Evaluation of bone healing following Er:YAG laser ablation in rat calvaria compared with bur drilling

The Er:YAG laser is currently used for bone ablation. However, the effect of Er:YAG laser irradiation on bone healing remains unclear. The aim of this study was to investigate bone healing following ablation by laser irradiation as compared with bur drilling. Rat calvarial bone was ablated using Er:YAG laser or bur with water coolant. Er:YAG laser effectively ablated bone without major thermal changes. In vivo micro‐computed tomography analysis revealed that laser irradiation showed significantly higher bone repair ratios than bur drilling. Scanning electron microscope analysis showed more fibrin deposition on laser‐ablated bone surfaces. Microarray analysis followed by gene set enrichment analysis revealed that IL6/JAK/STAT3 signaling and inflammatory response gene sets were enriched in bur‐drilled bone at 6 hours, whereas the E2F targets gene set was enriched in laser‐irradiated bone. Additionally, Hspa1a and Dmp1 expressions were increased and Sost expression was decreased in laser‐irradiated bone compared with bur‐drilled bone. In granulation tissue formed after laser ablation, Alpl and Gblap expressions increased compared to bur‐drilled site. Immunohistochemistry showed that osteocalcin‐positive area was increased in the laser‐ablated site. These results suggest that Er:YAG laser might accelerate early new bone formation with advantageous surface changes and cellular responses for wound healing, compared with bur‐drilling.      

Intestinal Bile Acids Induce a Morphotype Switch in Vancomycin-Resistant Enterococcus that Facilitates Intestinal Colonization

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Intracellular mobilization of Ca by the insect steroid hormone 20-hydroxyecdysone during programmed cell death in silkworm anterior silk glands

20-Hydroxyecdysone (20E) triggers programmed cell death (PCD) and regulates de novo gene expression in the anterior silk glands (ASGs) of the silkworm Bombyx mori. PCD is mediated via a nongenomic pathway that includes Ca²⁺ as a second messenger and the activation of protein kinase C/caspase-3-like protease; however, the steps leading to a concomitant buildup of intracellular Ca²⁺ are unknown. We employed pharmacological tools to identify the components of this pathway. ASGs were cultured in the presence of 1 μM 20E and one of the following inhibitors: a G-protein-coupled receptor (GPCR) inhibitor, a phospholipase C (PLC) inhibitor, an inositol 1,4,5-trisphosphate receptor (IP₃R) antagonist, and an L- or T-type Ca²⁺ channel blocker. The T-type Ca²⁺ channel blocker inhibited 20E-induced nuclear and DNA fragmentation; in contrast, PCD was induced by 20E in Ca²⁺-free medium, indicating that the source of Ca²⁺ is an intracellular reservoir. The IP₃R antagonist inhibited nuclear and DNA fragmentation, suggesting that the endoplasmic reticulum may be the Ca²⁺ source. Finally, the GPCR and PLC inhibitors effectively blocked nuclear and DNA fragmentation. Our results indicate that 20E increases the intracellular level of Ca²⁺ by activating IP₃R, and that this effect may be brought about by the serial activation of GPCR, PLC, and IP₃.     

Process of reductive evolution during 10 years in plasmids of a non-insect-transmissible phytoplasma

A non-insect-transmissible phytoplasma strain (OY-NIM) was obtained from insect-transmissible strain OY-M by plant grafting using no insect vectors. In this study, we analyzed for the gene structure of plasmids during its maintenance in plant tissue culture for 10 years. OY-M strain has one plasmid encoding orf3 gene which is thought to be involved in insect transmissibility. The gradual loss of OY-NIM plasmid sequence was observed in subsequent steps: first, the promoter region of orf3 was lost, followed by the loss of then a large region including orf3, and finally the entire plasmid was disappeared. In contrast, no mutation was found in a pseudogene on OY-NIM chromosome in the same period, indicating that OY-NIM plasmid evolved more rapidly than the chromosome-encoded gene tested. Results revealed an actual evolutionary process of OY plasmid, and provide a model for the stepwise process in reductive evolution of plasmids by environmental adaptation. Furthermore, this study indicates the great plasticity of plasmids throughout the evolution of phytoplasma.     

Gingival Fibroblasts as a Promising Source of Induced Pluripotent Stem Cells

Background

Induced pluripotent stem (iPS) cells efficiently generated from accessible tissues have the potential for clinical applications. Oral gingiva, which is often resected during general dental treatments and treated as biomedical waste, is an easily obtainable tissue, and cells can be isolated from patients with minimal discomfort.

Methodology/Principal Findings

We herein demonstrate iPS cell generation from adult wild-type mouse gingival fibroblasts (GFs) via introduction of four factors (Oct3/4, Sox2, Klf4 and c-Myc; GF-iPS-4F cells) or three factors (the same as GF-iPS-4F cells, but without the c-Myc oncogene; GF-iPS-3F cells) without drug selection. iPS cells were also generated from primary human gingival fibroblasts via four-factor transduction. These cells exhibited the morphology and growth properties of embryonic stem (ES) cells and expressed ES cell marker genes, with a decreased CpG methylation ratio in promoter regions of Nanog and Oct3/4. Additionally, teratoma formation assays showed ES cell-like derivation of cells and tissues representative of all three germ layers. In comparison to mouse GF-iPS-4F cells, GF-iPS-3F cells showed consistently more ES cell-like characteristics in terms of DNA methylation status and gene expression, although the reprogramming process was substantially delayed and the overall efficiency was also reduced. When transplanted into blastocysts, GF-iPS-3F cells gave rise to chimeras and contributed to the development of the germline. Notably, the four-factor reprogramming efficiency of mouse GFs was more than 7-fold higher than that of fibroblasts from tail-tips, possibly because of their high proliferative capacity.

