In chronic inflammatory diseases the anti-inflammatory effect of glucocorticoids (GCs) is often decreased, leading to GC resistance. Inflammation is related with increased levels of reactive oxygen species (ROS), leading to oxidative stress which is thought to contribute to the development of GC resistance. Plant-derived compounds such as flavonoids are known for their ability to protect against ROS. In this exploratory study we screened a broad range of food-derived bioactives for their antioxidant and anti-inflammatory effects in order to investigate whether their antioxidant effects are associated with the ability to preserve the anti-inflammatory effects of cortisol. The anti-inflammatory potency of the tested compounds was assessed by measuring the oxidative stress–induced GC resistance in human macrophage-like cells. Cells were pre-treated with H₂O₂ (800 µM) with and without bioactives and then exposed to lipopolysaccharides (LPS) (10 ng/mL) and cortisol (100 nM). The level of inflammation was deducted from the concentration of interleukin-8 (IL-8) in the medium. Intracellular oxidative stress was measured using the fluorescent probe 2′,7′-dichlorofluorescein (DCFH). We found that most of the dietary bioactives display antioxidant and anti-inflammatory action through the protection of the cortisol response. All compounds, except for quercetin, revealing antioxidant activity also protect the cortisol response. This indicates that the antioxidant activity of compounds plays an important role in the protection of the GC response. However, next to the antioxidant activity of the bioactives, other mechanisms also seem to be involved in this protective, anti-inflammatory effect. Full article

Senescence accelerated mice (SAM) are a group of mice that show aging-related diseases, and SAM prone 10 (SAMP10) show spontaneous brain atrophy and defects in learning and memory. Our previous report showed that the thymus and the percentage of T lymphocytes are abnormal in the SAMP10, but it was unclear whether the bone marrow-derived mesenchymal stroma cells (BMMSCs) were abnormal, and whether they played an important role in regenerative medicine. We thus compared BMMSCs from SAMP10 and their control, SAM-resistant (SAMR1), in terms of cell cycle, oxidative stress, and the expression of PI3K and mitogen-activated protein kinase (MAPK). Our cell cycle analysis showed that cell cycle arrest occurred in the G0/G1 phase in the SAMP10. We also found increased reactive oxygen stress and decreased PI3K and MAPK on the BMMSCs. These results suggested the BMMSCs were abnormal in SAMP10, and that this might be related to the immune system dysfunction in these mice. Full article

The mechanism of gap junction enhancer (PQ1) induced cytotoxicity is thought to be attributed to the change in connexin 43 (Cx43) expression; therefore, the effects of Cx43 modulation in cell survival were investigated in mammary carcinoma cells (FMC2u) derived from a malignant neoplasm of a female FVB/N-Tg(MMTV-PyVT)634Mul/J (PyVT) transgenic mouse. PQ1 was determined to have an IC₅₀ of 6.5 µM in FMC2u cells, while inducing an upregulation in Cx43 expression. The effects of Cx43 modulation in FMC2u cell survival was determined through transfection experiments with Cx43 cDNA, which induced an elevated level of protein expression similar to that seen with PQ1 exposure, or siRNA to silence Cx43 protein expression. Overexpression or silencing of Cx43 led to a reduction or an increase in cell viability, respectively. The mitogen-activated protein kinase (MAPK) family has been implicated in the regulation of cell survival and cell death; therefore, the gap junctional intercellular communication (GJIC)-independent function of PQ1 and Cx43 in the Raf/Mitogen-activated protein kinase/ERK kinase/extracellular-signal-regulated kinase (Raf-MEK-ERK) cascade of cellular survival and p38 MAPK-dependent pathway of apoptosis were explored. PQ1 treatment activated p44/42 MAPK, while the overexpression of Cx43 resulted in a reduced expression. This suggests that PQ1 affects the Raf-MEK-ERK cascade independent of Cx43 upregulation. Both overexpression of Cx43 and PQ1 treatment stimulated an increase in the phosphorylated form of p38-MAPK, reduced levels of the anti-apoptotic protein Bcl-2, and increased the cleavage of pro-caspase-3. Silencing of Cx43 protein expression led to a reduction in the phosphorylation of p38-MAPK and an increase in Bcl-2 expression. The mechanism behind PQ1-induced cytotoxicity in FMC2u mammary carcinoma cells is thought to be attributed to the change in Cx43 expression. Furthermore, PQ1-induced apoptosis through the upregulation of Cx43 may depend on p38 MAPK, highlighting that the effect of PQ1 on gap junctions as well as cellular survival via a MAPK-dependent pathway. Full article

