Int. J. Mol. Sci., Volume 17, Issue 1 (January 2016) – 134 articles

For healing of critically sized bone defects, biocompatible and angiogenesis supporting implants are favorable. Murine osteoblasts showed equal proliferation behavior on the polymers poly-ε-caprolactone (PCL) and poly-(3-hydroxybutyrate)/poly-(4-hydroxybutyrate) (P(3HB)/P(4HB)). As vitality was significantly better for PCL, it was chosen as a suitable coating material for further experiments. Titanium implants with 600 µm pore size were evaluated and found to be a good implant material for bone, as primary osteoblasts showed a vitality and proliferation onto the implants comparable to well bottom (WB). Pure porous titanium implants and PCL coated porous titanium implants were compared using Live Cell Imaging (LCI) with Green fluorescent protein (GFP)-osteoblasts. Cell count and cell covered area did not differ between the implants after seven days. To improve ingrowth of blood vessels into porous implants, proangiogenic factors like Vascular Endothelial Growth Factor (VEGF) and High Mobility Group Box 1 (HMGB1) were incorporated into PCL coated, porous titanium and magnesium implants. An angiogenesis assay was performed to establish an in vitro method for evaluating the impact of metallic implants on angiogenesis to reduce and refine animal experiments in future. Incorporated concentrations of proangiogenic factors were probably too low, as they did not lead to any effect. Magnesium implants did not yield evaluable results, as they led to pH increase and subsequent cell death. Full article

Previous reports suggest that neuroendocrine disturbances in patients with traumatic brain injury (TBI) or aneurysmal subarachnoid hemorrhage (SAH) may still develop or resolve months or even years after the trauma. We investigated a cohort of n = 168 patients (81 patients after TBI and 87 patients after SAH) in whom hormone levels had been determined at various time points to assess the course and pattern of hormonal insufficiencies. Data were analyzed using three different criteria: (1) patients with lowered basal laboratory values; (2) patients with lowered basal laboratory values or the need for hormone replacement therapy; (3) diagnosis of the treating physician. The first hormonal assessment after a median time of three months after the injury showed lowered hormone laboratory test results in 35% of cases. Lowered testosterone (23.1% of male patients), lowered estradiol (14.3% of female patients) and lowered insulin-like growth factor I (IGF-I) values (12.1%) were most common. Using Criterion 2, a higher prevalence rate of 55.6% of cases was determined, which correlated well with the prevalence rate of 54% of cases using the physicians’ diagnosis as the criterion. Intraindividual changes (new onset insufficiency or recovery) were predominantly observed for the somatotropic axis (12.5%), the gonadotropic axis in women (11.1%) and the corticotropic axis (10.6%). Patients after TBI showed more often lowered IGF-I values at first testing, but normal values at follow-up (p < 0.0004). In general, most patients remained stable. Stable hormone results at follow-up were obtained in 78% (free thyroxine (fT4) values) to 94.6% (prolactin values). Full article

Mangroves are critical marine resources for their remarkable ability to tolerate seawater. Antioxidant enzymes play an especially significant role in eliminating reactive oxygen species and conferring abiotic stress tolerance. In this study, a cytosolic copper/zinc superoxide dismutase (SaCSD1) cDNA of Sonneratia alba, a mangrove species with high salt tolerance, was successfully cloned and then expressed in Escherichia coli Rosetta-gami (designated as SaCSD1). SaCSD1 comprised a complete open reading frame (ORF) of 459 bp which encoded a protein of 152 amino acids. Its mature protein is predicted to be 15.32 kDa and the deduced isoelectric point is 5.78. SaCSD1 has high sequence similarity (85%–90%) with the superoxide dismutase (CSD) of some other plant species. SaCSD1 was expressed with 30.6% yield regarding total protein content after being introduced into the pET-15b (Sma I) vector for expression in Rosetta-gami and being induced with IPTG. After affinity chromatography on Ni-NTA, recombinant SaCSD1 was obtained with 3.2-fold purification and a specific activity of 2200 U/mg. SaCSD1 showed good activity as well as stability in the ranges of pH between 3 and 7 and temperature between 25 and 55 °C. The activity of recombinant SaCSD1 was stable in 0.25 M NaCl, Dimethyl Sulphoxide (DMSO), glycerol, and chloroform, and was reduced to a great extent in β-mercaptoethanol, sodium dodecyl sulfate (SDS), H₂O_2, and phenol. Moreover, the SaCSD1 protein was very susceptive to pepsin digestion. Real-time Quantitative Polymerase Chain Reaction (PCR) assay demonstrated that SaCSD1 was expressed in leaf, stem, flower, and fruit organs, with the highest expression in fruits. Under 0.25 M and 0.5 M salt stress, the expression of SaCSD1 was down-regulated in roots, but up-regulated in leaves. Full article

Understanding the relationship between genetic variants and traits of economic importance in aquaculture species is pertinent to selective breeding programmes. High-throughput sequencing technologies have enabled the discovery of large numbers of SNPs in Atlantic salmon, and high density SNP arrays now exist. A previous genome-wide association study (GWAS) using a high density SNP array (132K SNPs) has revealed the polygenic nature of early growth traits in salmon, but has also identified candidate SNPs showing suggestive associations with these traits. The aim of this study was to test the association of the candidate growth-associated SNPs in a separate population of farmed Atlantic salmon to verify their effects. Identifying SNP-trait associations in two populations provides evidence that the associations are true and robust. Using a large cohort (N = 1152), we successfully genotyped eight candidate SNPs from the previous GWAS, two of which were significantly associated with several growth and fillet traits measured at harvest. The genes proximal to these SNPs were identified by alignment to the salmon reference genome and are discussed in the context of their potential role in underpinning genetic variation in salmon growth. Full article

Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion. Full article

Plant phenolic compounds are common dietary antioxidants that possess antioxidant and anti-inflammatory properties. Flaxseed (FS) has been reported to be radioprotective in murine models of oxidative lung damage. Flaxseed’s protective properties are attributed to its main biphenolic lignan, secoisolariciresinol diglucoside (SDG). SDG is a free radical scavenger, shown in cell free systems to protect DNA from radiation-induced damage. The objective of this study was to investigate the in vitro radioprotective efficacy of SDG in murine lung cells. Protection against irradiation (IR)-induced DNA double and single strand breaks was assessed by γ-H2AX labeling and alkaline comet assay, respectively. The role of SDG in modulating the levels of cytoprotective enzymes was evaluated by qPCR and confirmed by Western blotting. Additionally, effects of SDG on clonogenic survival of irradiated cells were evaluated. SDG protected cells from IR-induced death and ameliorated DNA damage by reducing mean comet tail length and percentage of γ-H2AX positive cells. Importantly, SDG significantly increased gene and protein levels of antioxidant HO-1, GSTM1 and NQO1. Our results identify the potent radioprotective properties of the synthetic biphenolic SDG, preventing DNA damage and enhancing the antioxidant capacity of normal lung cells; thus, rendering SDG a potential radioprotector against radiation exposure. Full article

Programmed cell death 4 (PDCD4) is one multi-functional tumor suppressor inhibiting neoplastic transformation and tumor invasion. The role of PDCD4 in tumorigenesis has attracted more attention and has been systematically elucidated in cutaneous tumors. However, the normal biological function of PDCD4 in skin is still unclear. In this study, for the first time, we find that tumor suppressor PDCD4 is uniquely induced in a cell density-dependent manner in keratinocytes. To determine the potential role of PDCD4 in keratinocyte cell biology, we show that knockdown of PDCD4 by siRNAs can promote cell proliferation in lower cell density and partially impair contact inhibition in confluent HaCaT cells, indicating that PDCD4 serves as an important regulator of keratinocytes proliferation and contact inhibition in vitro. Further, knockdown of PDCD4 can induce upregulation of cyclin D1, one key regulator of the cell cycle. Furthermore, the expression patterns of PDCD4 in normal skin, different hair cycles and the process of wound healing are described in detail in vivo, which suggest a steady-state regulatory role of PDCD4 in epidermal homeostasis and wound healing. These findings provide a novel molecular mechanism for keratinocytes’ biology and indicate that PDCD4 plays a role in epidermal homeostasis. Full article

Fibroblast growth factor 21 (FGF21) plays an important role in the treatment of disease associated with muscle insulin resistance which is characterized by various factors, such as intramuscular triglyceride (IMT) content. Studies have also shown that FGF21 inhibits triglyceride synthesis in vivo. However, the precise mechanism whereby FGF21 regulates triglyceride metabolism in intramuscular fat (IMF), which may influence the muscle insulin sensitivity, is not clearly understood. In order to understand the role of FGF21 in IMF deposition, we performed FGF21 overexpression in IMF cells by stable transfection. Our results showed that FGF21 inhibited the key adipogenesis gene mRNA expression of peroxisome proliferator-activated receptor gamma (PPARG), CCAAT/enhancer-binding protein (CEBP) family by reducing lysine-specific demethylase 1 (LSD1) expression which led to significant decline in lipid accumulation, and the result was confirmed by Western blot. Moreover, triggered by FGF21, parts of the adipokines—fatty acid-binding protein 4 (FABP4), glucose transporter 4 (GLUT4), adiponectin (ADIPOQ), and perilipin (PLIN1)—were also down-regulated. Furthermore, FGF21 gene expression was suppressed by transcription factor CEBP beta (CEBPB) which contributed strongly to triglyceride synthesis. Taken together, our study is the first to experimentally demonstrate FGF21 emerging as an efficient blockade of adipogenesis in IMF, thus also providing a new understanding of the mechanism whereby FGF21 improves insulin sensitivity. Full article

Pasteurella multocida (P. multocida) is an animal pathogen of worldwide economic significance that causes fowl cholera in poultry and wild birds. Global gene regulators, including PhoP are important in regulating bacterial virulence and are good targets for developing attenuated vaccines against many pathogenic bacteria. However, the biological significance of phoP gene has not been identified in P. multocida. Here, we identified the phoP gene in P. multocida, and we evaluated the roles of phoP in P. multocida by deleting the phoP gene. The P. multocida phoP mutant exhibited similar growth curves and lipopolysaccharide and outer membrane protein profiles but displayed defective polymyxin resistance in vitro compared with the parent strain. Additionally, the phoP deletion resulted in decreased virulence. The LD₅₀ of the ΔphoP mutant was 32- and 154-fold higher than the parent strain via the oral and intranasal routes, respectively. Transcriptome sequencing analysis showed that 161 genes were up-regulated and 173 genes were down-regulated in the absence of the phoP gene. Finally, the immunogenicity and protective efficacy of the ΔphoP mutant were evaluated. Immunized ducks produced significantly higher levels of serum IgY and bile IgA compared to the control ducks, and immunization with the ΔphoP mutant conferred 54.5% protection efficiency against challenge with the virulent P. multocida. This work provides a platform to dissect the function of phoP and develop a new vaccine against P. multocida. Full article

The prior knowledge of protein structural class may offer useful clues on understanding its functionality as well as its tertiary structure. Though various significant efforts have been made to find a fast and effective computational approach to address this problem, it is still a challenging topic in the field of bioinformatics. The position-specific score matrix (PSSM) profile has been shown to provide a useful source of information for improving the prediction performance of protein structural class. However, this information has not been adequately explored. To this end, in this study, we present a feature extraction technique which is based on gapped-dipeptides composition computed directly from PSSM. Then, a careful feature selection technique is performed based on support vector machine-recursive feature elimination (SVM-RFE). These optimal features are selected to construct a final predictor. The results of jackknife tests on four working datasets show that our method obtains satisfactory prediction accuracies by extracting features solely based on PSSM and could serve as a very promising tool to predict protein structural class. Full article

Human lung cancer is highly invasive and the most malignant among human tumors. Adenocarcinoma as a specific type of non-small cell lung cancer occurs with high frequency and is also highly resistant to radiation therapy. Thus, how to avoid radiation resistance and improve radiotherapy effectiveness is a crucial question. In the present study, human lung cancer A549 and H1299 cells were irradiated using γ-rays from a Co60 irradiator. Protein expression was detected by Western blotting. Cell cycle and apoptosis were measured by flow cytometry. Surviving fraction was determined by colony formation assay. γH2AX and 53BP1 foci formation were examined by fluorescence microscopy. In the results, we show that CHMP4C, a subunit of Endosomal sorting complex-III (ESCRT-III), is involved in radiation-induced cellular response. Radiation-induced Aurora B expression enhances CHMP4C phosphorylation in non-small cell lung cancer (NSCLC) cells, maintaining cell cycle check-point and cellular viability as well as resisting apoptosis. CHMP4C depletion enhances cellular sensitivity to radiation, delays S-phase of cell cycle and reduces ionizing radiation (IR)-induced γH2AX foci formation. We found that Aurora B targets CHMP4C and inhibition of Aurora B exhibits similar effects with silencing of CHMP4C in radioresistance. We also confirm that CHMP4C phosphorylation is elevated after IR both in p53-positive and-negative cells, indicating that the close correlation between CHMP4C and Aurora B signaling pathway in mediating radiation resistance is not p53 dependent. Together, our work establishes a new function of CHMP4C in radiation resistance, which will offer a potential strategy for non-small cell lung cancer by disrupting CHMP4C. Full article

Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins. Full article

Protein-Protein Interactions (PPIs) play a vital role in most cellular processes. Although many efforts have been devoted to detecting protein interactions by high-throughput experiments, these methods are obviously expensive and tedious. Targeting these inevitable disadvantages, this study develops a novel computational method to predict PPIs using information on protein sequences, which is highly efficient and accurate. The improvement mainly comes from the use of the Rotation Forest (RF) classifier and the Local Phase Quantization (LPQ) descriptor from the Physicochemical Property Response (PR) Matrix of protein amino acids. When performed on three PPI datasets including Saccharomyces cerevisiae, Homo sapiens, and Helicobacter pylori, we obtained good results of average accuracies of 93.8%, 97.96%, and 89.47%, which are much better than in previous studies. Extensive validations have also been explored to evaluate the performance of the Rotation Forest ensemble classifier with the state-of-the-art Support Vector Machine classifier. These promising results indicate that the proposed method might play a complementary role for future proteomics research. Full article

The human Argonaute 2 (hAgo2) protein is a key player of RNA interference (RNAi). Upon complex formation with small non-coding RNAs, the protein initially interacts with the 5′-end of a given guide RNA through multiple interactions within the MID domain. This interaction has been reported to show a strong bias for U and A over C and G at the 5′-position. Performing molecular dynamics simulations of binary hAgo2/OH–guide–RNA complexes, we show that hAgo2 is a highly flexible protein capable of binding to guide strands with all four possible 5′-bases. Especially, in the case of C and G this is associated with rather large individual conformational rearrangements affecting the MID, PAZ and even the N-terminal domains to different degrees. Moreover, a 5′-G induces domain motions in the protein, which trigger a previously unreported interaction between the 5′-base and the L2 linker domain. Combining our in silico analyses with biochemical studies of recombinant hAgo2, we find that, contrary to previous observations, hAgo2 is capable of functionally accommodating guide strands regardless of the 5′-base. Full article

The cell nucleus contains a number of membrane-less organelles or intra-nuclear compartments. These compartments are dynamic structures representing liquid-droplet phases which are only slightly denser than the bulk intra-nuclear fluid. They possess different functions, have diverse morphologies, and are typically composed of RNA (or, in some cases, DNA) and proteins. We analyzed 3005 mouse proteins localized in specific intra-nuclear organelles, such as nucleolus, chromatin, Cajal bodies, nuclear speckles, promyelocytic leukemia (PML) nuclear bodies, nuclear lamina, nuclear pores, and perinuclear compartment and compared them with ~29,863 non-nuclear proteins from mouse proteome. Our analysis revealed that intrinsic disorder is enriched in the majority of intra-nuclear compartments, except for the nuclear pore and lamina. These compartments are depleted in proteins that lack disordered domains and enriched in proteins that have multiple disordered domains. Moonlighting proteins found in multiple intra-nuclear compartments are more likely to have multiple disordered domains. Protein-protein interaction networks in the intra-nuclear compartments are denser and include more hubs compared to the non-nuclear proteins. Hubs in the intra-nuclear compartments (except for the nuclear pore) are enriched in disorder compared with non-nuclear hubs and non-nuclear proteins. Therefore, our work provides support to the idea of the functional importance of intrinsic disorder in the cell nucleus and shows that many proteins associated with sub-nuclear organelles in nuclei of mouse cells are enriched in disorder. This high level of disorder in the mouse nuclear proteins defines their ability to serve as very promiscuous binders, possessing both large quantities of potential disorder-based interaction sites and the ability of a single such site to be involved in a large number of interactions. Full article

Interleukin (IL)-10, an anti-inflammatory cytokine, is expressed in the brain and can inhibit microglial activation. Herein, we utilized lipopolysaccharide (LPS)-induced inflammatory Parkinson’s disease (PD) cell model to determine whether microglia and astrocytes are necessary targets for IL-10 neuroprotection. Primary ventral mesencephalic (VM) cultures with different composition of neurons, microglia and astrocytes were prepared. The cells were exposed to IL-10 (15, 50 or 150 ng/mL) 1 h prior to LPS (50 ng/mL) treatment. LPS induced dopaminergic and non-dopaminergic neuronal loss in VM cultures, VM neuron-enriched cultures, and neuron-microglia co-cultures, but not in neuron-astrocyte co-cultures. IL-10 reduced LPS-induced neuronal loss particularly in single VM neuron cultures. Pro-inflammatory mediators (TNF-α, IL-1β, inducible nitric oxide synthase and cyclooxygenase-2) were upregulated in both neuron-microglia and neuron-astrocyte co-cultures by LPS. In contrast, neurotrophic factors (brain-derived neurotrophic factor, insulin-like growth factor-1 or glial cell-derived neurotrophic factor) were downregulated in neuron-microglia co-cultures, but upregulated in neuron-astrocyte co-cultures by LPS. IL-10 reduced both the increase in production of the pro-inflammatory mediators and the decrease in production of the neurotrophic factors induced by LPS. These results suggest that astrocytes can balance LPS neurotoxicity by releasing more neurotrophic factors and that IL-10 exerts neuroprotective property by an extensive action including direct on neurons and indirect via inhibiting microglial activation. Full article

This study aimed to elucidate the mechanisms explaining the persistence of anemia and resistance to recombinant human erythropoietin (rHuEPO) therapy in a rat model of chronic kidney disease (CKD)-associated anemia with formation of anti-rHuEPO antibodies. The remnant kidney rat model of CKD induced by 5/6 nephrectomy was used to test a long-term (nine weeks) high dose of rHuEPO (200 UI/kg bw/week) treatment. Hematological and biochemical parameters were evaluated as well as serum and tissue (kidney, liver and/or duodenum) protein and/or gene expression of mediators of erythropoiesis, iron metabolism and tissue hypoxia, inflammation, and fibrosis. Long-term treatment with a high rHuEPO dose is associated with development of resistance to therapy as a result of antibodies formation. In this condition, serum EPO levels are not deficient and iron availability is recovered by increased duodenal absorption. However, erythropoiesis is not stimulated, and the resistance to endogenous EPO effect and to rHuEPO therapy results from the development of a hypoxic, inflammatory and fibrotic milieu in the kidney tissue. This study provides new insights that could be important to ameliorate the current therapeutic strategies used to treat patients with CKD-associated anemia, in particular those that become resistant to rHuEPO therapy. Full article

Rheumatoid arthritis (RA), a common autoimmune disorder, is associated with a chronic inflammatory response and unbalanced bone metabolism within the articular microenvironment. Adiponectin, an adipokine secreted by adipocytes, is involved in multiple functions, including lipid metabolism and pro-inflammatory activity. However, the mechanism of adiponectin performance within arthritic inflammation remains unclear. In this study, we observed the effect of adiponectin on the expression of oncostatin M (OSM), a pro-inflammatory cytokine, in human osteoblastic cells. Pretreatment of cells with inhibitors of phosphatidylinositol 3-kinase (PI3K), Akt, and nuclear factor (NF)-κB reduced the adiponectin-induced OSM expression in osteoblasts. Stimulation of the cells with adiponectin increased phosphorylation of PI3K, Akt, and p65. Adiponectin treatment of osteoblasts increased OSM-luciferase activity and p65 binding to NF-κB on the OSM promoter. Our results indicate that adiponectin increased OSM expression via the PI3K, Akt, and NF-κB signaling pathways in osteoblastic cells, suggesting that adiponectin is a novel target for arthritis treatment. Full article

Recent studies have revealed the high cytotoxicity of p-menthane derivatives against human tumor cells. In this study, the substance perillaldehyde 8,9-epoxide, a p-menthane class derivative obtained from (S)-(−)-perillyl alcohol, was selected in order to assess antitumor activity against experimental sarcoma 180 tumors. Toxicological effects related to the liver, spleen, kidneys and hematology were evaluated in mice submitted to treatment. The tumor growth inhibition rate was 38.4%, 58.7%, 35.3%, 45.4% and 68.1% at doses of 100 and 200 mg/kg/day for perillaldehyde 8,9-epoxide, perillyl alcohol and 25 mg/kg/day for 5-FU intraperitoneal treatments, respectively. No toxicologically significant effect was found in liver and kidney parameters analyzed in Sarcoma 180-inoculated mice treated with perillaldehyde 8,9-epoxide. Histopathological analyses of the liver, spleen, and kidneys were free from any morphological changes in the organs of the animals treated with perillaldehyde 8,9-epoxide. In conclusion, the data suggest that perillaldehyde 8,9-epoxide possesses significant antitumor activity without systemic toxicity for the tested parameters. By comparison, there was no statistical difference for the antitumor activity between perillaldehyde 8,9-epoxide and perillyl alcohol. Full article

As the first defence for cells to counteract the toxicity of active oxygen, superoxide dismutase (SOD) plays an important role in the response of living organisms to stress and cell differentiation. One extracellular Cu-ZnSOD (ecCu-ZnSOD), and two MnSODs, were identified based on the Volvariella volvacea genome sequence. All three genes have complicated alternative splicing modes during transcription; only when the fourth intron is retained can the Vv_Cu-Znsod1 gene be translated into a protein sequence with SOD functional domains. The expression levels of the three sod genes in the pilei are higher than in the stipe. The Vv_Cu-Znsod1 and the Vv_Mnsod2 are co-expressed in different developmental stages of the fruiting body, with the highest level of expression in the pilei of the egg stage, and they show a significant, positive correlation with the efficiency of karyogamy, indicating the potential role of these two genes during karyogamy. The expression of the ecCu-Znsod and two Vv_Mnsod genes showed a significant up-regulated when treated by cold stress for one hour; however, the lack of the intracellular Cu-ZnSOD encoding gene (icCu-Znsod) and the special locus of the ecCu-Znsod gene initiation codon suggested a possible reason for the autolysis phenomenon of V. volvacea in cold conditions. Full article

Serum deprivation (SD) is well known to induce G0/G1 cell cycle arrest and apoptosis in various cells. In the present study, we firstly found that SD could induce G1 arrest and the differentiation of human osteoblastic MG-63 cells, as evidenced by the increase of osteoblastic differentiation markers, such as bone morphogenetic protein-2 (BMP-2), osteocalcin and runt-related transcription factor 2 (Runx2). In parallel, gene expression of human GM3 synthase (hST3Gal V) catalyzing ganglioside GM3 biosynthesis was upregulated by SD in MG-63 cells. The 5′-flanking region of the hST3Gal V gene was functionally characterized to elucidate transcriptional regulation of hST3Gal V in SD-induced MG-63 cells. Promoter analysis using 5′-deletion constructs of the hST3Gal V gene demonstrated that the −432 to −177 region functions as the SD-inducible promoter. Site-directed mutagenesis revealed that the Runx2 binding sites located side-by-side at positions −232 and −222 are essential for the SD-induced expression of hST3Gal V in MG-63 cells. In addition, the chromatin immunoprecipitation assay also showed that Runx2 specifically binds to the hST3Gal V promoter region containing Runx2 binding sites. These results suggest that SD triggers upregulation of hST3Gal V gene expression through Runx2 activation by BMP signaling in MG-63 cells. Full article

Astrocytes have critical roles in immune defense, homeostasis, metabolism, and synaptic remodeling and function in the central nervous system (CNS); however, excessive activation of astrocytes with increased intermediate filaments following neuronal trauma, infection, ischemia, stroke, and neurodegenerative diseases results in a pro-inflammatory environment and promotes neuronal death. As an important neurotrophic factor, the secretion of endogenous basic fibroblast growth factor (bFGF) contributes to the protective effect of neuronal cells, but the mechanism of bFGF in reactive astrogliosis is still unclear. In this study, we demonstrated that exogenous bFGF attenuated astrocyte activation by reducing the expression of glial fibrillary acidic protein (GFAP) and other markers, including neurocan and vimentin, but not nestin and decreased the levels of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), via the regulation of the upstream toll-like receptor 4/nuclear factor κB (TLR4/NFκB) signaling pathway. Our study suggests that the function of bFGF is not only related to the neuroprotective and neurotrophic effect but also involved in the inhibition of excessive astrogliosis and glial scarring after neuronal injury. Full article

