a major drawback remains its toxicity to healthy tissue and the development of multi-drug resistance during prolonged treatment. This is why in our work we aimed to improve DOX delivery and reduce the toxicity by chemical conjugation with a new nanoplatform based on polymalic acid. For delivery into recipient cancer cells, DOX was conjugated via pH-sensitive hydrazone linkage along with polyethylene glycol (PEG) to a biodegradable, non-toxic and non-immunogenic nanoconjugate platform: poly(β-L-malic acid) (PMLA). DOX-nanoconjugates were found stable under physiological conditions and shown to successfully inhibit in vitro cancer cell growth of several invasive breast carcinoma cell lines such as MDA-MB-231 and MDA-MB- 468 and of primary glioma cell lines such as U87MG and U251. Full article

Wax esters are long-chain esters that have been widely applied in premium lubricants, parting agents, antifoaming agents and cosmetics. In this study, the biocatalytic preparation of a specific wax ester, cetyl octanoate, is performed in n-hexane using two commercial immobilized lipases, i.e., Lipozyme^® RMIM (Rhizomucor miehei) and Novozym^® 435 (Candida antarctica). Response surface methodology (RSM) and 5-level-4-factor central composite rotatable design (CCRD) are employed to evaluate the effects of reaction time (1–5 h), reaction temperature (45–65 °C), substrate molar ratio (1–3:1), and enzyme amount (10%–50%) on the yield of cetyl octanoate. Using RSM to optimize the reaction, the maximum yields reached 94% and 98% using Lipozyme^® RMIM and Novozym^® 435, respectively. The optimum conditions for synthesis of cetyl octanoate by both lipases are established and compared. Novozym^® 435 proves to be a more efficient biocatalyst than Lipozyme^® RMIM. Full article

The radioprotective effect of anthocyanin extracted from Lonicera caerulea var. edulis (ALC), was studied in ICR mice. Different doses of ALC were intragastrically administered to mice once a day, prior to radiation. After two weeks, the mice received a one-time 5 Gy whole body ⁶⁰Coγ radiation. The spleen index, thymus index, activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), malondialdehyde (MDA) content, and glutathione (GSH) content in liver tissue were measured. Compared with the radiation control group, the levels of MDA in all ALC treated groups decreased significantly (p < 0.05). Moreover, the GSH content, activities of SOD and GSH-Px in liver tissue were enhanced significantly (p < 0.05) in all ALC groups. These results demonstrate that ALC may be a potential radioprotector, and a further study of the molecular mechanism is needed for further application. Full article

The aggregation of β-amyloid peptide (Aβ) into fibrils plays an important role in the pathogenesis of Alzheimer’s disease (AD). Metal ions including copper and zinc are closely connected to the precipitation and toxicity of Aβ. In this study, a surface plasmon resonance (SPR) biosensor was constructed to investigate the interactions between Aβ and metal ions. Aβ peptide was immobilized on the SPR chip surface through a preformed alkanethiol self-assembled monolayer (SAM). Our observations indicate that the immobilized Aβ undergoes a conformational change upon exposure to the metal ions. A difference in metal binding affinity between Aβ_1–28 and Aβ_1–42 was also detected. The results suggest that SPR is an effective method to characterize the interactions between Aβ and metal ions. Full article

Polymorphic microsatellite markers were developed for the Ibero-North African, strict gypsophyte Lepidium subulatum to unravel the effects of habitat fragmentation in levels of genetic diversity, genetic structure and gene flow among its populations. Using 454 pyrosequencing 12 microsatellite loci including di- and tri-nucleotide repeats were characterized in L. subulatum. They amplified a total of 80 alleles (2–12 alleles per locus) in a sample of 35 individuals of L. subulatum, showing relatively high levels of genetic diversity, H_O = 0.645, H_E = 0.627. Cross-species transferability of all 12 loci was successful for the Iberian endemics Lepidium cardamines, Lepidium stylatum, and the widespread, Lepidium graminifolium and one species each of two related genera, Cardaria draba and Coronopus didymus. These microsatellite primers will be useful to investigate genetic diversity, population structure and to address conservation genetics in species of Lepidium. Full article

Modified polystyrene with Pt(IV)–azomethine (APS–Sch–Pt) was synthesized by means of condensation and demonstrated to be a promising enzyme support by studying the enzymatic properties of glucose oxidase enzyme (GOx) immobilized on it. The characteristics of the immobilized glucose oxidase (APS–Sch–Pt–GOx) enzyme showed two optimum pH values that were pH = 4.0 and pH = 7. The insertion of stable Pt(IV)–azomethine spacers between the polystyrene backbone and the immobilized GOx, (APS–Sch–Pt–GOx), increases the enzymes’ activity and improves their affinity towards the substrate even at pH = 4. The influence of temperature, reusability and storage capacity on the free and immobilized glucose oxidase enzyme was investigated. The storage stability of the immobilized glucose oxidase was shown to be eleven months in dry conditions at +4 °C. Full article

The ability of macroencapsulated Saccharomyces cerevisiae CBS8066 to withstand readily and not readily in situ convertible lignocellulose-derived inhibitors was investigated in anaerobic batch cultivations. It was shown that encapsulation increased the tolerance against readily convertible furan aldehyde inhibitors and to dilute acid spruce hydrolysate, but not to organic acid inhibitors that cannot be metabolized anaerobically. Gene expression analysis showed that the protective effect arising from the encapsulation is evident also on the transcriptome level, as the expression of the stress-related genes YAP1, ATR1 and FLR1 was induced upon encapsulation. The transcript levels were increased due to encapsulation already in the medium without added inhibitors, indicating that the cells sensed low stress level arising from the encapsulation itself. We present a model, where the stress response is induced by nutrient limitation, that this helps the cells to cope with the increased stress added by a toxic medium, and that superficial cells in the capsules degrade convertible inhibitors, alleviating the inhibition for the cells deeper in the capsule. Full article

