Biochemical Alterations in White Matter Tracts of the Aging Mouse Brain Revealed by FTIR Spectroscopy Imaging.

White matter degeneration in the central nervous system (CNS) has been correlated with a decline in cognitive function during aging. Ultrastructural examination of the aging human brain shows a loss of myelin, yet little is known about molecular and biochemical changes that lead to myelin degeneration. In this study, we investigate myelination across the lifespan in C57BL/6 mice using electron microscopy and Fourier transform infrared (FTIR) spectroscopic imaging to better understand the relationship between structural and biochemical changes in CNS white matter tracts. A decrease in the number of myelinated axons was associated with altered lipid profiles in the corpus callosum of aged mice. FTIR spectroscopic imaging revealed alterations in functional groups associated with phospholipids, including the lipid acyl, lipid ester and phosphate vibrations. Biochemical changes in white matter were observed prior to structural changes and most predominant in the anterior regions of the corpus callosum. This was supported by biochemical analysis of fatty acid composition that demonstrated an overall trend towards increased monounsaturated fatty acids and decreased polyunsaturated fatty acids with age. To further explore the molecular mechanisms underlying these biochemical alterations, gene expression profiles of lipid metabolism and oxidative stress pathways were investigated. A decrease in the expression of several genes involved in glutathione metabolism suggests that oxidative damage to lipids may contribute to age-related white matter degeneration.

Immunological and antibiofilm property of haemocyanin purified from grooved tiger shrimp (Penaeus semisulcatus): An in vitro and in silico approach.

Haemocyanin (Hc) is a non-specific innate immune protein present in the haemolymph of arthropods and molluscs. In the current study, we characterized the structural and immunological properties of Hc from grooved tiger shrimp, Penaeus semisulcatus. Hc was isolated from the haemolymph of P. semisulcatus by gel filtration column chromatography using Sephadex G-100. High-performance liquid chromatography of the purified Hc emerged as a single peak through a retention time of 3.3 min demonstrating the homogeneity nature of the protein. X-ray diffraction analysis revealed a distinct peak at 31.7° indicating the crystalline character of the purified Hc. Circular dichroism spectra of the purified Hc displayed negative ellipticity bands close to 225 nm and 208 nm representing ß-sheet secondary structure. The purified Hc agglutinated sheep RBCs, yeast Saccharomyces cerevisiae and fungal Candida albicans. In addition, the purified Hc displayed antibacterial activity against Gram-positive bacteria (Bacillus thuringiensis and Bacillus pumilis) and Gram-negative bacteria (Vibrio parahaemolyticus and Vibrio alginolyticus) with a minimal inhibitory concentration of 50 µg/ml. Antibiofilm activity revealed the potential of purified Hc to inhibit biofilm formation of both Gram-positive and Gram-negative bacterial pathogens. Furthermore, live/dead staining of biofilms demonstrated the reduced viability of bacterial cells after exposure to the purified Hc. In silico molecular modeling was carried out using the sequence of Hc from SwissProt and molecular docking was performed with the cell surface components found in Gram-positive and Gram-negative bacteria. Overall our study demonstrates the involvement of Hc in the native immune reaction of P. semisulcatus by eliciting pathogen recognition. Thus, Hc could enhance disease resistance against pathogenic infection in shrimp aquaculture.

Htra1 is a Novel Transcriptional Target of RUNX2 That Promotes Osteogenic Differentiation.

BACKGROUND/AIMS: Runt-related transcription factor 2 (Runx2) is a master regulator of osteogenic differentiation, but most of the direct downstream targets of RUNX2 during osteogenesis are unknown. Likewise, High-temperature requirement factor A1 (HTRA1) is a serine protease expressed in bone, yet the role of Htra1 during osteoblast differentiation remains elusive. We investigated the role of Htra1 in osteogenic differentiation and the transcriptional regulation of Htra1 by RUNX2 in primary mouse mesenchymal progenitor cells. METHODS: Overexpression of Htra1 was carried out in primary mouse mesenchymal progenitor cells to evaluate the extent of osteoblast differentiation. Streptavidin agarose pulldown assay, chromatin immunoprecipitation assay, and dual luciferase assay were carried out to investigate the interaction of RUNX2 protein at the Htra1 promoter during osteoblast differentiation. RESULTS: Overexpression of Htra1 increased the production of mineralized bone matrix, upregulating several osteoblast genes, such as Sp7 transcription factor (Sp7) and Alkaline phosphatase, liver/bone/kidney (Alpl). In addition, Htra1 upregulated osteogenesis-related signalling genes, such as Fibroblast growth factor 9 (Fgf9) and Vascular endothelial growth factor A (Vegfa). A series of experiments confirmed Htra1 as a direct RUNX2 transcriptional target. Overexpression of Runx2 resulted in the upregulation of Htra1 mRNA and protein. Chromatin immunoprecipitation and streptavidin agarose pull-down assays showed that RUNX2 binds a proximal -400 bp region of the Htra1 promoter during osteogenic differentiation. Dual luciferase assays confirmed that RUNX2 activates the proximal Htra1 promoter during osteogenic differentiation. Mutation of putative RUNX2 binding sites revealed that RUNX2 interacts with the Htra1 promoter at -252 bp and -84 bp to induce Htra1 expression. CONCLUSION: We demonstrate that Htra1 is a positive regulator of osteogenic differentiation, showing for the first time that Htra1 is a direct downstream target of RUNX2.