Autoimmune regulator (Aire) can promote the ectopic expression of peripheral tissue-restricted antigens (TRAs) in thymic medullary epithelial cells (mTECs), which leads to the deletion of autoreactive T cells and consequently prevents autoimmune diseases. However, the functions of Aire in the periphery, such as in dendritic cells (DCs), remain unclear. This study’s aim was to investigate the effect of Aire-overexpressing DCs (Aire cells) on the functions of CD4⁺ T cells and the treatment of type 1 diabetes (T1D). We demonstrated that Aire cells upregulated the mRNA levels of the tolerance-related molecules CD73, Lag3, and FR4 and the apoptosis of CD4⁺ T cells in STZ-T1D mouse-derived splenocytes. Furthermore, following insulin stimulation, Aire cells decreased the number of CD4⁺ IFN-γ⁺ T cells in both STZ-T1D and WT mouse-derived splenocytes and reduced the expression levels of TCR signaling molecules (Ca²⁺ and p-ERK) in CD4⁺ T cells. We observed that Aire cells-induced CD4⁺ T cells could delay the development of T1D. In summary, Aire-expressing DCs inhibited TCR signaling pathways and decreased the quantity of CD4⁺IFN-γ⁺ autoreactive T cells. These data suggest a mechanism for Aire in the maintenance of peripheral immune tolerance and provide a potential method to control autoimmunity by targeting Aire. Full article

Testis-specific lactate dehydrogenase (LDH-C₄) is one of the lactate dehydrogenase (LDH) isozymes that catalyze the terminal reaction of pyruvate to lactate in the glycolytic pathway. LDH-C₄ in mammals was previously thought to be expressed only in spermatozoa and testis and not in other tissues. Plateau pika (Ochotona curzoniae) belongs to the genus Ochotona of the Ochotonidea family. It is a hypoxia-tolerant species living in remote mountain areas at altitudes of 3000–5000 m above sea level on the Qinghai-Tibet Plateau. Surprisingly, Ldh-c is expressed not only in its testis and sperm, but also in somatic tissues of plateau pika. To shed light on the function of LDH-C₄ in somatic cells, Ldh-a, Ldh-b, and Ldh-c of plateau pika were subcloned into bacterial expression vectors. The pure enzymes of Lactate Dehydrogenase A₄ (LDH-A₄), Lactate Dehydrogenase B₄ (LDH-B₄), and LDH-C₄ were prepared by a series of expression and purification processes, and the three enzymes were identified by the method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (PAGE). The enzymatic kinetics properties of these enzymes were studied by Lineweaver-Burk double-reciprocal plots. The results showed the Michaelis constant (Km) of LDH-C₄ for pyruvate and lactate was 0.052 and 4.934 mmol/L, respectively, with an approximate 90 times higher affinity of LDH-C₄ for pyruvate than for lactate. At relatively high concentrations of lactate, the inhibition constant (Ki) of the LDH isoenzymes varied: LDH-A₄ (Ki = 26.900 mmol/L), LDH-B₄ (Ki = 23.800 mmol/L), and LDH-C₄ (Ki = 65.500 mmol/L). These data suggest that inhibition of lactate by LDH-A₄ and LDH-B₄ were stronger than LDH-C₄. In light of the enzymatic kinetics properties, we suggest that the plateau pika can reduce reliance on oxygen supply and enhance its adaptation to the hypoxic environments due to increased anaerobic glycolysis by LDH-C₄. Full article

In order to develop more effective immunological strategies to prevent vibriosis of farmed marine fish in Hong Kong and southern China, various vaccine preparations including formalin-, phenol-, chloroform- and heat-killed whole cell bacterins and subcellular lipopolysaccharides (LPS), as well as different administration routes, were investigated. Fish immunized with the subcellular LPS exhibited the best protection [Relative Percent of Survival (RPS) = 100], while fish immunized with whole cell bacterins displayed varying degrees of protection (RPS ranged from 28 to 80), in descending order: formalin-killed > phenol-killed > heat-killed > chloroform-killed bacterins. Regarding various administration routes, fish immunized with two intraperitoneal (i.p.) injections exhibited the best protection, and the RPS values were 100 or 85 upon higher or lower doses of pathogenic V. alginolyticus challenges. Both oral vaccination and a combination of injection/immersion trial were also effective, which achieved relatively high protection (the RPS values ranged from 45 to 64.3). However, two hyperosmotic immersions could not confer satisfactory protection, especially when fish were exposed to the severe pathogenic bacteria challenge. Marked elevations of serum agglutinating antibody titer were detected in all immunized fish. Macrophage phagocytosis was enhanced significantly, especially in the fish immunized by formalin- and phenol-killed bacterins through various administration routes. Both adaptive (specific antibody) and innate (phagocytic activity) immunity elicited by different immunization strategies were in parallel with the degree of protection offered by each of them. Although all vaccination trials had no significant effect on the serum hematocrit and hemoglobin levels, the circulating lymphocyte counts were significantly elevated in the fish immunized with LPS, formalin- and phenol-killed bacterins. Serum cortisol levels appeared to be reduced in all immunized fish except the trial of hyperosmotic immersion, which indicated the stressful impact on vaccinated fish. Full article

Colorectal cancer (CRC) is one of the most common malignancies worldwide with substantial mortality and morbidity. Alisertib (ALS) is a selective Aurora kinase A (AURKA) inhibitor with unclear effect and molecular interactome on CRC. This study aimed to evaluate the molecular interactome and anticancer effect of ALS and explore the underlying mechanisms in HT29 and Caco-2 cells. ALS markedly arrested cells in G₂/M phase in both cell lines, accompanied by remarkable alterations in the expression level of key cell cycle regulators. ALS induced apoptosis in HT29 and Caco-2 cells through mitochondrial and death receptor pathways. ALS also induced autophagy in HT29 and Caco-2 cells, with the suppression of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR), but activation of 5′ AMP-activated protein kinase (AMPK) signaling pathways. There was a differential modulating effect of ALS on p38 MAPK signaling pathway in both cell lines. Moreover, induction or inhibition of autophagy modulated basal and ALS-induced apoptosis in both cell lines. ALS potently suppressed epithelial to mesenchymal transition (EMT) in HT29 and Caco-2 cells. Collectively, it suggests that induction of cell cycle arrest, promotion of apoptosis and autophagy, and suppression of EMT involving mitochondrial, death receptor, PI3K/Akt/mTOR, p38 MAPK, and AMPK signaling pathways contribute to the cancer cell killing effect of ALS on CRC cells. Full article

Radish cytoplasmic male sterility (CMS) has been widely used for breeding in Raphanus and Brassica genera. However, the detailed regulation network of the male sterility remains to be determined. Our previous work has shown that the abnormalities in a CMS radish appeared shortly after the tetrad stage when microspores were malformed and the tapetal cells grew abnormally large. In this work, histological analysis shows that anthers are at the tetrad stage when the radish buds are about 1.5 mm in length. Furthermore, a high throughput RNA sequencing technology was employed to characterize the transcriptome of radish buds with length about 1.5 mm from two CMS lines possessing the CMS-inducing orf138 gene and corresponding near-isogenic maintainer lines. A total of 67,140 unigenes were functionally annotated. Functional terms for these genes are significantly enriched in 55 Gene Ontology (GO) groups and 323 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The transcriptome detected transcripts for 72 out of a total of 79 protein genes encoded in the chloroplast genome from radish. In contrast, the radish mitochondrial genome contains 34 protein genes, but only 16 protein transcripts were detected from the transcriptome. The transcriptome comparison between CMS and near-isogenic maintainer lines revealed 539 differentially expressed genes (DEGs), indicating that the false positive rate for comparative transcriptome profiling was clearly decreased using two groups of CMS/maintainer lines with different nuclear background. The level of 127 transcripts was increased and 412 transcripts were decreased in the CMS lines. No change in levels of transcripts except CMS-inducing orf138 was identified from the mitochondrial and chloroplast genomes. Some DEGs which would be associated with the CMS, encoding MYB and bHLH transcription factors, pentatricopeptide repeat (PPR) proteins, heat shock transcription factors (HSFs) and heat shock proteins (HSPs), are discussed. The transcriptome dataset and comparative analysis will provide an important resource for further understanding anther development, the CMS mechanism and to improve molecular breeding in radish. Full article

Astaxanthin, a potent antioxidant carotenoid, plays a major role in modulating the immune response. In this study, we examined the immunomodulatory effects of astaxanthin on cytokine production in primary cultured lymphocytes both in vitro and ex vivo. Direct administration of astaxanthin (70–300 nM) did not produce cytotoxicity in lipopolysaccharide (LPS, 100 µg/ mL)- or concanavalin A (Con A, 10 µg/ mL)-activated lymphocytes, whereas astaxanthin alone at 300 nM induced proliferation of splenic lymphocytes (p < 0.05) in vitro. Although astaxanthin, alone or with Con A, had no apparent effect on interferon (INF-γ) and interleukin (IL-2) production in primary cultured lymphocytes, it enhanced LPS-induced INF-γ production. In an ex vivo experiment, oral administration of astaxanthin (0.28, 1.4 and 7 mg/kg/day) for 14 days did not cause alterations in the body or spleen weights of mice and also was not toxic to lymphocyte cells derived from the mice. Moreover, treatment with astaxanthin significantly increased LPS-induced lymphocyte proliferation ex vivo but not Con A-stimulated lymphocyte proliferation ex vivo. Enzyme linked immunosorbent assay (ELISA) analysis revealed that administration of astaxanthin significantly enhanced INF-γ production in response to both LPS and Con A stimulation, whereas IL-2 production increased only in response to Con A stimulation. Also, astaxanthin treatment alone significantly increased IL-2 production in lymphocytes derived from mice, but did not significantly change production of INF-γ. These findings suggest that astaxanthin modulates lymphocytic immune responses in vitro, and that it partly exerts its ex vivo immunomodulatory effects by increasing INF-γ and IL-2 production without inducing cytotoxicity. Full article

A molecular dynamics simulation has been performed on the greenhouse gases carbon dioxide and methane dissolved in a sodium chloride aqueous solution, as a simple model of seawater. A carbon dioxide molecule is also treated as a hydrogen carbonate ion. The structure, coordination number, diffusion coefficient, shear viscosity, specific heat, and thermal conductivity of the solutions have been discussed. The anomalous behaviors of these properties, especially the negative pressure dependence of thermal conductivity, have been observed in the higher-pressure region. Full article

Human leukocytic antigen-B27 heavy chain (HLA-B27 HC) has the tendency to fold slowly, in turn gradually forming a homodimer, (B27-HC)₂ via a disulfide linkage to activate killer cells and T-helper 17 cells and inducing endoplasmic reticulum (ER) stress to trigger the IL-23/IL-17 axis for pro-inflammatory reactions. All these consequences lead to the pathogenesis of ankylosing spondylitis (AS). Sulfasalazine (SSA) is a common medication used for treatment of patients with AS. However, the effects of SSA treatment on (B27-HC)₂ formation and on suppression of IL-23/IL-17 axis of AS patients remain to be determined. In the current study, we examine the (B27-HC)₂ of peripheral blood mononuclear cells (PBMC), the mean grade of sarcoiliitis and lumbar spine Bath Ankylosing Spondylitis Radiology Index (BASRI) scores of 23 AS patients. The results indicated that AS patients without (B27-HC)₂ on PBMC showed the lower levels of mean grade of sarcoiliitis and the lumbar spine BASRI scores. In addition, after treatment with SSA for four months, the levels of (B27-HC)₂ on PBMCs were significantly reduced. Cytokines mRNA levels, including TNFα, IL-17A, IL-17F and IFNγ, were also significantly down-regulated in PBMCs. However, SSA treatment did not affect the levels of IL-23 and IL-23R mRNAs. Full article

Well known for its anti-oxidative and anti-inflammation properties, curcumin is a polyphenol found in the rhizome of Curcuma longa. In this study, we evaluated the effects of curcumin on behavioral recovery, glial scar formation, tissue preservation, axonal sprouting, and inflammation after spinal cord injury (SCI) in male Wistar rats. The rats were randomized into two groups following a balloon compression injury at the level of T9–T10 of the spinal cord, namely vehicle- or curcumin-treated. Curcumin was applied locally on the surface of the injured spinal cord immediately following injury and then given intraperitoneally daily; the control rats were treated with vehicle in the same manner. Curcumin treatment improved behavioral recovery within the first week following SCI as evidenced by improved Basso, Beattie, and Bresnahan (BBB) test and plantar scores, representing locomotor and sensory performance, respectively. Furthermore, curcumin treatment decreased glial scar formation by decreasing the levels of MIP1α, IL-2, and RANTES production and by decreasing NF-κB activity. These results, therefore, demonstrate that curcumin has a profound anti-inflammatory therapeutic potential in the treatment of spinal cord injury, especially when given immediately after the injury. Full article

