Establishment and characterization of SV40 large T antigen-immortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation

Bovine brain cell lines with specific characteristics are useful in vitro experimental systems for molecular and cellular investigation of the interactions between bovine specific neuropathogenic agents and the host. Here, we established two novel cell lines from cultures of cryopreserved fetal bovine brain tissue by the transfection of SV40 large T antigen. Both cell lines showed cobblestone morphology in DMEM/F12 medium supplemented with 10% fetal bovine serum, epidermal growth factor and basic fibroblast growth factor. They were immunostained with endothelial marker, Von Willebrand Factor. Endothelial properties, such as capillary-like tube formation on matrigel and the incorporation of DiI-AcLDL were confirmed with these cells. Removal of growth factors increased the number of cells expressing alpha-smooth muscle actin, suggesting the potential of these cell lines to differentiate into smooth muscle cells. This study suggests an efficient protocol to immortalize brain endothelial cell lines from fetal bovine brain tissue culture.     

Scaffolds for Skeletal Regeneration

Although current treatment modalities for bone defects include autograft, allograft, and artificial bone substitutes, they have problems concerning invasiveness, safety, and performance, respectively, calling for development of innovative artificial bones with better handling and mechanical strength, better control of external and internal structures, and better biodegradability and osteo-inductive ability. We propose to fabricate novel high-performance artificial bones using 3D inkjet printer based on the image data of bone defect/deformity. Shape precisely fitting to the defect/deformity, internal structure facilitating cell invasion, and good biodegradability are achieved. Bioactive substances can be incorporated by printing in combination with drug delivery system to induce bone regeneration at the desired locations. These osteo-inductive artificial bones will help efficiently treat various types of bone defect/deformity in a less invasive and safe manner.     

Serotonin Transporter Gene Polymorphism Modulates Activity and Connectivity within an Emotional Arousal Network of Healthy Men during an Aversive Visceral Stimulus

Background and Aims

The 5-hydroxytryptamine transporter gene-linked polymorphic region (5-HTTLPR) has been linked to increased stress responsiveness and negative emotional states. During fearful face recognition individuals with the s allele of 5-HTTLPR show greater amygdala activation. We aimed to test the hypothesis that the 5-HTTLPR polymorphism differentially affects connectivity within brain networks during an aversive visceral stimulus.

Methods

Twenty-three healthy male subjects were enrolled. DNA was extracted from the peripheral blood. The genotype of 5-HTTLPR was determined using polymerase chain reaction. Subjects with the s/s genotype (n = 13) were compared to those with the l allele (genotypes l/s, l/l, n = 10). Controlled rectal distension from 0 to 40 mmHg was delivered in random order using a barostat. Radioactive H2[^15-O] saline was injected at time of distension followed by positron emission tomography (PET). Changes in regional cerebral blood flow (rCBF) were analyzed using partial least squares (PLS) and structural equation modeling (SEM).

Results

During baseline, subjects with s/s genotype demonstrated a significantly increased negative influence of pregenual ACC (pACC) on amygdala activity compared to l-carriers. During inflation, subjects with s/s genotype demonstrated a significantly greater positive influence of hippocampus on amygdala activity compared to l-carriers.

Conclusion

In male Japanese subjects, individuals with s/s genotype show alterations in the connectivity of brain regions involved in stress responsiveness and emotion regulation during aversive visceral stimuli compared to those with l carriers.     

Bright light exposure augments cognitive behavioral therapy for panic and posttraumatic stress disorders: a pilot randomized control trial

Sleep and Biological Rhythms - Bright light (BL) therapy is clinically utilized for treatment of sleep–wake disorders through the manipulation of circadian oscillation. It is also extended to...     