Many phytochemicals have been recognized to have potential therapeutic efficacy in cancer treatment. In this study, we investigated ethyl gallate (EG) for possible proapoptotic effects in the human promyelocytic leukemia cell line, HL-60. We examined cell viability, morphological changes, DNA content and fragmentation, and expression of apoptosis-related proteins for up to 48 h after EG treatment. The results showed that EG induced morphological changes and DNA fragmentation and reduced HL-60 cell viability in a dose-dependent and time-dependent manner. Western blotting analysis indicated that EG-mediated HL-60 apoptosis mainly occurred through the mitochondrial pathway, as shown by the release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G), as well as the upregulation of Bcl-2-associated X protein (Bax). EG also activated the death receptor-dependent pathway of apoptosis by enhancing the expression of caspases-8, -9, and -3 and the Bcl-2 interacting domain (Bid). Collectively, our results showed that EG induces apoptosis in HL-60 via mitochondrial-mediated pathways. Full article

Severe myocardial dysfunction and tissue damage resulting from ischemia/reperfusion (I/R) is a common clinical scenario in patients with certain types of heart diseases and therapies such as thrombolysis, percutaneous coronary intervention, coronary artery bypass grafting, and cardiac transplantation. The underlining mechanism of endogenous cardiac protection after I/R injury has been a focus of current research. Growing evidences suggests that soluble receptor for advanced glycation end-products (sRAGE) has a cardioprotective effect; however, its role in I/R injury remains unclear. We hypothesized that exogenous administration of sRAGE during hypoxia/reoxygenation (H/R) induces cardioprotection by inhibiting cardiomyocyte apoptosis via multiple signals, involving mitochondrial membrane potential (MMP), the mitochondrial permeability transition pore (mPTP), mitochondrial cytochrome c, caspase-3, Bcl-2 and Bax. Neonatal rat cardiomyocytes underwent hypoxia for 3-h followed by 2-h reoxygenation or were treated with sRAGE for 10 min before H/R. Compared with H/R alone, sRAGE pretreatment reduced H/R-induced cardiomyocyte apoptosis from 27.9% ± 5.9% to 9.4% ± 0.7% (p < 0.05). In addition, sRAGE treatment significantly inhibited H/R-induced mitochondrial depolarization and mPTP opening, reduced mitochondrial cytochrome c leakage, caspase-3 and caspase-9 activity, and decreased the ratio of Bax to Bcl-2. Therefore, we conclude that the exogenous administration of sRAGE during H/R is involved in cardioprotection by inhibiting apoptosis via the mitochondrial pathway, which, if further confirmed in vivo, may have important clinical implications during H/R. Full article

Colloidal Cu@CuAlO₂-Al₂O₃ bimetallic nanoparticles were prepared by a gamma irradiation method in an aqueous system in the presence of polyvinyl pyrrolidone (PVP) and isopropanol respectively as a colloidal stabilizer and scavenger of hydrogen and hydroxyl radicals. The gamma irradiation was carried out in a ⁶⁰Co gamma source chamber with different doses up to 120 kGy. The formation of Cu@CuAlO₂-Al₂O₃ nanoparticles was observed initially by the change in color of the colloidal samples from colorless to brown. Fourier transform infrared spectroscopy (FTIR) confirmed the presence of bonds between polymer chains and the metal surface at all radiation doses. Results of transmission electron microscopy (TEM), energy dispersive X-ray spectrometry (EDX), and X-ray diffraction (XRD) showed that Cu@CuAlO₂-Al₂O₃ nanoparticles are in a core-shell structure. By controlling the absorbed dose and precursor concentration, nanoclusters with different particle sizes were obtained. The average particle diameter increased with increased precursor concentration and decreased with increased dose. This is due to the competition between nucleation, growth, and aggregation processes in the formation of nanoclusters during irradiation. Full article

The poor outcome of advanced ovarian cancer under conventional therapy stimulated the exploration of new strategies to improve therapeutic efficacy. In our preclinical in vitro study we investigated a combination of targeted therapy and immunotherapy. Combination treatment with the anti-EGFR-antibody Cetuximab, related tyrosine kinase inhibitors (TKI) and cytolytic NK cells was tested against different ovarian cancer cell lines and primary tumour cells cultured from patient ascites. We found that selected ovarian cancer cells were susceptible to cetuximab and anti-EGFR-TKI-treatment, while the majority of cell lines were resistant to single or combination treatment with both substances. In addition, most ovarian cancer cells displayed low susceptibility to natural cytotoxicity of unstimulated NK cells. Notably, NK cytotoxicity against resistant ovarian cancer cells could be effectively enhanced by addition of Cetuximab mediating antibody-dependent cellular cytotoxicity (ADCC). Neither natural cytotoxicity nor ADCC of NK cells were negatively affected by the presence of TKIs. ADCC could be further increased when NK cells were pre-stimulated with monocytes and the immunostimulatory mycobacterial protein PstS-1. Our data suggest that targeted antibody therapy could be beneficial even against resistant tumour cells by augmenting supplementary cytolytic NK functions. Future studies should evaluate the combination of targeted therapy and immunotherapeutic approaches in patients with advanced ovarian cancer being resistant to standard treatment. Full article