Insomnia is a prevalent disorder with deleterious effects such as decreased quality of life, and a predisposition to a number of psychiatric disorders. Fortunately, numerous approved hypnotic treatments are available. This report reviews the state of the art of pharmacotherapy with a reference to cognitive behavioral therapy for insomnia (CBT-I) as well. It provides the clinician with a guide to all the Food and Drug Administration (FDA) approved hypnotics (benzodiazepines, nonbenzodiazepines, ramelteon, low dose sinequan, and suvorexant) including potential side effects. Frequently, chronic insomnia lasts longer than 2 years. Cognizant of this and as a result of longer-term studies, the FDA has approved all hypnotics since 2005 without restricting the duration of use. Our manuscript also reviews off-label hypnotics (sedating antidepressants, atypical antipsychotics, anticonvulsants and antihistamines) which in reality, are more often prescribed than approved hypnotics. The choice of which hypnotic to choose is discussed partially being based on which segment of sleep is disturbed and whether co-morbid illnesses exist. Lastly, we discuss recent label changes required by the FDA inserting a warning about “sleep-related complex behaviors”, e.g., sleep-driving for all hypnotics. In addition, we discuss FDA mandated dose reductions for most zolpidem preparations in women due to high zolpidem levels in the morning hours potentially causing daytime carry-over effects. Full article

All living organisms synchronize biological functions with environmental changes; melatonin plays a vital role in regulating daily and seasonal variations. Due to rhythmic activity of the timezyme aralkylamine N-acetyltransferase (AANAT), the blood level of melatonin increases at night and decreases during daytime. Whereas other vertebrates have a single form of AANAT, bony fishes possess various isoforms of aanat genes, though the reasons are still unclear. Here, we have taken advantage of multiple unpublished teleost aanat sequences to explore and expand our understanding of the molecular evolution of aanat in fish. Our results confirm that two rounds of whole-genome duplication (WGD) led to the existence of three fish isoforms of aanat, i.e., aanat1a, aanat1b, and aanat2; in addition, gene loss led to the absence of some forms from certain special fish species. Furthermore, we suggest the different roles of two aanat1s in amphibious mudskippers, and speculate that the loss of aanat1a, may be related to terrestrial vision change. Several important sites of AANAT proteins and regulatory elements of aanat genes were analyzed for structural comparison and functional forecasting, respectively, which provides insights into the molecular evolution of the differences between AANAT1 and AANAT2. Full article

Superparamagnetic iron-oxide nanoparticles (SPIONs) show great promise for multiple applications in biomedicine. While a number of studies have examined their safety profile, the toxicity of these particles on reproductive organs remains uncertain. The goal of this study was to evaluate the cytotoxicity of starch-coated, aminated, and PEGylated SPIONs on a cell line derived from Chinese Hamster ovaries (CHO-K1 cells). We evaluated the effect of particle diameter (50 and 100 nm) and polyethylene glycol (PEG) chain length (2k, 5k and 20k Da) on the cytotoxicity of SPIONs by investigating cell viability using the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine B (SRB) assays. The kinetics and extent of SPION uptake by CHO-K1 cells was also studied, as well as the resulting generation of intracellular reactive oxygen species (ROS). Cell toxicity profiles of SPIONs correlated strongly with their cellular uptake kinetics, which was strongly dependent on surface properties of the particles. PEGylation caused a decrease in both uptake and cytotoxicity compared to aminated SPIONs. Interestingly, 2k Da PEG-modifed SPIONs displayed the lowest cellular uptake and cytotoxicity among all studied particles. These results emphasize the importance of surface coatings when engineering nanoparticles for biomedical applications. Full article

The development of stimulus-responsive photosensitizer delivery systems that carry a high payload of photosensitizers is of great importance in photodynamic therapy. In this study, redox-responsive polysilsesquioxane nanoparticles (PSilQNPs) built by a reverse microemulsion approach using 5,10,15,20-tetrakis(carboxyphenyl) porphyrin (TCPP) silane derivatives as building blocks, were successfully fabricated. The structural properties of TCPP-PSilQNPs were characterized by dynamic light scattering (DLS)/ζ-potential, scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). The photophysical properties were determined by UV-vis and fluorescence spectroscopy. The quantity of singlet oxygen generated in solution was measured using 1,3-diphenylisobenzofuran. The redox-responsive release of TCPP molecules was successfully demonstrated in solution in the presence of a reducing agent. The internalization of TCPP-PSilQNPs in cancer cells was investigated using laser scanning confocal microscopy. Phototoxicity experiments in vitro showed that the redox-responsive TCPP-PSilQNPs exhibited an improved phototherapeutic effect on cervical cancer cells compared to a non-responsive TCPP-PSilQNP control material. Full article

Tea plant is known to be a hyper-accumulator of fluoride (F). Over-intake of F has been shown to have adverse effects on human health, e.g., dental fluorosis. Thus, understanding the mechanisms fluoride accumulation and developing potential approaches to decrease F uptake in tea plants might be beneficial for human health. In the present study, we found that pretreatment with the anion channel inhibitor NPPB reduced F accumulation in tea plants. Simultaneously, we observed that NPPB triggered Ca²⁺ efflux from mature zone of tea root and significantly increased relative CaM in tea roots. Besides, pretreatment with the Ca²⁺ chelator (EGTA) and CaM antagonists (CPZ and TFP) suppressed NPPB-elevated cytosolic Ca²⁺ fluorescence intensity and CaM concentration in tea roots, respectively. Interestingly, NPPB-inhibited F accumulation was found to be significantly alleviated in tea plants pretreated with either Ca²⁺ chelator (EGTA) or CaM antagonists (CPZ and TFP). In addition, NPPB significantly depolarized membrane potential transiently and we argue that the net Ca²⁺ and H⁺ efflux across the plasma membrane contributed to the restoration of membrane potential. Overall, our results suggest that regulation of Ca²⁺-CaM and plasma membrane potential depolarization are involved in NPPB-inhibited F accumulation in tea plants. Full article

Chromatin structure affects the extent of DNA damage and repair. Thus, it has been shown that heterochromatin is more protective against DNA double strand breaks (DSB) formation by ionizing radiation (IR); and that DNA DSB repair may proceed differently in hetero- and euchromatin regions. Human embryonic stem cells (hESC) have a more open chromatin structure than differentiated cells. Here, we study the effect of chromatin structure in hESC on initial DSB formation and subsequent DSB repair. DSB were scored by comet assay; and DSB repair was assessed by repair foci formation via 53BP1 antibody staining. We found that in hESC, heterochromatin is confined to distinct regions, while in differentiated cells it is distributed more evenly within the nuclei. The same dose of ionizing radiation produced considerably more DSB in hESC than in differentiated derivatives, normal human fibroblasts; and one cancer cell line. At the same time, the number of DNA repair foci were not statistically different among these cells. We showed that in hESC, DNA repair foci localized almost exclusively outside the heterochromatin regions. We also noticed that exposure to ionizing radiation resulted in an increase in heterochromatin marker H3K9me3 in cancer HT1080 cells, and to a lesser extent in IMR90 normal fibroblasts, but not in hESCs. These results demonstrate the importance of chromatin conformation for DNA protection and DNA damage repair; and indicate the difference of these processes in hESC. Full article

The aim of this work was to study the interaction of sulpiride with human serum albumin (HSA) and bovine serum albumin (BSA) through the fluorescence quenching technique. As sulpiride molecules emit fluorescence, we have developed a simple mathematical model to discriminate the quencher fluorescence from the albumin fluorescence in the solution where they interact. Sulpiride is an antipsychotic used in the treatment of several psychiatric disorders. We selectively excited the fluorescence of tryptophan residues with 290 nm wavelength and observed the quenching by titrating HSA and BSA solutions with sulpiride. Stern-Volmer graphs were plotted and quenching constants were estimated. Results showed that sulpiride form complexes with both albumins. Estimated association constants for the interaction sulpiride–HSA were 2.20 (±0.08) × 10⁴ M⁻¹, at 37 °C, and 5.46 (±0.20) × 10⁴ M⁻¹, at 25 °C. Those for the interaction sulpiride-BSA are 0.44 (±0.01) × 10⁴ M⁻¹, at 37 °C and 2.17 (±0.04) × 10⁴ M⁻¹, at 25 °C. The quenching intensity of BSA, which contains two tryptophan residues in the peptide chain, was found to be higher than that of HSA, what suggests that the primary binding site for sulpiride in albumin should be located next to the sub domain IB of the protein structure. Full article

The current work reports the synthesis, spectroscopic studies, antiradical and antiproliferative properties of four ruthenium(III) complexes of heterocyclic tridentate Schiff base bearing a simple 2′,4′-dihydroxyacetophenone functionality and ethylenediamine as the bridging ligand with RCHO moiety. The reaction of the tridentate ligands with RuCl₃·3H₂O lead to the formation of neutral complexes of the type [Ru(L)Cl₂(H₂O)] (where L = tridentate NNO ligands). The compounds were characterized by elemental analysis, UV-vis, conductivity measurements, FTIR spectroscopy and confirmed the proposed octahedral geometry around the Ru ion. The Ru(III) compounds showed antiradical potentials against 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radicals, with DPPH scavenging capability in the order: [(PAEBOD)RuCl₂] > [(BZEBOD)RuCl₂] > [(MOABOD)RuCl₂] > [Vit. C] > [rutin] > [(METBOD)RuCl₂], and ABTS radical in the order: [(PAEBOD)RuCl₂] < [(MOABOD)RuCl₂] < [(BZEBOD)RuCl₂] < [(METBOD)RuCl₂]. Furthermore, in vitro anti-proliferative activity was investigated against three human cancer cell lines: renal cancer cell (TK-10), melanoma cancer cell (UACC-62) and breast cancer cell (MCF-7) by SRB assay. Full article

Chitin fiber is the load-bearing component in natural chitin-based materials. In these materials, chitin is always partially deacetylated to different levels, leading to diverse material properties. In order to understand how the acetyl group enhances the fracture resistance capability of chitin fiber, we constructed atomistic models of chitin with varied acetylation degree and analyzed the hydrogen bonding pattern, fracture, and stress-strain behavior of these models. We notice that the acetyl group can contribute to the formation of hydrogen bonds that can stabilize the crystalline structure. In addition, it is found that the specimen with a higher acetylation degree presents a greater resistance against fracture. This study describes the role of the functional group, acetyl groups, in crystalline chitin. Such information could provide preliminary understanding of nanomaterials when similar functional groups are encountered. Full article

Acute liver failure (ALF) is a kind of complicated syndrome. Furthermore, adipose-derived mesenchymal stem cells (ADMSCs) can serve as a useful cell resource for autotransplantation due to their abundance and micro-invasive accessability. However, it is unknown how ALF will influence the characteristics of ADMSCs and whether ADMSCs from patients suffering from end-stage liver diseases are potential candidates for autotransplantation. This study was designed to compare various properties of ALF-derived ADMSCs with normal ADMSCs in pig models, with regard to their cellular morphology, cell proliferative ability, cell apoptosis, expression of surface antigens, mitochondrial and lysosomal activities, multilineage potency, and expression of liver-specific genes. Our results showed that ALF does not influence the stem cell characteristics and cell activities of ADMSCs. Intriguingly, the expression levels of several liver-specific genes in ALF-derived ADMSCs are higher than in normal ADMSCs. In conclusion, our findings indicate that the stem cell characteristics and cell activities of ADMSCs were not altered by ALF and these cells can serve as a new source for regenerative medicine. Full article

Earthworms express, as most animals, metallothioneins (MTs)—small, cysteine-rich proteins that bind d¹⁰ metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd₇wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by ¹H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo. Full article

Borderline personality disorder (BPD) is a serious psychic disease with a high risk for suicide. DNA methylation is a hallmark for aberrant epigenetic regulation and could be involved in the etiology of BPD. Previously, it has been reported that increased DNA methylation of neuropsychiatric genes is found in the blood of patients with BPD compared to healthy controls. Here, we analyzed DNA methylation patterns of the ribosomal RNA gene (rDNA promoter region and 5′-external transcribed spacer/5′ETS) and the promoter of the proline rich membrane anchor 1 gene (PRIMA1) in peripheral blood samples of 24 female patients (mean age (33 ± 11) years) diagnosed with DSM-IV BPD and in 11 female controls (mean age (32 ± 7) years). A significant aberrant methylation of rDNA and PRIMA1 was revealed for BPD patients using pyrosequencing. For the promoter of PRIMA1, the average methylation of six CpG sites was 1.6-fold higher in BPD patients compared to controls. In contrast, the methylation levels of the rDNA promoter region and the 5′ETS were significantly lower (0.9-fold) in patients with BPD compared to controls. Thus, for nine CpGs located in the rDNA promoter region and for four CpGs at the 5′ETS decreased methylation was found in peripheral blood of patients compared to controls. Our results suggest that aberrant methylation of rDNA and PRIMA1 is associated with the pathogenesis of BPD. Full article