To determine the effect of adipose-derived stem cells (ADSCs) added to bone marrow-derived mesenchymal stem cell (MSC) sheets on bone formation at an ectopic site. We isolated MSCs and ADSCs from the same rabbits. We then prepared MSC sheets for implantation with or without ADSCs subcutaneously in the backs of severe combined immunodeficiency (SCID) mice. We assessed bone formation at eight weeks after implantation by micro-computed tomography and histological analysis. In osteogenic medium, MSCs grew to form multilayer sheets containing many calcium nodules. MSC sheets without ADSCs formed bone-like tissue; although neo-bone and cartilage-like tissues were sparse and unevenly distributed by eight weeks after implantation. In comparison, MSC sheets with ADSCs promoted better bone regeneration as evidenced by the greater density of bone, increased mineral deposition, obvious formation of blood vessels, large number of interconnected ossified trabeculae and woven bone structures, and greater bone volume/total volume within the composite constructs. Our results indicate that although sheets of only MSCs have the potential to form tissue engineered bone at an ectopic site, the addition of ADSCs can significantly increase the osteogenic potential of MSC sheets. Thus, the combination of MSC sheets with ADSCs may be regarded as a promising therapeutic strategy to stimulate bone regeneration. Full article

Structurally similar double-point modified analogues of 1,25-dihydroxyvitamin D₂ (1,25D₂) were screened in vitro for their pro-differentiating activity against the promyeloid cell line HL60. Their affinities towards human full length vitamin D receptor (VDR) and metabolic stability against human vitamin D 24-hydroxylase (CYP24A1) were also tested. The analogues (PRI-1730, PRI-1731, PRI-1732, PRI-1733 and PRI-1734) contained 5,6-trans modification of the A-ring and of the triene system, additional hydroxyl or unsaturation at C-22 in the side chain and reversed absolute configuration (24-epi) at C-24 of 1,25D₂. As presented in this paper, introduction of selected structural modifications simultaneously in two distinct parts of the vitamin D molecule resulted in a divergent group of analogues. Analogues showed lower VDR affinity in comparison to that of the parent hormones, 1,25D₂ and 1,25D₃, and they caused effective HL60 cell differentiation only at high concentrations of 100 nM and above. Unexpectedly, introducing of a 5,6-trans modification combined with C-22 hydroxyl and 24-epi configuration switched off entirely the cell differentiation activity of the analogue (PRI-1734). However, this analogue remained a moderate substrate for CYP24A1, as it was metabolized at 22%, compared to 35% for 1,25D₂. Other analogues from this series were either less (12% for PRI-1731 and PRI-1733) or more (52% for PRI-1732) resistant to the enzymatic deactivation. Although the inactive analogue PRI-1734 failed to show VDR antagonism, when tested in HL60 cells, its structure might be a good starting point for our design of a vitamin D antagonist. Full article

Small RNAs are essential for a variety of cellular functions. Argonaute (AGO) proteins are associated with all of the different classes of small RNAs, and are indispensable in small RNA-mediated regulatory pathways. AGO proteins have been identified in various types of stem cells in diverse species from plants and animals. This review article highlights recent progress on how AGO proteins and AGO-bound small RNAs regulate the self-renewal and differentiation of distinct stem cell types, including pluripotent, germline, somatic, and cancer stem cells. Full article

PROK1-V67I has been shown to play a role as a modifier gene in the PROK1-PROKR system of human early pregnancy. To explore the related modifier mechanism of PROK1-V67I, we carried out a comparison study at the gene expression level and the cell function alternation of V67I, and its wild-type (WT), in transiently-transfected cells. We, respectively, performed quantitative RT-PCR and ELISA assays to evaluate the protein and/or transcript level of V67I and WT in HTR-8/SV neo, JAR, Ishikawa, and HEK293 cells. Transiently V67I- or WT-transfected HTR-8/SV neo and HEK293 cells were used to investigate cell function alternations. The transcript and protein expressions were down-regulated in all cell lines, ranging from 20% to 70%, compared with WT. There were no significant differences in the ligand activities of V67I and WT with regard to cell proliferation, cell invasion, calcium influx, and tubal formation. Both PROK1 alleles promoted cell invasion and intracellular calcium mobilization, whereas they had no significant effects on cell proliferation and tubal formation. In conclusion, the biological effects of PROK1-V67I on cell functions are similar to those of WT, and the common variant of V67I may act as a modifier in the PROK1-PROKR system through down-regulation of PROK1 expression. This study may provide a general mechanism that the common variant of V67I, modifying the disease severity of PROK1-related pathophysiologies. Full article