The hyaluronate molecule is a negatively charged polysaccharide that performs a plethora of physiological functions in many cell tissues depending on its conformation. In the present paper, molecular modeling at three levels of theory and two basis sets was used to gain a deeper insight in the complex molecular structure of calcium(II) and copper(II) hyaluronate. Simulation results were compared with the experimental data (EXAFS or X-ray). It was found that B3LYP does not properly reproduce the experimental data while the HF and M06 methods do. Simulation data confirm that the N-acetyl group of the N-acetylglucosamine residue does not participate in the coordination bonding to the calcium(II) or copper(II) ion, as evident from the experimental data. Full article

Porcine reproductive and respiratory syndrome (PRRS) is a disease produced by the (PRRS) virus, characterized by endemic evolution in the majority of countries, which remains in actuality being a permanent threat to health and economic free farms, as well as for those infected. The aim of this study was to evaluate the genetic diversity of Romanian PRRSV isolates from the four most important pig farms in Romania by comparing the nucleotide sequences obtained for ORF5 and ORF7 with a wide range of sequences from GenBank belonging to the main types of PRRSV; the type 1. Eighteen different sequences were obtained for ORF5 gene and 10 for ORF7 gene. One Romanian isolate (Rom3) was found in three of the four different investigated farms. The phylogenetic analysis revealed that the Romanian PRRSV nucleotide sequences clustered in three groups within the subtype 1 of the virus. The analysis of amino acid sequences evidenced for GP5 and N-nucleocapsid proteins confirmed that the Romanian virus belonged to type 1. Full article

Little is known about the interactions of single nucleotide polymorphisms (SNPs) and overweight/obesity on blood pressure levels. The present study was undertaken to detect 10 lipid-related gene SNPs and their interactions with overweight/obesity on blood pressure levels. Genotyping of ATP-binding cassette transporter A1 (ABCA-1) V825I, acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) rs1044925, low density lipoprotein receptor (LDL-R) AvaII hepatic lipase gene (LIPC) −250G > A, endothelial lipase gene (LIPG) 584C > T, methylenetetrahydrofolate reductase (MTHFR) 677C > T, the E3 ubiquitin ligase myosin regulatory light chain-interacting protein (MYLIP) rs3757354, proprotein convertase subtilisin-like kexin type 9 (PCSK9) E670G, peroxisome proliferator-activated receptor delta (PPARD) +294T > C, and Scavenger receptor class B type 1 (SCARB1) rs5888 was performed in 978 normal weight and 751 overweight/obese subjects. The interactions were detected by factorial regression analysis. The genotypes of ACAT-1 AC, LIPC GA and AA, and SCARB1 TT; LDL-R A-A- and LIPC GA; and SCARB1 TT were interacted with overweight/obesity to increase systolic, diastolic blood pressure (SBP, DBP) and pulse pressure (PP) levels; respectively. The genotypes of ACAT-1 CC; ACAT-1 AA and CC were interacted with overweight/obesity to decrease SBP, PP levels (p < 0.01–0.001); respectively. The differences in blood pressure levels between normal weight and overweight/obese subjects might partly result from different interactions of several SNPs and overweight/obesity. Full article

Nine new microsatellite markers (SSR) were isolated from Salvia officinalis L. A total of 125 alleles, with 8 to 21 alleles per locus, were detected in a natural population from the east Adriatic coast. The observed heterozygosity, expected heterozygosity, and polymorphic information content ranged from 0.46 to 0.83, 0.73 to 0.93 and 0.70 to 0.92, respectively. New microsatellite markers, as well as previously published markers, were tested for cross-amplification in Salvia brachyodon Vandas, a narrow endemic species known to be present in only two localities on the Balkan Peninsula. Out of 30 microsatellite markers tested on the natural S. brachyodon population, 15 were successfully amplified. To obtain evidence of recent bottleneck events in the populations of both species, observed genetic diversity (H_E) was compared to the expected genetic diversity at mutation-drift equilibrium (H_EQ) and calculated from the observed number of alleles using a two-phased mutation model (TPM). Recent bottleneck events were detected only in the S. brachyodon population. This result suggests the need to reconsider the current threat category of this endemic species. Full article

Single-molecule fluorescence spectroscopy has now been widely used to investigate complex dynamic processes which would normally be obscured in an ensemble-averaged measurement. In this report we studied photophysical behaviors of single fluorophores in proximity to zinc oxide nanostructures by single-molecule fluorescence spectroscopy and time-correlated single-photon counting (TCSPC). Single fluorophores on ZnO surfaces showed enhanced fluorescence brightness to various extents compared with those on glass; the single-molecule time trajectories also illustrated pronounced fluctuations of emission intensities, with time periods distributed from milliseconds to seconds. We attribute fluorescence fluctuations to the interfacial electron transfer (ET) events. The fluorescence fluctuation dynamics were found to be inhomogeneous from molecule to molecule and from time to time, showing significant static and dynamic disorders in the interfacial electron transfer reaction processes. Full article

Monocytes and macrophages are part of the first-line defense against bacterial, fungal, and viral infections during host immune responses; they express high levels of proinflammatory cytokines and cytotoxic molecules, including nitric oxide, reactive oxygen species, and their reaction product peroxynitrite. Peroxynitrite is a short-lived oxidant and a potent inducer of cell death. Honey, in addition to its well-known sweetening properties, is a natural antioxidant that has been used since ancient times in traditional medicine. We examined the ability of Gelam honey, derived from the Gelam tree (Melaleuca spp.), to scavenge peroxynitrite during immune responses mounted in the murine macrophage cell line RAW 264.7 when stimulated with lipopolysaccharide/interferon-γ (LPS/IFN-γ) and in LPS-treated rats. Gelam honey significantly improved the viability of LPS/IFN-γ-treated RAW 264.7 cells and inhibited nitric oxide production—similar to the effects observed with an inhibitor of inducible nitric oxide synthase (1400W). Furthermore, honey, but not 1400W, inhibited peroxynitrite production from the synthetic substrate 3-morpholinosydnonimine (SIN-1) and prevented the peroxynitrite-mediated conversion of dihydrorhodamine 123 to its fluorescent oxidation product rhodamine 123. Honey inhibited peroxynitrite synthesis in LPS-treated rats. Thus, honey may attenuate inflammatory responses that lead to cell damage and death, suggesting its therapeutic uses for several inflammatory disorders. Full article