The goal to successful wound healing is essentially to immobilize and recruit appropriate numbers of host stem or progenitor cells to the wound area. In this study, we developed a chitosan nanofiber-immobilized neuropeptide substance-P (SP), which mediates stem cell mobilization and migration, onto the surfaces of nanofibers using a peptide-coupling agent, and evaluated its biological effects on stem cells. The amount of immobilized SP on chitosan nanofibers was modulated over the range of 5.89 ± 3.27 to 75.29 ± 24.31 ng when reacted with 10 to 500 ng SP. In vitro migration assays showed that SP-incorporated nanofibers induced more rapid migration of human mesenchymal stem cells on nanofibers compared to pristine samples. Finally, the conjugated SP evoked a minimal foreign body reaction and recruited a larger number of CD29- and CD44-positive stem cells into nanofibers in a mouse subcutaneous pocket model. Full article

Gastric cancer (GC) has significant morbidity and mortality worldwide and especially in China. Its molecular pathogenesis has not been thoroughly elaborated. The acknowledged biomarkers for diagnosis, prognosis, recurrence monitoring and treatment are lacking. Proteins from matched pairs of human GC and adjacent tissues were analyzed by a coupled label-free Mass Spectrometry (MS) approach, followed by functional annotation with software analysis. Nano-LC-MS/MS, quantitative real-time polymerase chain reaction (qRT-PCR), western blot and immunohistochemistry were used to validate dysregulated proteins. One hundred forty-six dysregulated proteins with more than twofold expressions were quantified, 22 of which were first reported to be relevant with GC. Most of them were involved in cancers and gastrointestinal disease. The expression of a panel of four upregulated nucleic acid binding proteins, heterogeneous nuclear ribonucleoprotein hnRNPA2B1, hnRNPD, hnRNPL and Y-box binding protein 1 (YBX-1) were validated by Nano-LC-MS/MS, qRT-PCR, western blot and immunohistochemistry assays in ten GC patients’ tissues. They were located in the keynotes of a predicted interaction network and might play important roles in abnormal cell growth. The label-free quantitative proteomic approach provides a deeper understanding and novel insight into GC-related molecular changes and possible mechanisms. It also provides some potential biomarkers for clinical diagnosis. Full article

Organic gem-dihydroperoxides (DHPs) and their derived peroxides have attracted a great deal of attention as potential anti-cancer agents. However, the precise mechanism of their inhibitory effect on tumors is unknown. To determine the mechanism of the inhibitory effects of DHPs, we examined the effects of DHPs on leukemia K562 cells. As a result, certain DHPs used in this study exhibited growth-inhibitory activity according to a clear structure-activity relationship. The most potent DHP, 12AC3O, induced apoptosis in K562 cells, but not in peripheral blood monocytes (PBMCs) or fibroblast cells. 12AC3O induced apoptosis through the intrinsic mitochondrial pathway and thereafter through the extrinsic pathway. The activity of the former pathway was partly attenuated by a JNK inhibitor. Interestingly, 12AC3O induced apoptosis by trapping a large amount of ROS, leading to an extremely lower intracellular ROS level compared with that in the cells in the steady-state condition. These results suggest that an appropriate level of intracellular ROS was necessary for the maintenance of cancer cell growth. DHPs may have a potential to be a novel anti-cancer agent with minimum adverse effects on normal cells. Full article

MicroRNAs are 18–22 nucleotides long, non-coding RNAs that bind transcripts with complementary sequences leading to either mRNA degradation or translational suppression. However, the inherent differences in preferred mode of miRNA regulation among cells of different origin have not been examined. In our previous transcriptome profiling studies, we observed that post-transcriptional regulation can differ substantially depending on the cell in context. Here we examined mechanistic differences in the regulation of a let-7a targeted (wild type) or resistant (mutant) engineered renilla transcript across various mammalian cell lines of diverse origin. Dual luciferase assays show that compared to mutant (mut), the reporter gene containing wild type (wt) let-7a binding sites was efficiently suppressed upon transfection in various cell lines. Importantly, the strength of miRNA regulation varied across the cell lines. Total RNA analysis demonstrates that wt renilla mRNA was expressed to similar or higher levels compared to mut suggesting that translation repression is a predominant mode of miRNA regulation. Nonetheless, transcript degradation was observed in some cell lines. Ago-2 immunoprecipitation show that miRNA repressed renilla mRNA are associated with functional mi-RISC (miRNA-RNA induced silencing complex). Given the immense potential of miRNA as a therapeutic option, these findings highlight the necessity to thoroughly examine the mode of mRNA regulation in order to achieve the beneficial effects in targeting cells. Full article

Inflammation and oxidative stress are involved in age-related macular degeneration (AMD) and possibly associated with an activation of neuronal apoptosis inhibitor protein/class II transcription activator of the Major Histocompatibility Complex (MHC)/heterokaryon incompatibility/telomerase-associated protein 1, leucine-rich repeat or nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing 3 (NLRP3) inflammasome. In the present study, we used a translational approach to address this hypothesis. In patients with AMD, we observed increased mRNA levels of NLRP3, pro-interleukin-1 beta (IL-1β) and pro-IL-18 in AMD lesions of the retinal pigment epithelium (RPE) and photoreceptor. In vitro, a similar increase was evoked by oxidative stress or lipopolysaccharide (LPS) stimulation in the adult retinal pigment epithelium (ARPE-19) cell line, and the increase was reduced in siRNA transfected cells to knockdown NLRP3. Ultrastructural studies of ARPE-19 cells showed a swelling of the cytoplasm, mitochondrial damage, and occurrence of autophagosome-like structures. NLRP3 positive dots were detected within autophagosome-like structures or in the extracellular space. Next, we used a mouse model of AMD, Ccl2/Cx3cr1 double knockout on rd8 background (DKO rd8) to ascertain the in vivo relevance. Ultrastructural studies of the RPE of these mice showed damaged mitochondria, autophagosome-like structures, and cytoplasmic vacuoles, which are reminiscent of the pathology seen in stressed ARPE-19 cells. The data suggest that the NLRP3 inflammasome may contribute in AMD pathogenesis. Full article

Vitamin D is a lipid soluble steroid hormone with pleiotropic biological properties, including regulation of cell proliferation, differentiation and apoptosis. As to these desirable anticancer actions, 1,25-dihydroxyvitamins D and analogs have been reported to inhibit the proliferation and to induce differentiation of a wide variety of cancer cell types, including human malignant melanoma. However, there is a need for novel and more efficacious vitamin D analogs, and how best to design such is still an open issue. A series of double point modified (DPM) analogs of 1,25-dihydroxyvitamin D₂ (1,25(OH)₂D₂) induced differentiation of the vitamin D receptor (VDR) positive A375 and VDR negative SK-MEL 188b human malignant melanoma cell lines. Surprisingly, the dose of 1,25(OH)₂D₂ required to inhibit the proliferation of the A375 melanoma cell line by was several fold lower than that required in the case of 1,25(OH)₂D₃. To evaluate the impact of the modification in the side chain (additional 22-hydroxyl) and in the A-ring (5,6-trans modification), the regular side-chain of vitamin D₂ or D₃ was retained in the structure of our analogs. As expected, 5,6-trans modification was advantageous to enhancing the anti-proliferative activity of analogs, but not as a single point modification (SPM). Very unexpectedly, the additional 22-hydroxyl in the side-chain reduced significantly the anti-proliferative activity of both the natural and 5,6-trans series analogs. Finally, an induction of pigmentation in melanoma SK-MEL 188b cells was observed to sensitized cells to the effect of vitamin D analogs. Full article

Missense mutations in spermine synthase (SpmSyn) protein have been shown to cause the Snyder-Robinson syndrome (SRS). Depending on the location within the structure of SpmSyn and type of amino acid substitution, different mechanisms resulting in SRS were proposed. Here we focus on naturally occurring amino acid substitutions causing SRS, which are situated away from the active center of SpmSyn and thus are not directly involved in the catalysis. Two of the mutations, M35R and P112L, are reported for the first time in this study. It is demonstrated, both experimentally and computationally, that for such mutations the major effect resulting in dysfunctional SpmSyn is the destabilization of the protein. In vitro experiments indicated either no presence or very little amount of the mutant SpmSyn in patient cells. In silico modeling predicted that all studied mutations in this work destabilize SpmSyn and some of them abolish homo-dimer formation. Since dimerization and structural stability are equally important for the wild type function of SpmSyn, it is proposed that the SRS caused by mutations occurring in the N-domain of SpmSyn is a result of dysfunctional mutant proteins being partially unfolded and degraded by the proteomic machinery of the cell or being unable to form a homo-dimer. Full article

Six new 9-hydroxybriarane diterpenoids, briarenolides ZI–ZVI (1–6), were isolated from a gorgonian coral Briareum sp. The structures of briaranes 1–6 were elucidated by spectroscopic methods and by comparison of their spectroscopic data with those of related analogues. Briarenolides ZII (2) and ZVI (6) were found to significantly inhibit the expression of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Full article

Limited comprehension of aneurysm pathology has led to inconclusive results from clinical trials. miRNAs are key regulators of post-translational gene modification and are useful tools in elucidating key features of aneurysm pathogenesis in distinct entities of abdominal and popliteal aneurysms. Here, surgically harvested specimens from 19 abdominal aortic aneurysm (AAA) and 8 popliteal artery aneurysm (PAA) patients were analyzed for miRNA expression and histologically classified regarding extracellular matrix (ECM) remodeling and inflammation. DIANA-based computational target prediction and pathway enrichment analysis verified our results, as well as previous ones. miRNA-362, -19b-1, -194, -769, -21 and -550 were significantly down-regulated in AAA samples depending on degree of inflammation. Similar or inverse regulation was found for miR-769, 19b-1 and miR-550, -21, whereas miR-194 and -362 were unaltered in PAA. In situ hybridization verified higher expression of miR-550 and -21 in PAA compared to AAA and computational analysis for target genes and pathway enrichment affirmed signal transduction, cell-cell-interaction and cell degradation pathways, in line with previous results. Despite the vague role of miRNAs for potential diagnostic and treatment purposes, the number of candidates from tissue signature studies is increasing. Tissue morphology influences subsequent research, yet comparison of distinct entities of aneurysm disease can unravel core pathways. Full article

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions. Full article

Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients. Full article

Maternal hormones are essential for the normal fetal development during pregnancy. Autoimmune thyroid disease is a frequent pathology in our iodine replete region. The aim of this study is to evaluate the occurrence of subclinical hypothyroidism (SCH) in cases with known autoimmune thyroid disease, which were in a euthyroid state prior to pregnancy, and to assess the association between supplemental treatments administered and the outcome of the pregnancy. The study is a prospective interventional controlled study. The two cohorts comprise the interventional group, consisting of 109 pregnant women with known autoimmune asymptomatic thyroid disease, without any levothyroxine (LT4) treatment and an aged-matched control group, with an unknown thyroid disease. After the pregnancy, a monthly evaluation of TSH, FT3, and FT4 was performed. Offspring evaluation was made at birth time. 88.8% of the women developed SCH in the first four weeks of pregnancy. Average LT4 doses increased as the pregnancy progressed. The monthly adjustment was 12.5 or 25 μg. All SCH cases developed in the first trimester of pregnancy. There was no significant difference regarding the gestational week, weight, or length at birth between the interventional group and controls, when TSH values were in the optimal range, during the whole pregnancy. Premature birth was described in one case in the interventional group. Full article

Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β₁-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in Ras^G12V-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development. Full article

Anaerobic digestion using mixed-culture with broader choice of pretreatments for hydrogen (H₂) production was investigated. Pretreatment of wastewater sludge by five methods, such as heat, acid, base, microwave and chloroform was conducted using crude glycerol (CG) as substrate. Results for heat treatment (100 °C for 15 min) showed the highest H₂ production across the pretreatment methods with 15.18 ± 0.26 mmol/L of medium at 30 °C in absence of complex media and nutrient solution. The heat-pretreated inoculum eliminated H₂ consuming bacteria and produced twice as much as H₂ as compared to other pretreatment methods. The fermentation conditions, such as CG concentration (1.23 to 24 g/L), percentage of inoculum size (InS) (1.23% to 24% v/v) along with initial pH (2.98 to 8.02) was tested using central composite design (CCD) with H₂ production as response parameter. The maximum H₂ production of 29.43 ± 0.71 mmol/L obtained at optimum conditions of 20 g/L CG, 20% InS and pH 7. Symbiotic correlation of pH over CG and InS had a significant (p-value: 0.0011) contribution to H₂ production. The mixed-culture possessed better natural acclimatization activity for degrading CG, at substrate inhibition concentration and provided efficient inoculum conditions in comparison to mono- and co-culture systems. The heat pretreatment step used across mixed-culture system is simple, cheap and industrially applicable in comparison to mono-/co-culture systems for H₂ production. Full article