Background: Allergic reactions can result in life-threatening situations resulting in high economic costs and morbidity. Therefore, more effective reagents are needed for allergy treatment. A causal relationship has been suggested to exist between the intake of omega-3/6 fatty acids, such as docosahexanoic acid (DHA), eicosapentanoic acid (EPA), docosapentanoic acid (DPA) and arachidonic acid (AA), and atopic individuals suffering from allergies. In allergic cascades, the hallmark cytokine IL-4 bind to IL-4 receptor (IL-4R) and IL-13 binds to IL-13 receptor (IL-13R), this activates the STAT6 phosphorylation pathway leading to gene activation of allergen-specific IgE antibody production by B cells. The overall aim of this study was to characterize omega-3/6 fatty acids and their effects on STAT6 signaling pathway that results in IgE production in allergic individuals. Methods: The fatty acids were tested in vitro with a HEK-Blue IL-4/IL-13 reporter cell line model, transfected with a reporter gene that produces an enzyme, secreted embryonic alkaline phosphatase (SEAP). SEAP acts as a substitute to IgE when cells are stimulated with bioactive cytokines IL-4 and/or IL-13. Results: We have successfully used DHA, EPA and DPA in our studies that demonstrated a decrease in SEAP secretion, as opposed to an increase in SEAP secretion with AA treatment. A statistical Student’s t-test revealed the significance of the results, confirming our initial hypothesis. Conclusion: We have successfully identified and characterised DHA, EPA, DPA and AA in our allergy model. While AA was a potent stimulator, DHA, EPA and DPA were potential inhibitors of IL-4R/IL-13R signalling, which regulates the STAT6 induced pathway in allergic cascades. Such findings are significant in the future design of dietary therapeutics for the treatment of allergies. Full article

Apobec-1 complementation factor (A1CF) is a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family, which participates in site-specific posttranscriptional RNA editing of apolipoprotein B (apoB) transcript. The posttranscriptional editing of apoB mRNA by A1CF in the small intestine is required for lipid absorption. Apart from the intestine, A1CF mRNA is also reported to be highly expressed in the kidneys. However, it is remained unknown about the functions of A1CF in the kidneys. The aim of this paper is to explore the potential functions of A1CF in the kidneys. Our results demonstrated that in C57BL/6 mice A1CF was weakly expressed in embryonic kidneys from E15.5dpc while strongly expressed in mature kidneys after birth, and it mainly existed in the tubules of inner cortex. More importantly, we identified A1CF negatively regulated the process of epithelial-mesenchymal transition (EMT) in kidney tubular epithelial cells. Our results found ectopic expression of A1CF up-regulated the epithelial markers E-cadherin, and down-regulated the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in NRK52e cells. In addition, knockdown of A1CF enhanced EMT contrary to the overexpression effect. Notably, the two A1CF variants led to the similar trend in the EMT process. Taken together, these data suggest that A1CF may be an antagonistic factor to the EMT process of kidney tubular epithelial cells. Full article

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight (BB) in rice (Oryza sativa L.). In this study, we investigated the genome-wide transcription patterns of two Xoo strains (KACC10331 and HB1009), which showed different virulence patterns against eight rice cultivars, including IRBB21 (carrying Xa21). In total, 743 genes showed a significant change (p-value < 0.001 in t-tests) in their mRNA expression levels in the HB1009 (K3a race) strain compared with the Xoo KACC10331 strain (K1 race). Among them, four remarkably enriched GO terms, DNA binding, transposition, cellular nitrogen compound metabolic process, and cellular macromolecule metabolic process, were identified in the upregulated genes. In addition, the expression of 44 genes was considerably higher (log2 fold changes > 2) in the HB1009 (K3a race) strain than in the Xoo KACC10331 (K1 race) strain. Furthermore, 13 and 12 genes involved in hypersensitive response and pathogenicity (hrp) and two-component regulatory systems (TCSs), respectively, were upregulated in the HB1009 (K3a race) strain compared with the Xoo KACC10331 (K1 race) strain, which we determined using either quantitative real-time PCR analysis or next-generation RNA sequencing. These results will be helpful to improve our understanding of Xoo and to gain a better insight into the Xoo–rice interactions. Full article

α-Lipoic acid (LA) contains a chiral carbon and exists as two enantiomers (R-α-lipoic acid (RLA) and S-α-lipoic acid (SLA)). We previously demonstrated that oral bioavailability of RLA is better than that of SLA. This difference arose from the fraction absorbed multiplied by gastrointestinal availability (F_a × F_g) and hepatic availability (F_h) in the absorption phase. However, it remains unclear whether F_a and/or F_g are involved in enantioselectivity. In this study, Caco-2 cells and Madin–Darby canine kidney strain II cells were used to assess the enantioselectivity of membrane permeability. LA was actively transported from the apical side to basal side, regardless of the differences in its steric structure. Permeability rates were proportionally increased in the range of 10–250 µg LA/mL, and the permeability coefficient did not differ significantly between enantiomers. Hence, we conclude that enantioselective pharmacokinetics arose from the metabolism (F_h or F_g × F_h), and definitely not from the membrane permeation (F_a) in the absorption phase. Full article