Fluctuations in serum autofluorescence (AF) intensity have recently been widely used as markers of certain diseases such as cancer. To determine the diagnostic value of serum AF intensity for liver fibrosis in rats, we induced liver fibrosis by subcutaneous injection of carbon tetrachloride into rats. The rat serum AF intensities were detected at the excitation wavelength of 337 nm and the emission wavelength of 512 nm. The degree of liver fibrosis was evaluated by Van Gieson’s staining. The relationship between serum AF intensity and the degree of liver fibrosis was analyzed by Spearman and Pearson Correlation. The diagnostic sensitivity and specificity of the serum AF was determined by analyzing the receiver operating characteristic (ROC) curves. Our results show that the serum AF intensity in the rat liver fibrosis model increased when compared with control rats eight weeks and twelve weeks post induction of liver fibrosis. However, there was no significant difference in serum AF intensity between fibrotic and control rats at four week post induction. Furthermore, serum AF intensity correlated positively with the severity of the degree of hepatic fibrosis. ROC analysis further suggested that serum AF intensity is a valid marker for staging fibrosis. Therefore, it may potentially be developed as a novel diagnostic tool for hepatic fibrosis. Full article

Rb₁-hydrolyzing β-glucosidase from Aspergillus niger KCCM 11239 was studied to develop a bioconversion process for minor ginsenosides. The specific activity of the purified enzyme was 46.5 times greater than that of the crude enzyme. The molecular weight of the native enzyme was estimated to be approximately 123 kDa. The optimal pH of the purified enzyme was pH 4.0, and the enzyme proved highly stable over a pH range of 5.0–10.0. The optimal temperature was 70 °C, and the enzyme became unstable at temperatures above 60 °C. The enzyme was inhibited by Cu²⁺, Mg²⁺, Co²⁺, and acetic acid (10 mM). In the specificity tests, the enzyme was found to be active against ginsenoside Rb₁, but showed very low levels of activity against Rb₂, Rc, Rd, Re, and Rg₁. The enzyme hydrolyzed the 20-C,β-(1→6)-glucoside of ginsenoside Rb₁ to generate ginsenoside Rd and Rg₃, and hydrolyzed 3-C,β-(1→2)-glucoside to generate F₂. The properties of the enzyme indicate that it could be a useful tool in biotransformation applications in the ginseng industry, as well as in the development of novel drug compounds. Full article

Reactive oxygen species (ROS) are increasingly being implicated in the regulation of cellular signaling cascades. Intracellular ROS fluxes are associated with cellular function ranging from proliferation to cell death. Moreover, the importance of subtle, spatio-temporal shifts in ROS during localized cellular signaling events is being realized. Understanding the biochemical nature of the ROS involved will enhance our knowledge of redox-signaling. An ideal intracellular sensor should therefore resolve real-time, localized ROS changes, be highly sensitive to physiologically relevant shifts in ROS and provide specificity towards a particular molecule. For in vivo applications issues such as bioavailability of the probe, tissue penetrance of the signal and signal-to-noise ratio also need to be considered. In the past researchers have heavily relied on the use of ROS-sensitive fluorescent probes and, more recently, genetically engineered ROS sensors. However, there is a great need to improve on current methods to address the above issues. Recently, the field of molecular sensing and imaging has begun to take advantage of the unique physico-chemical properties of nanoparticles and nanotubes. Here we discuss the recent advances in the use of these nanostructures as alternative platforms for ROS sensing, with particular emphasis on intracellular and in vivo ROS detection and quantification. Full article

Due to the growing importance of cellular signaling mediated by reactive oxygen species (ROS), proteins that are reversibly modulated by these reactant molecules are of high interest. In this context, protein kinases and phosphatases, which act coordinately in the regulation of signal transduction through the phosphorylation and dephosphorylation of target proteins, have been described to be key elements in ROS-mediated signaling events. The major mechanism by which these proteins may be modified by oxidation involves the presence of key redox-sensitive cysteine residues. Protein kinase C (PKC) is involved in a variety of cellular signaling pathways. These proteins have been shown to contain a unique structural feature that is susceptible to oxidative modification. A large number of scientific studies have highlighted the importance of ROS as a second messenger in numerous cellular processes, including cell proliferation, gene expression, adhesion, differentiation, senescence, and apoptosis. In this context, the goal of this review is to discuss the mechanisms by which PKCs are modulated by ROS and how these processes are involved in the cellular response. Full article

Organisms and their cells are constantly exposed to environmental fluctuations. Among them are stressors, which can induce macromolecular damage that exceeds a set threshold, independent of the underlying cause. Stress responses are mechanisms used by organisms to adapt to and overcome stress stimuli. Different stressors or different intensities of stress trigger different cellular responses, namely induce cell repair mechanisms, induce cell responses that result in temporary adaptation to some stressors, induce autophagy or trigger cell death. Studies have reported life-prolonging effects of a wide variety of so-called stressors, such as oxidants, heat shock, some phytochemicals, ischemia, exercise and dietary energy restriction, hypergravity, etc. These stress responses, which result in enhanced defense and repair and even cross-resistance against multiple stressors, may have clinical use and will be discussed, while the emphasis will be on the effects/cross-effects of oxidants. Full article

