AKAP12 Mediates Barrier Functions of Fibrotic Scars during CNS Repair

The repair process after CNS injury shows a well-organized cascade of three distinct stages: inflammation, new tissue formation, and remodeling. In the new tissue formation stage, various cells migrate and form the fibrotic scar surrounding the lesion site. The fibrotic scar is known as an obstacle for axonal regeneration in the remodeling stage. However, the role of the fibrotic scar in the new tissue formation stage remains largely unknown. We found that the number of A-kinase anchoring protein 12 (AKAP12)-positive cells in the fibrotic scar was increased over time, and the cells formed a structure which traps various immune cells. Furthermore, the AKAP12-positive cells strongly express junction proteins which enable the structure to function as a physical barrier. In in vivo validation, AKAP12 knock-out (KO) mice showed leakage from a lesion, resulting from an impaired structure with the loss of the junction complex. Consistently, focal brain injury in the AKAP12 KO mice led to extended inflammation and more severe tissue damage compared to the wild type (WT) mice. Accordingly, our results suggest that AKAP12-positive cells in the fibrotic scar may restrict excessive inflammation, demonstrating certain mechanisms that could underlie the beneficial actions of the fibrotic scar in the new tissue formation stage during the CNS repair process.     

ERK mediates anti-apoptotic effect through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to DNA damage in normal human embryonic fibroblast (HEF) cells

Since anti-apoptotic effect of ERK has not been elucidated clearly in DNA-damage-induced cell death, the role of ERK was examined in normal HEF cells treated with mild DNA damage using etoposide or camptothecin. ERK was activated by DNA damage in HEF cells. PD98059 increased apoptosis and reduced DNA-damage-induced p21Waf1/Cip1/Sdi level. Depletion of p21Waf1/Cip1/Sdi induced cell death and PD98059 induced additional cell death. DNA-damage-induced increase in cytoplasmic localization and phosphorylation of threonine residues of p21Waf1/Cip1/Sdi was reversed by PD98059. Thus, the results suggest that ERK pathway mediates anti-apoptotic effects through phosphorylation and cytoplasmic localization of p21Waf1/Cip1/Sdi in response to mild DNA damage.     

Nectin: an adhesion molecule involved in formation of synapses

The nectin-afadin system is a novel cell-cell adhesion system that organizes adherens junctions cooperatively with the cadherin-catenin system in epithelial cells. Nectin is an immunoglobulin-like adhesion molecule, and afadin is an actin filament-binding protein that connects nectin to the actin cytoskeleton. Nectin has four isoforms (-1, -2, -3, and -4). Each nectin forms a homo-cis-dimer followed by formation of a homo-trans-dimer, but nectin-3 furthermore forms a hetero-trans-dimer with nectin-1 or -2, and the formation of each hetero-trans-dimer is stronger than that of each homo-trans-dimer. We show here that at the synapses between the mossy fiber terminals and dendrites of pyramidal cells in the CA3 area of adult mouse hippocampus, the nectin-afadin system colocalizes with the cadherin-catenin system, and nectin-1 and -3 asymmetrically localize at the pre- and postsynaptic sides of puncta adherentia junctions, respectively. During development, nectin-1 and -3 asymmetrically localize not only at puncta adherentia junctions but also at synaptic junctions. Inhibition of the nectin-based adhesion by an inhibitor of nectin-1 in cultured rat hippocampal neurons results in a decrease in synapse size and a concomitant increase in synapse number. These results indicate an important role of the nectin-afadin system in the formation of synapses.     

Conversion of 7-ketocholesterol to oxysterol metabolites by recombinant CYP27A1 and retinal pigment epithelial cells

Of the different oxygenated cholesterol metabolites, 7-ketocholesterol (7KCh) is considered a noxious oxy-sterol implicated in the development of certain pathologies, including those found in the eye. Here we elucidated whether sterol 27-hydroxylase cytochrome P450 27A1 (CYP27A1) is involved in elimination of 7KCh from the posterior part of the eye: the neural retina and underlying retinal pigment epithelium (RPE). We first established that the affinities of purified recombinant CYP27A1 for 7KCh and its endogenous substrate cholesterol are similar, yet 7KCh is metabolized at a 4-fold higher rate than cholesterol in the reconstituted system in vitro. Lipid extracts from bovine neural retina and RPE were then analyzed by isotope dilution GC-MS for the presence of the 7KCh-derived oxysterols. Two metabolites, 3β,27-dihydroxy-5-cholesten-7-one (7KCh-27OH) and 3β-hydroxy-5-cholesten-7-one-26-oic acid (7KCh-27COOH), were detected in the RPE but not in the neural retina. 7KCh-27OH was also formed when RPE homogenates were supplemented with NADPH and the mitochondrial redox system. Quantifications in human RPE showed that CYP27A1 is indeed expressed in the RPE at 2-4-fold higher levels than in the neural retina. The data obtained represent evidence for the role of CYP27A1 in retinal metabolism of 7KCh and suggest that, in addition to cholesterol removal, the functions of this enzyme could also include elimination of toxic endogenous compounds.     