RNA-binding Protein Quaking Stabilizes Sirt2 mRNA during Oligodendroglial Differentiation.

Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv ) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3' untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation.

Does Sirt2 Regulate Cholesterol Biosynthesis During Oligodendroglial Differentiation In Vitro and In Vivo?

Sirtuin2 (SIRT2) is a deacetylase enzyme predominantly expressed in myelinating glia of the central nervous system (CNS). We have previously demonstrated that Sirt2 expression enhances oligodendrocyte (OL) differentiation and arborization in vitro, but the molecular targets of SIRT2 in OLs remain speculative. SIRT2 has been implicated in cholesterol biosynthesis by promoting the nuclear translocation of sterol regulatory element binding protein (SREBP)-2. We investigated this further in CNS myelination by examining the role of Sirt2 in cholesterol biosynthesis in vivo and in vitro employing Sirt2 -/- mice, primary OL cells and CG4-OL cells. Our results demonstrate that expression of cholesterol biosynthetic genes in the CNS white matter or cholesterol content in purified myelin fractions did not differ between Sirt2 -/- and age-matched wild-type mice. Cholesterol biosynthetic gene expression profiles and total cholesterol content were not altered in primary OLs from Sirt2 -/- mice and in CG4-OLs when Sirt2 was either down-regulated with RNAi or overexpressed. In addition, Sirt2 knockdown or overexpression in CG4-OLs had no effect on SREBP-2 nuclear translocation. Our results indicate that Sirt2 does not impact the expression of genes encoding enzymes involved in cholesterol biosynthesis, total cholesterol content, or nuclear translocation of SREBP-2 during OL differentiation and myelination.

Organotypic Cultures from the Adult CNS: A Novel Model to Study Demyelination and Remyelination Ex Vivo.

Experimental models of multiple sclerosis (MS) have significantly advanced our understanding of pathophysiology and therapeutic interventions. Although in vivo rodent models are considered to most closely represent the complex cellular and molecular disease states of the human central nervous system (CNS), these can be costly to maintain and require long timelines. Organotypic slice cultures maintain the cytotypic organization observed in the intact CNS, yet provide many of the experimental advantages of in vitro cell culture models. Cerebellar organotypic cultures have proven useful for studying myelination and remyelination, but this model has only been established using early postnatal tissue. This young brain tissue allows for neuro development ex vivo to mimic the 'mature' CNS; however, there are many differences between postnatal and adult organotypic cultures. This may be particularly relevant to MS, as a major barrier to myelin regeneration is age. This paper describes a modified protocol to study demyelination and remyelination in adult cerebellar tissue, which has been used to demonstrate neuroprotection with omega-3 fatty acids. Thus, adult cerebellar organotypic cultures provide a novel ex vivo platform for screening potential therapies in myelin degeneration and repair.

Effect of ß-1, 3 glucan binding protein based zinc oxide nanoparticles supplemented diet on immune response and disease resistance in Oreochromis mossambicus against Aeromonas hydrophila.