Novel aerosol therapeutic modalities have been investigated for lung cancer. Inhaled gene therapy has presented safety and effectiveness previously in cystic fibrosis. However, safety concerns have been raised regarding the safety of non-viral vectors for inhaled gene therapy in lung cancer, and therefore small steps have been made towards this multifunctional treatment modality. During the last decade, numerous new nanocomplexes have been created and investigated as a safe gene delivery nano-vehicle. These formulations are multifunctional; they can be used as either local therapy or carrier for an effective inhaled gene therapy for lung cancer. Herein, we present current and future perspectives of nanocomplexes for inhaled gene therapy treatment in lung cancer. Full article

Antimicrobial peptides (AMPs) are naturally-occurring molecules that exhibit strong antibiotic properties against numerous infectious bacterial strains. Because of their unique mechanism of action, they have been touted as a potential source for novel antibiotic drugs. We present a summary of computational investigations in our lab aimed at understanding this unique mechanism of action, in particular the development of models that provide a quantitative connection between molecular-level biophysical phenomena and relevant biological effects. Our work is focused on protegrins, a potent class of AMPs that attack bacteria by associating with the bacterial membrane and forming transmembrane pores that facilitate the unrestricted transport of ions. Using fully atomistic molecular dynamics simulations, we have computed the thermodynamics of peptide-membrane association and insertion, as well as peptide aggregation. We also present a multi-scale analysis of the ion transport properties of protegrin pores, ranging from atomistic molecular dynamics simulations to mesoscale continuum models of single-pore electrodiffusion to models of transient ion transport from bacterial cells. Overall, this work provides a quantitative mechanistic description of the mechanism of action of protegrin antimicrobial peptides across multiple length and time scales. Full article

Premature senescence induced by DNA damage or oncogene is a critical mechanism of tumor suppression. Reactive oxygen species (ROS) have been implicated in the induction of premature senescence response. Several pathological disorders such as cancer, aging and age related neurological abnormalities have been linked to ROS deregulation. Here, we discuss how Damaged DNA binding Protein-2 (DDB2), a nucleotide excision repair protein, plays an important role in ROS regulation by epigenetically repressing the antioxidant genes MnSOD and Catalase. We further revisit a model in which DDB2 plays an instrumental role in DNA damage induced ROS accumulation, ROS induced premature senescence and inhibition of skin tumorigenesis. Full article

Chronic inflammation characterizing patients with inflammatory bowel disease (IBD) represents a major risk factor for the development of colorectal cancer. Mechanisms underlying this neoplastic transformation are not fully understood though studies in experimental models of colon carcinogenesis suggest that inflammatory cell-derived cytokines either directly or indirectly stimulate the uncontrolled growth of cancer cells. Nevertheless, under specific inflammatory conditions, immune cells can boost an anti-tumor immune response with the down-stream effect of eliminating dysplastic and cancerous cells. This review outlines the beneficial and detrimental role of inflammation in colon carcinogenesis. Full article

Changes in the developmental or metabolic state of plastids can trigger profound changes in the transcript profiles of nuclear genes. Many nuclear transcription factors were shown to be controlled by signals generated in the organelles. In addition to the many different compounds for which an involvement in retrograde signaling is discussed, accumulating evidence suggests a role for proteins in plastid-to-nucleus communication. These proteins might be sequestered in the plastids before they act as transcriptional regulators in the nucleus. Indeed, several proteins exhibiting a dual localization in the plastids and the nucleus are promising candidates for such a direct signal transduction involving regulatory protein storage in the plastids. Among such proteins, the nuclear transcription factor WHIRLY1 stands out as being the only protein for which an export from plastids and translocation to the nucleus has been experimentally demonstrated. Other proteins, however, strongly support the notion that this pathway might be more common than currently believed. Full article

New treatment strategies with erythropoietin (EPO) offer exciting opportunities to prevent the onset and progression of neurodegenerative disorders that currently lack effective therapy and can progress to devastating disability in patients. EPO and its receptor are present in multiple systems of the body and can impact disease progression in the nervous, vascular, and immune systems that ultimately affect disorders such as Alzheimer’s disease, Parkinson’s disease, retinal injury, stroke, and demyelinating disease. EPO relies upon wingless signaling with Wnt1 and an intimate relationship with the pathways of phosphoinositide 3-kinase (PI 3-K), protein kinase B (Akt), and mammalian target of rapamycin (mTOR). Modulation of these pathways by EPO can govern the apoptotic cascade to control b-catenin, glycogen synthase kinase-3b, mitochondrial permeability, cytochrome c release, and caspase activation. Yet, EPO and each of these downstream pathways require precise biological modulation to avert complications associated with the vascular system, tumorigenesis, and progression of nervous system disorders. Further understanding of the intimate and complex relationship of EPO and the signaling pathways of Wnt, PI 3-K, Akt, and mTOR are critical for the effective clinical translation of these cell pathways into robust treatments for neurodegenerative disorders. Full article

Here we review our development of, and results with, high resolution studies on global genome nucleotide excision repair (GGNER) in Saccharomyces cerevisiae. We have focused on how GGNER relates to histone acetylation for its functioning and we have identified the histone acetyl tranferase Gcn5 and acetylation at lysines 9/14 of histone H3 as a major factor in enabling efficient repair. We consider results employing primarily MFA2 as a model gene, but also those with URA3 located at subtelomeric sequences. In the latter case we also see a role for acetylation at histone H4. We then go on to outline the development of a high resolution genome-wide approach that enables one to examine correlations between histone modifications and the nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers throughout entire genomes. This is an approach that will enable rapid advances in understanding the complexities of how compacted chromatin in chromosomes is processed to access DNA damage and then returned to its pre-damaged status to maintain epigenetic codes. Full article