Maternal protein components change markedly during mammalian oogenesis. Many of these proteins have yet to be characterized and verified. In this study, a proteomics approach was used to evaluate changes in proteins during oogenesis in the Swamp Buffalo (Bubalus bubalis). Proteins from 500 immature oocytes and 500 in vitro matured oocytes were subjected to two-dimensional electrophoresis, and more than 400 spots were detected. Image analysis indicated that 17 proteins were differentially expressed between the two groups. Eight proteins were identified by mass spectrometry. In mature oocytes, three proteins were down-regulated: major vault protein (MVP), N-acetyllactosaminide β-1,6-N-acetylglucosaminyl-transferase (GCNT-2), and gem-associated protein (GEMIN)8, whereas five other proteins, heat shock protein (HSP)60, Ras-responsive element-binding protein 1 (RREB-1), heat shock cognate 71 kDa protein (HSC71), hemoglobin subunit α (HBA), and BMP-2-inducible protein kinase (BMP-2K), were up-regulated. The expression profiles of HSP60 and GEMIN8 were further verified by Western blotting. The changes in HSP60 protein expression demonstrate the increasing need for mitochondrial protein importation to facilitate macromolecular assembly during oocyte maturation. The down-regulation of GEMIN8 production implies that RNA splicing is impaired in mature oocytes. Full article

Prolactin family 7, subfamily d, member 1 (PRL7D1) is found in mouse placenta. Our recent work showed that PRL7D1 is also present in mouse testis Leydig cells, and the expression of PRL7D1 in the testis exhibits an age-related increase. In the present study, we generated transgenic mice with Leydig cell-specific PRL7D1 overexpression to explore its function during male reproduction. Prl7d1 male mice exhibited subfertility as reflected by reduced sperm counts and litter sizes. The testes from Prl7d1 transgenic mice appeared histologically normal, but the frequency of apoptotic germ cells was increased. Prl7d1 transgenic mice also had lower testosterone concentrations than wild-type mice. Mechanistic studies revealed that Prl7d1 transgenic mice have defects in the testicular expression of steroidogenic acute regulatory protein (STAR) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase cluster (HSD3B). Further studies revealed that PRL7D1 overexpression affected the expression of transferrin (TF) in Sertoli cells. These results suggest that PRL7D1 overexpression could lead to increased germ cell apoptosis and exert an inhibitory effect on testosterone production in Leydig cells by reducing the expression of certain steroidogenic-related genes. In addition, PRL7D1 appears to have important roles in the function of Sertoli cells, which, in turn, affects male fertility. We conclude that the expression level of PRL7D1 is associated with the reproductive function of male mice. Full article

The d-galactose (d-gal)-injected animal model, which is typically established by administering consecutive subcutaneous d-gal injections to animals for approximately six or eight weeks, has been frequently used for aging research. In addition, this animal model has been demonstrated to accelerate aging in the brain, kidneys, liver and blood cells. However, studies on aging in male reproductive organs that have used this animal model remain few. Therefore, the current study aimed to optimize a model of male reproductive aging by administering d-gal injections to male mice and to determine the possible mechanism expediting senescence processes during spermatogenesis. In this study, C57Bl/6 mice were randomized into five groups (each containing 8–10 mice according to the daily intraperitoneal injection of vehicle control or 100 or 200 mg/kg dosages of d-gal for a period of six or eight weeks). First, mice subjected to d-gal injections for six or eight weeks demonstrated considerably decreased superoxide dismutase activity in the serum and testis lysates compared to those in the control group. The lipid peroxidation in testis also increased in the d-gal-injected groups. Furthermore, the d-gal-injected groups exhibited a decreased ratio of testis weight/body weight and sperm count compared to the control group. The percentages of both immotile sperm and abnormal sperm increased considerably in the d-gal-injected groups compared to those of the control group. To determine the genes influenced by the d-gal injection during murine spermatogenesis, a c-DNA microarray was conducted to compare testicular RNA samples between the treated groups and the control group. The d-gal-injected groups exhibited RNA transcripts of nine spermatogenesis-related genes (Cycl2, Hk1, Pltp, Utp3, Cabyr, Zpbp2, Speer2, Csnka2ip and Katnb1) that were up- or down-regulated by at least two-fold compared to the control group. Several of these genes are critical for forming sperm-head morphologies or maintaining nuclear integration (e.g., cylicin, basic protein of sperm head cytoskeleton 2 (Cylc2), casein kinase 2, alpha prime interacting protein (Csnka2ip) and katanin p80 (WD40-containing) subunit B1 (Katnb1)). These results indicate that d-gal-injected mice are suitable for investigating male reproductive aging. Full article

Photodynamic therapy (PDT) of the thoracic cavity can be performed in conjunction with surgery to treat cancers of the lung and its pleura. However, illumination of the cavity results in tissue exposure to a broad range of fluence rates. In a murine model of intrathoracic PDT, we studied the efficacy of 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH; Photochlor^®)-mediated PDT in reducing the burden of non-small cell lung cancer for treatments performed at different incident fluence rates (75 versus 150 mW/cm). To better understand a role for growth factor signaling in disease progression after intrathoracic PDT, the expression and activation of epidermal growth factor receptor (EGFR) was evaluated in areas of post-treatment proliferation. The low fluence rate of 75 mW/cm produced the largest reductions in tumor burden. Bioluminescent imaging and histological staining for cell proliferation (anti-Ki-67) identified areas of disease progression at both fluence rates after PDT. However, increased EGFR activation in proliferative areas was detected only after treatment at the higher fluence rate of 150 mW/cm. These data suggest that fluence rate may affect the activation of survival factors, such as EGFR, and weaker activation at lower fluence rate could contribute to a smaller tumor burden after PDT at 75 mW/cm. Full article

Astaxanthin is a coloring agent which is used as a feed additive in aquaculture nutrition. Recently, potential health benefits of astaxanthin have been discussed which may be partly related to its free radical scavenging and antioxidant properties. Our electron spin resonance (ESR) and spin trapping data suggest that synthetic astaxanthin is a potent free radical scavenger in terms of diphenylpicryl-hydrazyl (DPPH) and galvinoxyl free radicals. Furthermore, astaxanthin dose-dependently quenched singlet oxygen as determined by photon counting. In addition to free radical scavenging and singlet oxygen quenching properties, astaxanthin induced the antioxidant enzyme paroxoanase-1, enhanced glutathione concentrations and prevented lipid peroxidation in cultured hepatocytes. Present results suggest that, beyond its coloring properties, synthetic astaxanthin exhibits free radical scavenging, singlet oxygen quenching, and antioxidant activities which could probably positively affect animal and human health. Full article

Melanoma, one of the aggressive cancers, is known to be resistant to chemotherapy. Because of its aggressive nature, effectively inducing apoptosis is necessary to treat melanoma. Erythroid differentiation regulator 1 (Erdr1) is known to be a stress-related survival factor exhibiting anti-cancer effects in several cancers. However, little is known about the functions and underlying mechanisms of Erdr1 so far. To demonstrate the effect of Erdr1 in melanoma apoptosis, recombinant murine Erdr1 was injected into mice implanted with B16F10 melanoma cells. In vivo tumor growth was significantly inhibited in mice injected with Erdr1 compared to the control. In addition, the tumor from Erdr1-injected mice showed an increased level of apoptosis. Accordingly, apoptosis-regulating factors including anti-apoptotic marker Bcl-2 and pro-apoptotic marker Bax in the tumor tissues were examined. As expected, the decreased level of Bcl-2 and increased level of Bax were detected in tumors within the mice injected with Erdr1. Based on the in vivo study, the role of Erdr1 in tumor apoptosis was further tested by incubating it with cells of the murine melanoma cell line B16F10. Erdr1-induced apoptosis in B16F10 cells was observed. Additionally, Erdr1 downregulated STAT3 activity, inhibiting apoptosis via regulation of the Bcl-2 family. Overall, data demonstrate that Erdr1 induced murine melanoma apoptosis through the regulation of Bcl-2 and Bax. These findings suggest that Erdr1 is a novel regulator of apoptosis in melanoma. Full article

Monoamine oxidase (MAO) catalyzes the oxidative deamination of monoamines including dopamine (DA). MAO expression is elevated in Parkinson’s disease (PD). An increase in MAO activity is closely related to age, and this may induce neuronal degeneration in the brain due to oxidative stress. MAO (and particularly monoamine oxidase B (MAO-B)) participates in the generation of reactive oxygen species (ROS), such as hydrogen peroxide that are toxic to dopaminergic cells and their surroundings. Although the polyphenol-rich aqueous walnut extract (JSE; an extract of Juglandis Semen) has been shown to have various beneficial bioactivities, no study has been dedicated to see if JSE is capable to protect dopaminergic neurons against neurotoxic insults in models of PD. In the present study we investigated the neuroprotective potential of JSE against 1-methyl-4-phenylpyridinium (MPP⁺)- or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicities in primary mesencephalic cells and in a mouse model of PD. Here we show that JSE treatment suppressed ROS and nitric oxide productions triggered by MPP⁺ in primary mesencephalic cells. JSE also inhibited depletion of striatal DA and its metabolites in vivo that resulted in significant improvement in PD-like movement impairment. Altogether our results indicate that JSE has neuroprotective effects in PD models and may have potential for the prevention or treatment of PD. Full article

Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) gene plays a crucial role in maintaining genomic stability, tumorigenesis and myogenesis. However, little is known about the regulatory elements governing the transcription of porcine ROCK1 gene. In the current study, the transcription start site (TSS) was identified by 5’-RACE, and was found to differ from the predicted one. The region in ROCK1 promoter which is critical for promoter activity was investigated via progressive deletions. Site-directed mutagenesis indicated that the region from −604 to −554 bp contains responsive elements for Sp1. Subsequent experiments showed that ROCK1 promoter activity is enhanced by Sp1 in a dose-dependent manner, whereas treatment with specific siRNA repressed ROCK1 promoter activity. Electrophoretic mobility shift assay (EMSA), DNA pull down and chromatin immunoprecipitation (ChIP) assays revealed Sp1 can bind to this region. qRT-PCR and Western blotting research followed by overexpression or inhibition of Sp1 indicate that Sp1 can affect endogenous ROCK1 expression at both mRNA and protein levels. Overexpression of Sp1 can promote the expression of myogenic differentiation 1(MyoD), myogenin (MyoG), myosin heavy chain (MyHC). Taken together, we conclude that Sp1 positively regulates ROCK1 transcription by directly binding to the ROCK1 promoter region (from −604 to −532 bp) and may affect the process of myogenesis. Full article

Herbal compounds that have notable therapeutic effect upon Alzheimer's disease (AD) have frequently been found, despite the recent failure of late-stage clinical drugs. Icariin, which is isolated from Epimedium brevicornum, is widely reported to exhibit significant anti-AD effects in in vitro and in vivo studies. However, the molecular mechanism remains thus far unclear. In this work, the anti-AD mechanisms of icariin were investigated at a target network level assisted by an in silico target identification program (INVDOCK). The results suggested that the anti-AD effects of icariin may be contributed by: attenuation of hyperphosphorylation of tau protein, anti-inflammation and regulation of Ca²⁺ homeostasis. Our results may provide assistance in understanding the molecular mechanism and further developing icariin into promising anti-AD agents. Full article

Herb-induced liver injury (HILI) has recently attracted attention due to increasing reports of hepatotoxicity associated with use of phytotherapeutics. Here, we present data on HILI from the Berlin Case-Control Surveillance Study. The study was initiated in 2000 to investigate the serious toxicity of drugs including herbal medicines. Potential cases of liver injury were ascertained in more than 180 Departments of all 51 Berlin hospitals from October 2002 to December 2011. Drug or herb intake was assessed through a standardized face-to-face interview. Drug or herbal aetiology was assessed based on the updated Council for International Organizations of Medical Sciences scale. In ten of all 198 cases of hepatotoxicity included in the study, herbal aetiology was assessed as probable (once ayurvedic herb) or possible (Valeriana five times, Mentha piperita once, Pelargonium sidoides once, Hypericum perforatum once, Eucalyptus globulus once). Mean age was 56.4 ± 9.7 years, and the predominant pattern of liver injury was hepatocellular. No cases of acute liver failure or death were observed. This case series corroborates known risks for ayurvedic herbs, supports the suspected association between Valeriana use and liver injury, and indicates a hepatotoxic potential for herbs such as Pelargonium sidoides, Hypericum perforatum or Mentha piperita that were rarely associated with liver injury before. However, given that possible causality does not prove clinical significance, further studies in this field are needed. Full article

Spontaneous mutation in the lysosomal acid phosphatase 2 (Acp2) mouse (nax—naked-ataxia mutant mouse) correlates with severe cerebellar defects including ataxia, reduced size and abnormal lobulation as well as Purkinje cell (Pc) degeneration. Loss of Pcs in the nax cerebellum is compartmentalized and harmonized to the classic pattern of gene expression of the cerebellum in the wild type mouse. Usually, degeneration starts in the anterior and posterior zones and continues to the central and nodular zones of cerebellum. Studies have suggested that the p75 neurotrophin receptor (NTR) plays a role in Pc degeneration; thus, in this study, we investigated the p75NTR pattern and protein expression in the cerebellum of the nax mutant mouse. Despite massive Pc degeneration that was observed in the nax mouse cerebellum, p75NTR pattern expression was similar to the HSP25 pattern in nax mice and comparable with wild type sibling cerebellum. In addition, immunoblot analysis of p75NTR protein expression did not show any significant difference between nax and wild type sibling (p > 0.5). In comparison with wild type counterparts, p75NTR pattern expression is aligned with the fundamental cytoarchitecture organization of the cerebellum and is unchanged in the nax mouse cerebellum despite the severe neurodevelopmental disorder accompanied with Pc degeneration. Full article

Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae. Full article

CHR20 and CHR21 are a pair of stable diastereoisomers derived from genipin. These stereoisomers are activators of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). In the rat retinal ganglion (RGC-5) cell model these compounds are non-toxic. Treatment of RGC-5 with 750 μM of sodium nitroprusside (SNP) produces nitrosative stress. Both genipin derivatives, however, protect these cells against SNP-induced apoptic cell death, although CHR21 is significantly more potent than CHR20 in this regard. With Western blotting we showed that the observed neuroprotection is primarily due to the activation of protein kinase B (Akt)/eNOS and extracellular signal-regulated kinase (ERK1/2) signaling pathways. Therefore, LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or PD98059 (a MAPK-activating enzyme inhibitor) abrogated the protective effects of CHR20 and CHR21. Altogether, our results show that in our experimental setup neuroprotection by the diasteromeric pair is mediated through the PI3K/Akt/eNOS and ERK1/2 signaling pathways. Further studies are needed to establish the potential of these compounds to prevent ntric oxide (NO)-induced toxicity commonly seen in many neurodegenerative diseases. Full article

The ability to glycosylate surfaces has medical and diagnostic applications, but there is no technology currently recognized as being able to coat any surface without the need for prior chemical modification of the surface. Recently, a family of constructs called function-spacer-lipids (FSL) has been used to glycosylate cells. Because it is known that lipid-based material can adsorb onto surfaces, we explored the potential and performance of cell-labelling FSL constructs to “glycosylate” non-biological surfaces. Using blood group A antigen as an indicator, the performance of a several variations of FSL constructs to modify a large variety of non-biological surfaces was evaluated. It was found the FSL constructs when optimised could in a few seconds glycosylate almost any non-biological surface including metals, glass, plastics, rubbers and other polymers. Although the FSL glycan coating was non-covalent, and therefore temporary, it was sufficiently robust with appropriate selection of spacer and surface that it could capture anti-glycan antibodies, immobilize cells (via antibody), and withstand incubation in serum and extensive buffer washing, making it suitable for diagnostic and research applications. Full article

HtrA serine peptidase 2 (HtrA2), also named Omi, is a pro-apoptotic protein that exhibits dramatic changes in expression levels in a variety of disorders, including ischemia/reperfusion injury, cancer, and neurodegeneration. In our study, Omi/HtrA2 protein levels were high in the heart, brain, kidney and liver, with elevated heart/brain expression in aging mice. A similar expression pattern was observed at the mRNA level, which suggests that the regulation of Omi/HtrA2 is predominately transcriptional. Promoter binding by transcription factors is the main influencing factor of transcription, and to identify specific promoter elements that contribute to the differential expression of mouse Omi/HtrA2, we constructed truncated Omi/HtrA2 promoter/luciferase reporter vectors and analyzed their relative luciferase activity; it was greatest in the promoter regions at −1205~−838 bp and −146~+93 bp, with the −838~−649 bp region exhibiting negative regulatory activity. Bioinformatics analysis suggested that the Omi/HtrA2 gene promoter contains a CpG island at −709~+37 bp, and eight heat shock transcription factor 1 (HSF1) sites, two Sp1 transcription factor (SP1)sites, one activator protein (AP) site, seven p53 sites, and four YY1 transcription factor(YY1) sites were predicted in the core areas. Furthermore, we found that p53 and HSF1 specifically binds to the Omi/HtrA2 promoter using chromatin immunoprecipitation analysis. These results provide a foundation for understanding Omi/HtrA2 regulatory mechanisms, which could further understanding of HtrA-associated diseases. Full article

Despite the susceptibility to frequent intrinsic and extrinsic injuries, especially in the inner zone, the meniscus does not heal spontaneously owing to its poor vascularity. In this study, the effect of platelet-rich plasma (PRP), containing various growth factors, on meniscal mechanisms was examined under normal and post-traumatic inflammatory conditions. Isolated primary meniscal cells of New Zealand white (NZW) rabbits were incubated for 3, 10, 14 and 21 days with PRP(−), 10% PRP (PRP(+)), IL(+) or IL(+)PRP(+). The meniscal cells were collected and examined using reverse-transcription polymerase chain reaction (RT-PCR). Culture media were examined by immunoblot analyses for matrix metalloproteinases (MMP) catabolic molecules. PRP containing growth factors improved the cellular viability of meniscal cells in a concentration-dependent manner at Days 1, 4 and 7. However, based on RT-PCR, meniscal cells demonstrated dedifferentiation, along with an increase in type I collagen in the PRP(+) and in IL(+)PRP(+). In PRP(+), the aggrecan expression levels were lower than in the PRP(−) until Day 21. The protein levels of MMP-1 and MMP-3 were higher in each PRP group, i.e., PRP(+) and IL(+)PRP(+), at each culture time. A reproducible 2-mm circular defect on the meniscus of NZW rabbit was used to implant fibrin glue (control) or PRP in vivo. After eight weeks, the lesions in the control and PRP groups were occupied with fibrous tissue, but not with meniscal cells. This study shows that PRP treatment of the meniscus results in an increase of catabolic molecules, especially those related to IL-1α-induced inflammation, and that PRP treatment for an in vivo meniscus injury accelerates fibrosis, instead of meniscal cartilage. Full article

Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations—in human breast cell lines and breast tumor biopsies—we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression. Full article

Metformin is known to alleviate hepatosteatosis by inducing 5’ adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1) ad libitum feeding of a standard chow diet; (2) intraperitoneal injections of metformin 300 mg/kg; and (3) 3 g/day caloric restriction (CR). HepG2 cells were treated with palmitate (PA) plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER) stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins. Full article

The transference and reactivity of proanthocyanidins is an important issue that affects the technological processing of some fruits, such as grapes and apples. These processes are affected by proanthocyanidins bound to cell wall polysaccharides, which are present in high concentrations during the processing of the fruits. Therefore, the effective extraction of proanthocyanidins from fruits to their juices or derived products will depend on the ability to manage these associations, and, in this respect, enzymes that degrade these polysaccharides could play an important role. The main objective of this work was to test the role of pure hydrolytic enzymes (polygalacturonase and cellulose) and a commercial enzyme containing these two activities on the extent of proanthocyanidin-cell wall interactions. The results showed that the modification promoted by enzymes reduced the amount of proanthocyanidins adsorbed to cell walls since they contributed to the degradation and release of the cell wall polysaccharides, which diffused into the model solution. Some of these released polysaccharides also presented some reactivity towards the proanthocyanidins present in a model solution. Full article

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. The pectate lyase gene is essential for successful invasion of their host plants by plant-parasitic nematodes. To demonstrate the role of pectate lyase gene in the PWD process, RNA interference (RNAi) is used to analyze the function of the pectate lyase 1 gene in B. xylophilus (Bxpel1). The efficiency of RNAi was detected by real-time PCR. The result demonstrated that the quantity of B. xylophilus propagated with control solution treatment was 62 times greater than that soaking in double-stranded RNA (dsRNA) after B. xylophilus inoculation in Botrytis cinerea for the first generation (F1). The number of B. xylophilus soaking in control solution was doubled compared to that soaking in Bxpel1 dsRNA four days after inoculation in Pinus thunbergii. The quantity of B. xylophilus was reduced significantly (p < 0.001) after treatment with dsRNAi compared with that using a control solution treatment. Bxpel1 dsRNAi reduced the migration speed and reproduction of B. xylophilus in pine trees. The pathogenicity to P. thunbergii seedling of B. xylophilus was weaker after soaking in dsRNA solution compared with that after soaking in the control solution. Our results suggest that Bxpel1 gene is a significant pathogenic factor in the PWD process and this basic information may facilitate a better understanding of the molecular mechanism of PWD. Full article

Background: Cerebral amyloid angiopathy (CAA) is characterized by vascular deposition of amyloid β (Aβ) with a higher incidence of cerebral microbleeds (cMBs) and spontaneous hemorrhage. Since statins are known for their benefit in vascular disease we tested for the effect on CAA. Methods: APP23-transgenic mice received atorvastatin-supplemented food starting at the age of eight months (n = 13), 12 months (n = 7), and 16 months (n = 6), respectively. Controls (n = 16) received standard food only. At 24 months of age cMBs were determined with T2*-weighted 9.4T magnetic resonance imaging and graded by size. Results: Control mice displayed an average of 35 ± 18.5 cMBs (mean ± standard deviation), compared to 29.3 ± 9.8 in mice with eight months (p = 0.49), 24.9 ± 21.3 with 12 months (p = 0.26), and 27.8 ± 15.4 with 16 months of atorvastatin treatment (p = 0.27). In combined analysis treated mice showed lower absolute numbers (27.4 ± 15.6, p = 0.16) compared to controls and also after adjustment for cMB size (p = 0.13). Conclusion: Despite to a non-significant trend towards fewer cMBs our results failed to provide evidence for beneficial effects of long-term atorvastatin treatment in the APP23-transgenic mouse model of CAA. A higher risk for bleeding complications was not observed. Full article

As a chronic disorder, insomnia affects approximately 10% of the population at some time during their lives, and its treatment is often challenging. Since the antagonists of the H₁ receptor, a protein prevalent in human central nervous system, have been proven as effective therapeutic agents for treating insomnia, the H₁ receptor is quite possibly a promising target for developing potent anti-insomnia drugs. For the purpose of understanding the structural actors affecting the antagonism potency, presently a theoretical research of molecular interactions between 129 molecules and the H₁ receptor is performed through three-dimensional quantitative structure-activity relationship (3D-QSAR) techniques. The ligand-based comparative molecular similarity indices analysis (CoMSIA) model (Q² = 0.525, R²_ncv = 0.891, R²_pred = 0.807) has good quality for predicting the bioactivities of new chemicals. The cross-validated result suggests that the developed models have excellent internal and external predictability and consistency. The obtained contour maps were appraised for affinity trends for the investigated compounds, which provides significantly useful information in the rational drug design of novel anti-insomnia agents. Molecular docking was also performed to investigate the mode of interaction between the ligand and the active site of the receptor. Furthermore, as a supplementary tool to study the docking conformation of the antagonists in the H₁ receptor binding pocket, molecular dynamics simulation was also applied, providing insights into the changes in the structure. All of the models and the derived information would, we hope, be of help for developing novel potent histamine H₁ receptor antagonists, as well as exploring the H₁-antihistamines interaction mechanism. Full article

Metabolic syndrome (MetS) represents a cluster of metabolic derangements. Dyslipidemia is an important factor in MetS and is related to atrial fibrillation (AF). We hypothesized that very low density lipoproteins (VLDL) in MetS (MetS-VLDL) may induce atrial dilatation and vulnerability to AF. VLDL was therefore separated from normal (normal-VLDL) and MetS individuals. Wild type C57BL/6 male mice were divided into control, normal-VLDL (nVLDL), and MetS-VLDL (msVLDL) groups. VLDL (15 µg/g) and equivalent volumes of saline were injected via tail vein three times a week for six consecutive weeks. Cardiac chamber size and function were measured by echocardiography. MetS-VLDL significantly caused left atrial dilation (control, n = 10, 1.64 ± 0.23 mm; nVLDL, n = 7, 1.84 ± 0.13 mm; msVLDL, n = 10, 2.18 ± 0.24 mm; p < 0.0001) at week 6, associated with decreased ejection fraction (control, n = 10, 62.5% ± 7.7%, vs. msVLDL, n = 10, 52.9% ± 9.6%; p < 0.05). Isoproterenol-challenge experiment resulted in AF in young msVLDL mice. Unprovoked AF occurred only in elderly msVLDL mice. Immunohistochemistry showed excess lipid accumulation and apoptosis in msVLDL mice atria. These findings suggest a pivotal role of VLDL in AF pathogenesis for MetS individuals. Full article

This article focuses on correlating the column classification obtained from the method created at the Katholieke Universiteit Leuven (KUL), with the chromatographic resolution attained in biomedical separation. In the KUL system, each column is described with four parameters, which enables estimation of the F_KUL value characterising similarity of those parameters to the selected reference stationary phase. Thus, a ranking list based on the F_KUL value can be calculated for the chosen reference column, then correlated with the results of the column performance test. In this study, the column performance test was based on analysis of moclobemide and its two metabolites in human plasma by liquid chromatography (LC), using 18 columns. The comparative study was performed using traditional correlation of the F_KUL values with the retention parameters of the analytes describing the column performance test. In order to deepen the comparative assessment of both data sets, factor analysis (FA) was also used. The obtained results indicated that the stationary phase classes, closely related according to the KUL method, yielded comparable separation for the target substances. Therefore, the column ranking system based on the F_KUL-values could be considered supportive in the choice of the appropriate column for biomedical analysis. Full article

Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several chemical control agents, novel sustainable measures are required to control Saprolegnia infections in aquaculture. Previously, fungal community analysis by terminal restriction fragment length polymorphism (T-RFLP) revealed that the Ascomycota, specifically the genus Microdochium, was an abundant fungal phylum associated with salmon eggs from a commercial fish farm. Here, phylogenetic analyses showed that most fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride species. Phylogenetic and quantitative PCR analyses showed both a quantitative and qualitative difference in Trichoderma population between diseased and healthy salmon eggs, which was not the case for the Microdochium population. In vitro antagonistic activity of the fungi against Saprolegnia diclina was isolate-dependent; for most Trichoderma isolates, the typical mycoparasitic coiling around and/or formation of papilla-like structures on S. diclina hyphae were observed. These results suggest that among the fungal community associated with salmon eggs, Trichoderma species may play a role in Saprolegnia suppression in aquaculture. Full article

Alternative splicing of pre-mRNA helps to enhance the genetic diversity within mammalian cells by increasing the number of protein isoforms that can be generated from one gene product. This provides a great deal of flexibility to the host cell to alter protein function, but when dysregulation in splicing occurs this can have important impact on health and disease. Alternative splicing is widely used in the mammalian immune system to control the development and function of antigen specific lymphocytes. In this review we will examine the splicing of pre-mRNAs yielding key proteins in the immune system that regulate apoptosis, lymphocyte differentiation, activation and homeostasis, and discuss how defects in splicing can contribute to diseases. We will describe how disruption to trans-acting factors, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), can impact on cell survival and differentiation in the immune system. Full article

Recently, an increasing number of studies on natural antisense transcripts have been reported, especially regarding their classification, temporal and spatial expression patterns, regulatory functions and mechanisms. It is well established that natural antisense transcripts are produced from the strand opposite to the strand encoding a protein. Despite the pivotal roles of natural antisense transcripts in regulating the expression of target genes, the transcriptional mechanisms initiated by antisense promoters (ASPs) remain unknown. To date, nearly all of the studies conducted on this topic have focused on the ASP of a single gene of interest, whereas no study has systematically analyzed the locations of ASPs in the genome, ASP activity, or factors influencing this activity. This review focuses on elaborating on and summarizing the characteristics of ASPs to extend our knowledge about the mechanisms of antisense transcript initiation. Full article

High concentrations of nickel (Ni) are harmful to humans and animals. Ni targets a number of organs and produces multiple toxic effects. Apoptosis is important in Ni-induced toxicity of the kidneys, liver, nerves, and immune system. Apoptotic pathways mediated by reactive oxygen species (ROS), mitochondria, endoplasmic reticulum (ER), Fas, and c-Myc participate in Ni-induced cell apoptosis. However, the exact mechanism of apoptosis caused by Ni is still unclear. Understanding the mechanism of Ni-induced apoptosis may help in designing measures to prevent Ni toxicity. Full article

The emerge of personalized medicine demands high-quality human biospecimens with appropriate clinical annotation, especially in complex diseases such as cancer, neurodegenerative, cardiovascular, and metabolic alterations in which specimen heterogeneity and individual responses often complicate the development of precision therapeutic programs. In the growing field of extracellular vesicles (EVs) research, exosomes (EXOs)—a particular type of EVs—have been proposed as an advantageous diagnostic tool, as effective delivery vehicles and as therapeutic targets. However, the lack of consensus on isolation methods and rigorous criteria to characterize them puts the term EXO into question at the time that might explain some of the controversial results found in the literature. A lack of response in the biobank network to warrant standard optimized procedures for the isolation, characterization, and storage of EXOs will undoubtedly lead to a waste of resources and failure. This review is aimed at highlighting the increasing importance of EXOs for the clinic, especially in the cancer field, and at summarizing the initiatives taken to improve current isolation procedures, classification criteria, and storage conditions of EXOs as an effort to identify technological demands that biobank platforms face for the incorporation of EXOs and other extracellular vesicle fractions as valuable biospecimens for research. Full article

RUCAM (Roussel Uclaf Causality Assessment Method) or its previous synonym CIOMS (Council for International Organizations of Medical Sciences) is a well established tool in common use to quantitatively assess causality in cases of suspected drug induced liver injury (DILI) and herb induced liver injury (HILI). Historical background and the original work confirm the use of RUCAM as single term for future cases, dismissing now the term CIOMS for reasons of simplicity and clarity. RUCAM represents a structured, standardized, validated, and hepatotoxicity specific diagnostic approach that attributes scores to individual key items, providing final quantitative gradings of causality for each suspect drug/herb in a case report. Experts from Europe and the United States had previously established in consensus meetings the first criteria of RUCAM to meet the requirements of clinicians and practitioners in care for their patients with suspected DILI and HILI. RUCAM was completed by additional criteria and validated, assisting to establish the timely diagnosis with a high degree of certainty. In many countries and for more than two decades, physicians, regulatory agencies, case report authors, and pharmaceutical companies successfully applied RUCAM for suspected DILI and HILI. Their practical experience, emerging new data on DILI and HILI characteristics, and few ambiguous questions in domains such alcohol use and exclusions of non-drug causes led to the present update of RUCAM. The aim was to reduce interobserver and intraobserver variability, to provide accurately defined, objective core elements, and to simplify the handling of the items. We now present the update of the well accepted original RUCAM scale and recommend its use for clinical, regulatory, publication, and expert purposes to validly establish causality in cases of suspected DILI and HILI, facilitating a straightforward application and an internationally harmonized approach of causality assessment as a common basic tool. Full article

As an actively renewable tissue, changes in skin architecture are subjected to the regulation of stem cells that maintain the population of cells responsible for the formation of epidermal layers. Stems cells retain their self-renewal property and express biomarkers that are unique to this population. However, differential regulation of the biomarkers can initiate the pathway of terminal cell differentiation. Although, pockets of non-clarity in stem cell maintenance and differentiation in skin still exist, the influence of epigenetics in epidermal stem cell functions and differentiation in skin homeostasis and wound healing is clearly evident. The focus of this review is to discuss the epigenetic regulation of confirmed and probable epidermal stem cell biomarkers in epidermal stratification of normal skin and in diseased states. The role of epigenetics in wound healing, especially in diseased states of diabetes and cancer, will also be conveyed. Full article

2-Piperidineethanol (1) and its corresponding N-protected aldehyde (2) were used for the synthesis of several natural and synthetic compounds. The existence of a stereocenter at position 2 of the piperidine skeleton and the presence of an easily-functionalized group, such as the alcohol, set 1 as a valuable starting material for enantioselective synthesis. Herein, are presented both synthetic and enzymatic methods for the resolution of the racemic 1, as well as an overview of synthesized natural products starting from the enantiopure 1. Full article

Hypertension is an important component of metabolic syndrome. Adulthood hypertension and metabolic syndrome can be programmed in response to nutritional insults in early life. Peroxisome proliferator-activated receptors (PPARs) serve as a nutrient-sensing signaling linking nutritional programming to hypertension and metabolic syndrome. All three members of PPARs, PPARα, PPARβ/δ, and PPARγ, are expressed in the kidney and involved in blood pressure control. This review provides an overview of potential clinical applications of targeting on the PPARs in the kidney to prevent programmed hypertension and metabolic syndrome, with an emphasis on the following areas: mechanistic insights to interpret programmed hypertension; the link between the PPARs, nutritional insults, and programmed hypertension and metabolic syndrome; the impact of PPAR signaling pathway in a maternal high-fructose model; and current experimental studies on early intervention by PPAR modulators to prevent programmed hypertension and metabolic syndrome. Animal studies employing a reprogramming strategy via targeting PPARs to prevent hypertension have demonstrated interesting results. It is critical that the observed effects on developmental reprogramming in animal models are replicated in human studies, to halt the globally-growing epidemic of metabolic syndrome-related diseases. Full article

Adulthood hypertension can be programmed in response to a suboptimal environment in early life. However, developmental plasticity also implies that one can prevent hypertension in adult life by administrating appropriate compounds during early development. We have termed this reprogramming. While the risk of hypertension has been assessed in many mother-child cohorts of human developmental programming, interventions necessary to prove causation and provide a reprogramming strategy are lacking. Since the developing kidney is particularly vulnerable to environmental insults and blood pressure is determined by kidney function, renal programming is considered key in developmental programming of hypertension. Common pathways, whereby both genetic and acquired developmental programming converge into the same phenotype, have been recognized. For instance, the same reprogramming interventions aimed at shifting nitric oxide (NO)-reactive oxygen species (ROS) balance, such as perinatal citrulline or melatonin supplements, can be protective in both genetic and developmentally programmed hypertension. Furthermore, a significantly increased expression of gene Ephx2 (soluble epoxide hydrolase) was noted in both genetic and acquired animal models of hypertension. Since a suboptimal environment is often multifactorial, such common reprogramming pathways are a practical finding for translation to the clinic. This review provides an overview of potential clinical applications of reprogramming strategies to prevent programmed hypertension. We emphasize the kidney in the following areas: mechanistic insights from human studies and animal models to interpret programmed hypertension; identified risk factors of human programmed hypertension from mother-child cohorts; and the impact of reprogramming strategies on programmed hypertension from animal models. It is critical that the observed effects on developmental reprogramming in animal models are replicated in human studies. Full article

Neurodegenerative diseases (NDs) are among the most feared of the disorders that afflict humankind for the lack of specific diagnostic tests and effective treatments. Understanding the molecular, cellular, biochemical changes of NDs may hold therapeutic promise against debilitating central nerve system (CNS) disorders. In the present review, we summarized the clinical presentations and biology backgrounds of NDs, including Parkinson’s disease (PD), Huntington’s disease (HD), and Alzheimer’s disease (AD) and explored the role of molecular mechanisms, including dys-regulation of epigenetic control mechanisms, Ataxia-telangiectasia-mutated protein kinase (ATM), and neuroinflammation in the pathogenesis of NDs. Targeting these mechanisms may hold therapeutic promise against these devastating diseases. Full article

The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that is activated by several ligands leading to the activation of diverse signaling pathways controlling mainly proliferation, differentiation, and survival. The EGFR signaling axis has been shown to play a key role during liver regeneration following acute and chronic liver damage, as well as in cirrhosis and hepatocellular carcinoma (HCC) highlighting the importance of the EGFR in the development of liver diseases. Despite the frequent overexpression of EGFR in human HCC, clinical studies with EGFR inhibitors have so far shown only modest results. Interestingly, a recent study has shown that in human HCC and in mouse HCC models the EGFR is upregulated in liver macrophages where it plays a tumor-promoting function. Thus, the role of EGFR in liver diseases appears to be more complex than what anticipated. Further studies are needed to improve the molecular understanding of the cell-specific signaling pathways that control disease development and progression to be able to develop better therapies targeting major components of the EGFR signaling network in selected cell types. In this review, we compiled the current knowledge of EGFR signaling in different models of liver damage and diseases, mainly derived from the analysis of HCC cell lines and genetically engineered mouse models (GEMMs). Full article

microRNAs (miRNAs) are short ~22 nucleotides (nt) ribonucleic acids which post-transcriptionally regulate gene expression. miRNAs are key regulators of all cellular processes, and the correct expression of miRNAs in an organism is crucial for proper development and cellular function. As a result, the miRNA biogenesis pathway is highly regulated. In this review, we outline the basic steps of miRNA biogenesis and miRNA mediated gene regulation focusing on the role of RNA binding proteins (RBPs). We also describe multiple mechanisms that regulate the canonical miRNA pathway, which depends on a wide range of RBPs. Moreover, we hypothesise that the interaction between miRNA regulation and RBPs is potentially more widespread based on the analysis of available high-throughput datasets. Full article

Targeted radionuclide therapy is one of the most intensively developing directions of nuclear medicine. Unlike conventional external beam therapy, the targeted radionuclide therapy causes less collateral damage to normal tissues and allows targeted drug delivery to a clinically diagnosed neoplastic malformations, as well as metastasized cells and cellular clusters, thus providing systemic therapy of cancer. The methods of targeted radionuclide therapy are based on the use of molecular carriers of radionuclides with high affinity to antigens on the surface of tumor cells. The potential of targeted radionuclide therapy has markedly grown nowadays due to the expanded knowledge base in cancer biology, bioengineering, and radiochemistry. In this review, progress in the radionuclide therapy of hematological malignancies and approaches for treatment of solid tumors is addressed. Full article