Conclusions/Significance

These results suggest that GFs from the easily obtainable gingival tissues can be readily reprogrammed into iPS cells, thus making them a promising cell source for investigating the basis of cellular reprogramming and pluripotency for future clinical applications. In addition, high-quality iPS cells were generated from mouse GFs without Myc transduction or a specific system for reprogrammed cell selection.     

Treatment of Obesity by Personality Classification-Oriented Program

It is apparent that personality is related to the pathogenesis of obesity, and that understanding the personality of the patient may be a key to successful treatment of the disease. Using the Rorschach test and interviews by a psychiatrist, the types of personality were classified into four groups according to the healthiness of personalities. The judgment of healthiness was based mainly on the scores obtained from the Rorschach test This classification revealed that the occurrence of mental and physical symptoms during therapy with a very low calorie diet(VLCD)and subsequent rebound of body weight were more frequently observed in patients with relatively less healthy personalities. We used this classification to adapt our pro gram to treat obese patients. In this program, severe diet restrictions were applied to patients with relatively healthy personalities. These restrictions were applied with modifications to patients with less healthy personality, because severe restrictions would be possibly very stressful for them and would bring about an undesirable reaction. For strengthening the patients' motivation for therapy, the significance of body weight reduction was explained in different ways to patients with different types of personality. The target of body weight reduction, reward for patients with successful weight reduction, and the duration of therapy were setup differently for patients with different personalities types. The results showed that body weight rebound one or two years after treatment was reduced with the personality-oriented therapy pro gram compared to that observed with the previous conventional therapies. Also, the incidence of psychological problems was remarkably decreased.     

Gene expression and intracellular localization of somatolactin in the pituitary of rainbow trout

The gene expression and intracellular localization of somatolactin (SL), a putative pituitary hormone structurally related to both growth hormone and prolactin, were investigated in the pituitary of rainbow trout, Oncorhynchus mykiss. Using an in situ hybridization technique, we demonstrated the gene expression of the SL molecule in cells bordering the neurohypophysial tissue in the pars intermedia. These cells were identified immunocytochemically as SL-cells on the adjacent section. Electron-microscopic immunocytochemistry by means of the protein A-gold technique, also revealed that the SL-immunoreactivity was located mostly on the secretory granules in SL-cells. Our findings clearly indicate that SL is biosynthesized and stored in the granules in these cells.     

Genetic Analysis of the Capsid Region of Human Astrovirus Serotype 3 Isolated in Japan

The total capsid region of astrovirus serotype 3 was analyzed using five isolates including four from Japan and one from the United Kingdom. The nucleic acid and deduced amino acid sequences in the region were homologous (over 97%). However, the sequences of serotype 3 were different from those of serotypes 1, 2, 4, 5, 6 and 8, especially in the C terminus.     

Alteration of the T-cell receptor repertoire in A. CA mice expressing anEa d transgene

In an effort to generate an A.CA mouse expressing E^d, the Ea^d gene has been introduced into A. CA mice which lack the major histocompatibility complex (MHC) class II E molecule. Flow cytometric analysis shows cell surface expression of the E chain on lymphocytes and macrophages in the transgenic mice. Analysis of T-cell receptor (Tcr) genes deleted in some E-expressing mouse strains demonstrates that T cells expressingTcrb-VS are partially deleted in these transgenic mice while those expressingTcrb-V8 andTcrb-11 are not. In addition, the expressed E^d chain can promote Mycoplasma arthriditis mitogen (MAM)-induced T-cell proliferation. The expression of the Ea chain, presumably as an Aß ^fE^d heterodimer, can alter the peripheral T-cell repertoire and T-cell reactivity to a microbial superantigen.     

1,25-Dihydroxyvitamin D3 suppresses the expression of the VCAM-1 receptor, VLA-4 in human leukemic HL-60 cells

Very late antigen-4 (VLA-4) is the complex with alpha4 and beta1 integrins, which is the receptors to fibronectin and VCAM-1. We evaluate the effect of 1,25(OH)2D3 on the expression of VLA-4 in human leukemic HL-60, U937 cells and human melanoma A375 cells. Flow cytometric analysis demonstrate that the expression of alpha4 integrin is negatively regulated in the cell lines we studied. The expression of beta1 integrin is also decreased in HL-60 and U937 cells. The mRNA expression of alpha4 integrin is significantly decreased by the treatment with 1,25(OH)2D3, whereas 1,25(OH)2D3 does not alter the expression of beta1 mRNA. The adhesion assay demonstrate that the number of adherent cells treated with 1, 25(OH)2D3 is significantly lower than that untreated on VCAM-1-coated wells. Because VCAM-1 is highly expressed in the endothelial cells, it is possible that 1,25(OH)2D3 prevents the attachment of the cells from the endothelial cells in vivo.