Drug-eluting stents (DES) have reduced in-stent-restenosis drastically. Yet, the stent surface material directly interacts with cascades of biological processes leading to an activation of cellular defense mechanisms. To prevent adverse clinical implications, to date almost every patient with a coronary artery disease is treated with statins. Besides their clinical benefit, statins exert a number of pleiotropic effects on endothelial cells (ECs). Since maintenance of EC function and reduction of uncontrolled smooth muscle cell (SMC) proliferation represents a challenge for new generation DES, we investigated the effect of atorvastatin (ATOR) on human coronary artery cells grown on biodegradable polymers. Our results show a cell type-dependent effect of ATOR on ECs and SMCs. We observed polymer-dependent changes in IC₅₀ values and an altered ATOR-uptake leading to an attenuation of statin-mediated effects on SMC growth. We conclude that the selected biodegradable polymers negatively influence the anti-proliferative effect of ATOR on SMCs. Hence, the process of developing new polymers for DES coating should involve the characterization of material-related changes in mechanisms of drug actions. Full article

Myrosinases are β-thioglucoside glucohydrolases and serve as defense mechanisms against insect pests and pathogens by producing toxic compounds. AtTGG6 in Arabidopsis thaliana was previously reported to be a myrosinase pseudogene but specifically expressed in pollen. However, we found that AlTGG6, an ortholog to AtTGG6 in A. lyrata (an outcrossing relative of A. thaliana) was functional, suggesting that functional AtTGG6 alleles may still exist in A. thaliana. AtTGG6 alleles in 29 A. thaliana ecotypes were cloned and sequenced. Results indicate that ten alleles were functional and encoded Myr II type myrosinase of 512 amino acids, and myrosinase activity was confirmed by overexpressing AtTGG6 in Pichia pastoris. However, the 19 other ecotypes had disabled alleles with highly polymorphic frame-shift mutations and diversified sequences. Thirteen frame-shift mutation types were identified, which occurred independently many times in the evolutionary history within a few thousand years. The functional allele was expressed specifically in pollen similar to the disabled alleles but at a higher expression level, suggesting its role in defense of pollen against insect pests such as pollen beetles. However, the defense function may have become less critical after A. thaliana evolved to self-fertilization, and thus resulted in loss of function in most ecotypes. Full article

Traumatic brain injury (TBI) is a primary injury caused by external physical force and also a secondary injury caused by biological processes such as metabolic, cellular, and other molecular events that eventually lead to brain cell death, tissue and nerve damage, and atrophy. It is a common disease process (as opposed to an event) that causes disabilities and high death rates. In order to treat all the repercussions of this injury, treatment becomes increasingly complex and difficult throughout the evolution of a TBI. Using high-throughput microarray data, we developed a systems biology approach to explore potential molecular mechanisms at four time points post-TBI (4, 8, 24, and 72 h), using a controlled cortical impact (CCI) model. We identified 27, 50, 48, and 59 significant proteins as network biomarkers at these four time points, respectively. We present their network structures to illustrate the protein–protein interactions (PPIs). We also identified UBC (Ubiquitin C), SUMO1, CDKN1A (cyclindependent kinase inhibitor 1A), and MYC as the core network biomarkers at the four time points, respectively. Using the functional analytical tool MetaCore™, we explored regulatory mechanisms and biological processes and conducted a statistical analysis of the four networks. The analytical results support some recent findings regarding TBI and provide additional guidance and directions for future research. Full article

The potential of the hyperthermophilic β-glycosidase from Pyrococcus woesei (DSM 3773) for the synthesis of glycosides under microwave irradiation (MWI) at low temperatures was investigated. Transgalactosylation reactions with β-N-acetyl-d-glucosamine as acceptor substrate (GlcNAc-linker-tBoc) under thermal heating (TH, 85 °C) and under MWI at 100 and 300 W resulted in the formation of (Galβ(1,4)GlcNAc-linker-tBoc) as the main product in all reactions. Most importantly, MWI at temperatures far below the temperature optimum of the hyperthermophilic glycosidase led to higher product yields with only minor amounts of side products β(1,6-linked disaccharide and trisaccharides). At high acceptor concentrations (50 mM), transgalactosylation reactions under MWI at 300 W gave similar product yields when compared to TH at 85 °C. In summary, we demonstrate that MWI is useful as a novel experimental set-up for the synthesis of defined galacto-oligosaccharides. In conclusion, glycosylation reactions under MWI at low temperatures have the potential as a general strategy for regioselective glycosylation reactions of hyperthermophilic glycosidases using heat-labile acceptor or donor substrates. Full article