L-Arginine (Arg) is oxidized to L-citrulline and nitric oxide (NO) by the action of endothelial nitric oxide synthase (NOS). In contrast, protein-incorporated Arg residues can be methylated with subsequent proteolysis giving rise to methylarginine compounds, such as asymmetric dimethylarginine (ADMA) that competes with Arg for binding to NOS. Most ADMA is degraded by dimethylarginine dimethyaminohydrolase (DDAH), distributed widely throughout the body and regulates ADMA levels and, therefore, NO synthesis. In recent years, several studies have suggested that increased ADMA levels are a marker of atherosclerotic change, and can be used to assess cardiovascular risk, consistent with ADMA being predominantly absorbed by endothelial cells. NO is an important messenger molecule involved in numerous biological processes, and its activity is essential to understand both pathogenic and therapeutic mechanisms in kidney disease and renal transplantation. NO production is reduced in renal patients because of their elevated ADMA levels with associated reduced DDAH activity. These factors contribute to endothelial dysfunction, oxidative stress and the progression of renal damage, but there are treatments that may effectively reduce ADMA levels in patients with kidney disease. Available data on ADMA levels in controls and renal patients, both in adults and children, also are summarized in this review. Full article

Lung cancer has long been recognized as an extremely heterogeneous disease, since its development is unique in every patient in terms of clinical characterizations, prognosis, response and tolerance to treatment. Personalized medicine refers to the use of markers to predict which patient will most likely benefit from a treatment. In lung cancer, the well-developed epidermal growth factor receptor (EGFR) and the newly emerging EML4-anaplastic lymphoma kinase (ALK) are important therapeutic targets. This review covers the basic mechanism of EGFR and EML4-ALK activation, the predictive biomarkers, the mechanism of resistance, and the current targeted tyrosine kinase inhibitors. The efficacy of EGFR and ALK targeted therapies will be discussed in this review by summarizing the prospective clinical trials, which were performed in biomarker-based selected patients. In addition, the revolutionary sequencing and systems strategies will also be included in this review since these technologies will provide a comprehensive understanding in the molecular characterization of cancer, allow better stratification of patients for the most appropriate targeted therapies, eventually resulting in a more promising personalized treatment. The relatively low incidence of EGFR and ALK in non-Asian patients and the lack of response in mutant patients limit the application of the therapies targeting EGFR or ALK. Nevertheless, it is foreseeable that the sequencing and systems strategies may offer a solution for those patients. Full article

Organisms are continuously exposed to DNA damaging agents, consequently, cells have developed an intricate system known as the DNA damage response (DDR) in order to detect and repair DNA lesions. This response has to be rapid and accurate in order to keep genome integrity. It has been observed that the condensation state of chromatin hinders a proper DDR. However, the condensation state of chromatin is not the only barrier to DDR. In this review, we have collected data regarding the presence of DDR factors on micronuclear DNA lesions that indicate that micronuclei are almost incapable of generating an effective DDR because of defects in their nuclear envelope. Finally, considering the recent observations about the reincorporation of micronuclei to the main bulk of chromosomes, we suggest that, under certain circumstances, micronuclei carrying DNA damage might be a source of chromosome instability. Full article

α-Synucleinopathies are neurodegenerative disorders that are characterized by progressive decline of motor and non-motor dysfunctions. α-Synuclein (αS) has been shown to play a causative role in neurodegeneration, but the pathogenic mechanisms are still unclear. Thus, there are no radical therapies that can halt or reverse the disease’s progression. β-Synuclein (βS), the non-amyloidogenic homologue of αS, ameliorates the neurodegeneration phenotype of αS in transgenic (tg) mouse models, as well as in cell free and cell culture systems, which suggests that βS might be a negative regulator of neurodegeneration caused by αS, and that “loss of function” of βS might be involved in progression of α-synucleinopathies. Alternatively, it is possible that “toxic gain of function” of wild type βS occurs during the pathogenesis of sporadic α-synucleinopathies, since tg mice expressing dementia with Lewy bodies-linked P123H βS develop progressive neurodegeneration phenotypes, such as axonal pathology and dementia. In this short review, we emphasize the aspects of “toxic gain of function” of wild type βS during the pathogenesis of sporadic α-synucleinopathies. Full article

In the past few years, growing attention has been devoted to the study of the interactions taking place in mixed systems of phospholipid membranes (for instance in the form of vesicles) and hard nanoparticles (NPs). In this context liposomes (vesicles) may serve as versatile carriers or as a model system for biological membranes. Research on these systems has led to the observation of novel hybrid structures whose morphology strongly depends on the charge, composition and size of the interacting colloidal species as well as on the nature (pH, ionic strength) of their dispersing medium. A central role is played by the phase behaviour of phospholipid bilayers which have a tremendous influence on the liposome properties. Another central aspect is the incorporation of nanoparticles into vesicles, which is intimately linked to the conditions required for transporting a nanoparticle through a membrane. Herein, we review recent progress made on the investigations of the interactions in liposome/nanoparticle systems focusing on the particularly interesting structures that are formed in these hybrid systems as well as their potential applications. Full article

Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts. Full article

Ovarian cancer is the most lethal gynecologic malignancy. Despite advances in chemotherapy, the five-year survival rate of advanced ovarian cancer patients with peritoneal metastasis remains around 30%. The most significant prognostic factor is stage, and most patients present at an advanced stage with peritoneal dissemination. There is often no clearly identifiable precursor lesion; therefore, the events leading to metastatic disease are poorly understood. This article reviews metastatic suppressor genes, the epithelial-mesenchymal transition (EMT), and the tumor microenvironment as they relate to ovarian cancer metastasis. Additionally, novel chemotherapeutic agents targeting the metastasis-related biochemical pathways are discussed. Full article