Dithienogermole‐Containing Small‐Molecule Solar Cells with 7.3% Efficiency: In‐Depth Study on the Effects of Heteroatom Substitution of Si with Ge

Two small molecule donor materials (DTGe(FBTTh₂)₂ and DTGe(FBTBFu)₂) incorporating the dithienogermole (DTGe) moiety with fluorobenzothiadiazole (FBT) and bithiophene (Th₂) or benzofuran (BFu) end‐capping groups are synthesized and their properties as donor materials in small molecule bulk heterojunction type (BHJ) solar cells are investigated. The DTGe(FBTTh₂)₂ with Th₂ end groups shows outstanding solar cell characteristics with efficiencies up to 6.4% using a standard BHJ architecture and 7.3% using a ZnO optical spacer, while the BFu end‐capped DTGe(FBTBFu)₂ has slightly wider band gaps and yields slightly higher open circuit voltage (V_OC) at the expense of short circuit current (J_SC) and fill factor (FF). In this study, the DTGe‐based molecules are systematically compared to the dithienosilole (DTSi)‐based analogues, which are currently among the highest power conversion efficiency (PCE) small molecule solar cell donor materials known. The J_SC produced by the DTGe molecule is found to be similar to, or slightly higher than the Si analogue, despite similar absorption characteristics, however, the PCE is similar to the Si analogues due to small decreases in V_OC and FF. This report marks the first small molecule BHJ based on a Ge‐containing heterocycle with PCE over 7%.     

Statistical power and sample size requirements for three level hierarchical cluster randomized trials

SUMMARY: Cluster randomized clinical trials (cluster-RCT), where the community entities serve as clusters, often yield data with three hierarchy levels. For example, interventions are randomly assigned to the clusters (level three unit). Health care professionals (level two unit) within the same cluster are trained with the randomly assigned intervention to provide care to subjects (level one unit). In this study, we derived a closed form power function and formulae for sample size determination required to detect an intervention effect on outcomes at the subject's level. In doing so, we used a test statistic based on maximum likelihood estimates from a mixed-effects linear regression model for three level data. A simulation study follows and verifies that theoretical power estimates based on the derived formulae are nearly identical to empirical estimates based on simulated data. Recommendations at the design stage of a cluster-RCT are discussed.     

FRZB as a key molecule in abdominal aortic aneurysm progression affecting vascular integrity

Abdominal aortic aneurysm (AAA), when ruptured, results in high mortality. The identification of molecular pathways involved in AAA progression is required to improve AAA prognosis. The aim of the present study was to assess the key genes for the progression of AAA and their functional role. Genomic and clinical data of three independent cohorts were downloaded from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) ({"type":"entrez-geo","attrs":{"text":"GSE57691","term_id":"57691"}}GSE57691, {"type":"entrez-geo","attrs":{"text":"GSE7084","term_id":"7084"}}GSE7084, and {"type":"entrez-geo","attrs":{"text":"GSE98278","term_id":"98278"}}GSE98278). To develop AAA diagnosis and progression-related differentially expressed genes (DEGs), we used a significance analysis of microarray (SAM). Spearman correlation test and gene set analysis were performed to identify potential enriched pathways for DEGs. Only the Frizzled-related protein (FRZB) gene and chromosome 1 open reading frame 24 (C1orf24) exhibited significant down-regulation in all analyses. With FRZB, the pathways were associated with RHO GTPase and elastin fiber formation. With C1orf24, the pathways were elastic fiber formation, extracellular matrix organization, and cell–cell communication. Since only FRZB was evolutionally conserved in the vertebrates, function of FRZB was validated using zebrafish embryos. Knockdown of frzb remarkably reduced vascular integrity in zebrafish embryos. We believe that FRZB is a key gene involved in AAA initiation and progression affecting vascular integrity.     

Evaluation of the genotoxic effects of a folk medicine, Petiveria alliacea (Anamu).

Crude extract from a plant known as Petiveria alliacea (Anamu) is used extensively as folk medicine in developing countries like Colombia, South America. Although the plant is known to contain toxic ingredients potential adverse health effects from its use have not been adequately evaluated. We investigated its genotoxic activities by conducting a sister chromatid exchange (SCE) assay using cells in vitro and in vivo. Lymphocytes from humans were treated at 24 h after initiation of culture for 6 h with alcohol extract from the folk medicine. Concentrations of 0, 10, 100, 250, 275, 500, 750, and 1000 micrograms/ml of the extract were used. Significant dose-dependent increase of SCE (3.7-7.4 SCE per cell) were observed (analysis of variances, p less than 0.01). Delay in cell proliferation but not inhibition of mitosis was also observed. In another experiment, mice were exposed once orally to 1x, 200x, 300x and 400x the human daily consumption dose of Anamu. The induction of sister chromatid exchanges in bone marrow cells were investigated. We observed a significant dose dependent increase of SCE compared with the saline control (2.15-4.53; p less than 0.01) and compared with the solvent control (3.04-4.53; p less than 0.01). Our data suggest, therefore, that the folk medicine contains mutagenic and potentially carcinogenic agents although the medicine is not a potent mutagen. Individuals who consume large amounts of this drug may be at risk for development of health problems. Further studies with cells from exposed individuals and from experimental animals should be conducted to provide a better evaluation of health risk from the use of this drug.