Recently, several immunostimulants such as ß-glucan, microbial and plant products have been used as dietary supplements to combat disease outbreaks in aquaculture. The present study investigates the potential of Portunus pelagicus ß-1, 3 glucan binding protein based zinc oxide nanoparticles (Ppß-GBP-ZnO NPs) supplemented diet on growth, immune response and disease resistance in Mozambique tilapia, Oreochromis mossambicus. The immune-related protein ß-GBP was purified from the haemolymph of P. pelagicus using Sephadex G-100 affinity column chromatography. Ppß-GBP-ZnO NPs was physico- chemically characterized and experimental feed was formulated. Fish were separately fed with commercial diet (control-group I) and Ppß-GBP (group II, III, IV), Ppß-GBP-ZnO NPs (group V, VI, VII), chem-ZnO NPs (VIII, IX, X) mixed diet at the concentration of 0.001%, 0.002% and 0.004% respectively. Triplicate groups of O. mossambicus were fed with experimental diets twice a day for 30 days. Fish receiving Ppß-GBP-ZnO NPs supplemented diet showed a significant increase (P < 0.05) in growth performance. Cellular immune responses (myeloperoxidase activity, lysozyme activity and reactive oxygen species activity) and humoral immune responses (complement activity, antiprotease activity and alkaline phosphatase activity) were evaluated at an interval of 15 days during the feeding trial. Results demonstrate that both cellular and humoral immune responses were substantially increased (P < 0.05) in fish fed with 0.004% of Ppß-GBP-ZnO NPs supplemented diet than others. Antibiofilm potential of Ppß-GBP-ZnO NPs against Aeromonas hydrophila was visualized through confocal laser scanning microscopy (CLSM), which reveals reduction in the preformed biofilm thickness to 10 µm  at the concentration of 50 µg/ml. Furthermore, after 30 days of feeding trial, fish were challenged with aquatic fish pathogen A. hydrophila (1 × 107 cells ml-1) through intraperitoneal injection. Challenge study displayed a reduced mortality rate in fish fed with diet containing Ppß-GBP-ZnO NPs. Thus our study suggests that dietary supplementation of Ppß-GBP-ZnO NPs at 0.004% may have a potential effect to enhance the immune system and survival of O. mossambicus.

Sirt2 epigenetically down-regulates PDGFRα expression and promotes CG4 cell differentiation.

We have previously found that Sirt2 enhanced the outgrowth of cellular processes and MBP expression in CG4 cells, where Sirt2 expression is suppressed by transcription factor Nkx2.2. However, the detailed mechanism of Sirt2 facilitating oligodendroglial cell differentiation remained unclear. In the present study, we observed that Sirt2 partially translocated into the nuclei when CG4 cells were induced to differentiate. Sirt2 was detected at the CpG island of PDGFRα promoter via ChIP assay during the cells differentiation process rather than during the state of growth. Sirt2 deacetylated protein(s) bound to the promoter of PDGFRα and simultaneously appeared to facilitate histone3 K27 tri-methylation, both of which are suppressive signatures on gene transcription activation. In bisulfate assay, we identified that Sirt2 significantly induced DNA methylation of PDGFRα promoter compared with the control. Consistently, Sirt2 overexpression down-regulated PDGFRα expression in CG4 cells. The knock-down of PDGFRα or Sirt2 over-expression repressed cell proliferation, but knock-down of Sirt2 promoted cell proliferation. Taken together, Sirt2 translocated into the nuclei while the cells initiated a differentiation process, facilitating CG4 cell differentiation partially through epigenetic modification and suppression of PDGFRα expression. The repression of PDGFRα expression mediated by Sirt2 appeared to facilitate a transition of cellular processes, i.e. from a proliferating progenitor state to a post-mitotic state in CG4 cells.

Bacillus thuringiensis coated zinc oxide nanoparticle and its biopesticidal effects on the pulse beetle, Callosobruchus maculatus.

Insect pests belonging to the genus Callosobruchus are the major cause of damage to stored pulse crops. Recently, nanotechnology has emerged as a promising tool for pest control. In the present study, we report for the first time the synthesis and biological evaluation of Bacillus thuringiensis coated zinc oxide nanoparticles (Bt-ZnO NPs) on the pulse beetle, Callosobruchus maculatus. The biologically synthesized Bt-ZnO NPs were extensively characterized using UV-Vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM) and Zeta potential. The bio-physical characterization revealed that the Bt-ZnO NPs has a hexagonal wurtzite structures with an average particle size of 20nm. In addition, zeta potential measurement demonstrated that the Bt-ZnO NPs are negatively charged (-12.7mV) and are moderately stable. The biopesticidal effect of Bt-ZnO NPs was tested against the pulse beetle, C. maculatus. Treatment with Bt-ZnO NPs reduced the fecundity (eggs laid) and hatchability of C. maculatus in a dose-dependent manner. A significant delay in the larval, pupal and total development period of C. maculatus was observed after treatment with Bt-ZnO NPs at 25µg/mL. Furthermore, Bt-ZnO NPs are highly effective in the control of C. maculatus and caused 100% mortality at 25µg/mL. The LC50 value was estimated to be 10.71µg/mL. In addition, treatment with Bt-ZnO NPs decreased the mid-gut α-amylase, cysteine protease, α-glucosidase and glutathione S-transferase (GST) activity in C. maculatus. Our results suggest that Bt-ZnO NPs are effective against C. maculatus and could be used as nanobiopesticides in the control of stored grain insect pests in the future.