Multiple sclerosis (MS) is a chronic disabling disease of the central nervous system commonly affecting young adults. Pathologically, there are patches of inflammation (plaques) with demyelination of axons and oligodendrocyte loss. There is a global latitude gradient in MS prevalence, and incidence of MS is increasing (particularly in females). These changes suggest a major role for environmental factors in causation of disease. We have reviewed the evidence and potential mechanisms of action for three exposures: vitamin D, Epstein Barr virus and cigarette smoking. Recent advances supporting gene-environment interactions are reviewed. Further research is needed to establish mechanisms of causality in humans and to explore preventative strategies. Full article

Neuroprotection aims to prevent salvageable neurons from dying. Despite showing efficacy in experimental stroke studies, the concept of neuroprotection has failed in clinical trials. Reasons for the translational difficulties include a lack of methodological agreement between preclinical and clinical studies and the heterogeneity of stroke in humans compared to homogeneous strokes in animal models. Even when the international recommendations for preclinical stroke research, the Stroke Academic Industry Roundtable (STAIR) criteria, were followed, we have still seen limited success in the clinic, examples being NXY-059 and haematopoietic growth factors which fulfilled nearly all the STAIR criteria. However, there are a number of neuroprotective treatments under investigation in clinical trials such as hypothermia and ebselen. Moreover, promising neuroprotective treatments based on a deeper understanding of the complex pathophysiology of ischemic stroke such as inhibitors of NADPH oxidases and PSD-95 are currently evaluated in preclinical studies. Further concepts to improve translation include the investigation of neuroprotectants in multicenter preclinical Phase III-type studies, improved animal models, and close alignment between clinical trial and preclinical methodologies. Future successful translation will require both new concepts for preclinical testing and innovative approaches based on mechanistic insights into the ischemic cascade. Full article

Oxidative stress plays a crucial role in many neurodegenerative conditions such as Alzheimer’s disease, amyotrophic lateral sclerosis and Parkinson’s as well as Huntington’s disease. Inflammation and oxidative stress are also thought to promote tissue damage in multiple sclerosis (MS). Recent data point at an important role of anti-oxidative pathways for tissue protection in chronic-progressive MS, particularly involving the transcription factor nuclear factor (erythroid-derived 2)-related factor 2 (Nrf2). Thus, novel therapeutics enhancing cellular resistance to free radicals could prove useful for MS treatment. Here, fumaric acid esters (FAE) are a new, orally available treatment option which had already been tested in phase II/III MS trials demonstrating beneficial effects on relapse rates and magnetic resonance imaging markers. In vitro, application of dimethylfumarate (DMF) leads to stabilization of Nrf2, activation of Nrf2-dependent transcriptional activity and abundant synthesis of detoxifying proteins. Furthermore, application of FAE involves direct modification of the inhibitor of Nrf2, Kelch-like ECH-associated protein 1. On cellular levels, the application of FAE enhances neuronal survival and protects astrocytes against oxidative stress. Increased levels of Nrf2 are detected in the central nervous system of DMF treated mice suffering from experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In EAE, DMF ameliorates the disease course and improves preservation of myelin, axons and neurons. Finally, Nrf2 is also up-regulated in the spinal cord of autopsy specimens from untreated patients with MS, probably as part of a naturally occurring anti-oxidative response. In summary, oxidative stress and anti-oxidative pathways are important players in MS pathophysiology and constitute a promising target for future MS therapies like FAE. Full article

Ubiquitin-like proteins (Ubls) confer diverse functions on their target proteins. The modified proteins are involved in various biological processes, including DNA replication, signal transduction, cell cycle control, embryogenesis, cytoskeletal regulation, metabolism, stress response, homeostasis and mRNA processing. Modifiers such as SUMO, ATG12, ISG15, FAT10, URM1, and UFM have been shown to modify proteins thus conferring functions related to programmed cell death, autophagy and regulation of the immune system. Putative modifiers such as Domain With No Name (DWNN) have been identified in recent times but not fully characterized. In this review, we focus on cellular processes involving human Ubls and their targets. We review current progress in targeting these modifiers for drug design strategies. Full article

Over recent decades, a deep understanding of pathways that repair DNA double strand breaks (DSB) has been gained from biochemical, structural, biophysical and cellular studies. DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two major DSB repair pathways, and both processes are now well understood. Recent work has demonstrated that the chromatin environment at a DSB significantly impacts upon DSB repair and that, moreover, dramatic modifications arise in the chromatin surrounding a DSB. Chromatin is broadly divided into open, transcriptionally active, euchromatin (EC) and highly compacted, transcriptionally inert, heterochromatin (HC), although these represent extremes of a spectrum. The HC superstructure restricts both DSB repair and damage response signaling. Moreover, DSBs within HC (HC-DSBs) are rapidly relocalized to the EC-HC interface. The damage response protein kinase, ataxia telangiectasia mutated (ATM), is required for HC-DSB repair but is dispensable for the relocalization of HC-DSBs. It has been proposed that ATM signaling enhances HC relaxation in the DSB vicinity and that this is a prerequisite for HC-DSB repair. Hence, ATM is essential for repair of HC-DSBs. Here, we discuss how HC impacts upon the response to DSBs and how ATM overcomes the barrier that HC poses to repair. Full article