Complete genome sequence of Leadbetterella byssophila type strain (4M15)

Leadbetterella byssophila Weon et al. 2005 is the type species of the genus Leadbetterella of the family Cytophagaceae in the phylum Bacteroidetes. Members of the phylum Bacteroidetes are widely distributed in nature, especially in aquatic environments. They are of special interest for their ability to degrade complex biopolymers. L. byssophila occupies a rather isolated position in the tree of life and is characterized by its ability to hydrolyze starch and gelatine, but not agar, cellulose or chitin. Here we describe the features of this organism, together with the complete genome sequence, and annotation. L. byssophila is already the 16(th) member of the family Cytophagaceae whose genome has been sequenced. The 4,059,653 bp long single replicon genome with its 3,613 protein-coding and 53 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

The modulation of hepatic adenosine triphosphate and inflammation by eicosapentaenoic acid during severe fibrotic progression in the SHRSP5/Dmcr rat model

AimsEicosapentaenoic acid (EPA) can ameliorate certain liver lesions involved in non-alcoholic steatohepatitis (NASH). A previous study has found that stroke-prone spontaneously hypertensive 5/Dmcr (SHRSP5/Dmcr) rats fed a high fat-cholesterol (HFC) diet developed fibrotic steatohepatitis with histological similarities to NASH. This study evaluated the potential effects and mechanisms of action of EPA supplementation using this rodent model.Main methodsMale rats were randomly assigned to groups that were fed with either the stroke-prone (SP) diet or HFC diet with or without EPA for 2, 8 and 14 weeks, respectively. The liver histopathology, biochemical features, mRNA and protein levels, and nuclear factor-κB (NF-κB) DNA binding activity were determined.Key findingsThe SP diet-fed rats presented normal livers. Conversely, the HFC diet-fed rats developed microvesicular/macrovesicular steatosis, inflammation, ballooning degeneration and severe fibrosis. At 2 weeks, the administration of EPA inhibited hepatic inflammatory recruitment by blocking the phosphorylation of inhibitor of κB-α (IκBα), which antagonizes the NF-κB activation pathway. The dietary supplementation of EPA for 8 weeks ameliorated hepatic triglyceride accumulation and macrovesicular steatosis by inhibiting the HFC diet-induced decrease in the protein levels of enzymes involved in fatty acid β-oxidation including carnitine palmitoyltransferase 1, very long chain acyl-CoA dehydrogenase and peroxisomal bifunctional protein. Although the administration of EPA elicited no histologically detectable effects on severe fibrosis at 14 weeks, it restored an HFC diet-induced decline in hepatic adenosine triphosphate (ATP) levels and suppressed ballooning degeneration, suggesting that EPA may inhibit HFC diet-induced ATP loss and cell death.SignificanceInitial amelioration of the inflammation and steatosis in the rats after EPA supplementation indicates a possibility to treat steatohepatitis. Additionally, this study provides new insights into the roles of EPA in hepatic ATP depletion and subsequent hepatocellular injury during severe fibrosis.     

Inhibition of insulin-like growth factor-1 (IGF-1) expression by prolonged transforming growth factor-β1 (TGF-β1) administration suppresses osteoblast differentiation

TGF-β1 can regulate osteoblast differentiation not only positively but also negatively. However, the mechanisms of negative regulation are not well understood. We previously established the reproducible model for studying the suppression of osteoblast differentiation by repeated or high dose treatment with TGF-β1, although single low dose TGF-β1 strongly induced osteoblast differentiation. The mRNA expression and protein level of insulin-like growth factor-1 (IGF-1) were remarkably decreased by repeated TGF-β1 administration in human periodontal ligament cells, human mesenchymal stem cells, and murine preosteoblast MC3T3-E1 cells. Repeated TGF-β1 administration subsequently decreased alkaline phosphatase (ALP) activity and mRNA expression of osteoblast differentiation marker genes, such as RUNX2, ALP, and bone sialoprotein (BSP). Additionally, repeated administration significantly reduced the downstream signaling pathway of IGF-1, such as Akt phosphorylation in these cells. Surprisingly, exogenous and overexpressed IGF-1 recovered ALP activity and mRNA expression of osteoblast differentiation marker genes even with repeated TGF-β1 administration. These facts indicate that the key mechanism of inhibition of osteoblast differentiation induced by repeated TGF-β1 treatment is simply due to the down-regulation of IGF-1 expression. Inhibition of IGF-1 signaling using small interfering RNA (siRNA) against insulin receptor substrate-1 (IRS-1) suppressed mRNA expression of RUNX2, ALP, BSP, and IGF-1 even with single TGF-β1 administration. This study showed that persistence of TGF-β1 inhibited osteoblast differentiation via suppression of IGF-1 expression and subsequent down-regulation of the PI3K/Akt pathway. We think this fact could open the way to use IGF-1 as a treatment tool for bone regeneration in prolonged inflammatory disease.