It has been a long-standing question how DNA damage repair proceeds in a nuclear environment where DNA is packaged into chromatin. Several decades of analysis combining in vitro and in vivo studies in various model organisms ranging from yeast to human have markedly increased our understanding of the mechanisms underlying chromatin disorganization upon damage detection and re-assembly after repair. Here, we review the methods that have been developed over the years to delineate chromatin alterations in response to DNA damage by focusing on the well-characterized Nucleotide Excision Repair (NER) pathway. We also highlight how these methods have provided key mechanistic insight into histone dynamics coupled to repair in mammals, raising new issues about the maintenance of chromatin integrity. In particular, we discuss how NER factors and central players in chromatin dynamics such as histone modifiers, nucleosome remodeling factors, and histone chaperones function to mobilize histones during repair. Full article

DNA repair in eukaryotic cells takes place in the context of chromatin, where DNA, including damaged DNA, is tightly packed into nucleosomes and higher order chromatin structures. Chromatin intrinsically restricts accessibility of DNA repair proteins to the damaged DNA and impacts upon the overall rate of DNA repair. Chromatin is highly responsive to DNA damage and undergoes specific remodeling to facilitate DNA repair. How damaged DNA is accessed, repaired and restored to the original chromatin state, and how chromatin remodeling coordinates these processes in vivo, remains largely unknown. ATP-dependent chromatin remodelers (ACRs) are the master regulators of chromatin structure and dynamics. Conserved from yeast to humans, ACRs utilize the energy of ATP to reorganize packing of chromatin and control DNA accessibility by sliding, ejecting or restructuring nucleosomes. Several studies have demonstrated that ATP-dependent remodeling activity of ACRs plays important roles in coordination of spatio-temporal steps of different DNA repair pathways in chromatin. This review focuses on the role of ACRs in regulation of various aspects of nucleotide excision repair (NER) in the context of chromatin. We discuss current understanding of ATP-dependent chromatin remodeling by various subfamilies of remodelers and regulation of the NER pathway in vivo. Full article

Genomic instability, a hallmark of cancer, occurs preferentially at specific genomic regions known as common fragile sites (CFSs). CFSs are evolutionarily conserved and late replicating regions with AT-rich sequences, and CFS instability is correlated with cancer. In the last decade, much progress has been made toward understanding the mechanisms of chromosomal instability at CFSs. However, despite tremendous efforts, identifying a cancer-associated CFS gene (CACG) remains a challenge and little is known about the function of CACGs at most CFS loci. Recent studies of FATS (for Fragile-site Associated Tumor Suppressor), a new CACG at FRA10F, reveal an active role of this CACG in regulating DNA damage checkpoints and suppressing tumorigenesis. The identification of FATS may inspire more discoveries of other uncharacterized CACGs. Further elucidation of the biological functions and clinical significance of CACGs may be exploited for cancer biomarkers and therapeutic benefits. Full article

Glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine; it has a variety of functions in the central nervous system. Brain GSH depletion is considered a preclinical sign in age-related neurodegenerative diseases, and it promotes the subsequent processes toward neurotoxicity. A neuroprotective mechanism accomplished by increasing GSH synthesis could be a promising approach in the treatment of neurodegenerative diseases. In neurons, cysteine is the rate-limiting substrate for GSH synthesis. Excitatory amino acid carrier 1 (EAAC1) is a neuronal cysteine/glutamate transporter in the brain. EAAC1 translocation to the plasma membrane promotes cysteine uptake, leading to GSH synthesis, while being negatively regulated by glutamate transport associated protein 3-18 (GTRAP3-18). Our recent studies have suggested GTRAP3-18 as an inhibitory factor for neuronal GSH synthesis. Inhibiting GTRAP3-18 function is an endogenous mechanism to increase neuron-specific GSH synthesis in the brain. This review gives an overview of EAAC1-mediated GSH synthesis, and its regulatory mechanisms by GTRAP3-18 in the brain, and a potential approach against neurodegeneration. Full article

In a previous study, we demonstrated that mouse adult F₁ offspring, exposed to a vitamin D deficiency during pregnancy, developed a less severe and delayed Experimental Autoimmune Encephalomyelitis (EAE), when compared with control offspring. We then wondered whether a similar response was observed in the subsequent generation. To answer this question, we assessed F₂ females whose F₁ parents (males or females) were vitamin D-deprived when developing in the uterus of F₀ females. Unexpectedly, we observed that the vitamin D deficiency affecting the F₀ pregnant mice induced a precocious and more severe EAE in the F₂ generation. This paradoxical finding led us to assess its implications for the epidemiology of Multiple Sclerosis (MS) in humans. Using the REFGENSEP database for MS trios (the patient and his/her parents), we collected the parents’ dates of birth and assessed a potential season of birth effect that could potentially be indicative of the vitamin D status of the pregnant grandmothers. A trend for a reduced number of births in the Fall for the parents of MS patients was observed but statistical significance was not reached. Further well powered studies are warranted to validate the latter finding. Full article

Aspergillosis remains to be a life-threatening complication in immunocompromised patients. However, Aspergillus infection can be observed in non-immunocompromised individuals in rare cases. We report a case of liver aspergilloma in a chronic aplastic anemia patient under relatively intact immune status. Therapeutic strategy for this rare condition was extensively discussed and caspofungin acetate single agent first-line therapy was applied after careful consideration. Encouraging clinical and radiologic improvements were achieved in response to the antifungal salvage. Our long-term follow-up study also revealed a favorable prognosis. Based on this experience, we suggest caspofungin acetate as first-line therapy for treatment plans of liver aspergilloma. Full article

A set of polymorphic loci was characterised using an enrichment library for the Australian alpine specialist, the chameleon grasshopper (Kosciuscola tristis), an atypical grasshopper known for its remarkable temperature-controlled colour change. The number of alleles per locus ranged from three to 20 and observed heterozygosity from 0.16 to 0.76. These are the first microsatellite markers for a non-endangered Australian alpine animal and will inform questions of gene flow across the sky islands of this unique and threatened region. Full article