Real-Time Polarization-Resolved Imaging of Living Tissues Based on Two-Photon Excitation Spinning-Disk Confocal Microscopy

Laser scanning microscopy using high-peak-power ultrashort near infrared light pulses can visualize biological microstructures by utilizing non-linear optical processes, such as multi-photon excitation and sum frequency generation. Here we introduced a polarization-resolving detection methodology for a laser scanning microscopy system equipped with a spinning-disk confocal scanner. The developed system achieved high-speed intravital imaging of living tissues with resolving their signals to orthogonally polarized components. First, we applied the system to a liposomal vesicle labeled with the fluorescent lipophilic dye and confirmed the orientation map of the lipid bilayer. Next, by detecting polarization-resolved second harmonic generation signals, the structural orientations of the collagen fibers in fixed mouse tissues were visualized without exogenous or genetic fluorophore labeling. Finally, we demonstrated in vivo polarization-resolved second harmonic generation imaging of the collagen fibers in the mouse skeletal muscles at a 56 Hz temporal resolution. We expect that our developed methodology can achieve real-time visualization, thus, revealing the conformational changes of supramolecular structures in living animals

Single Nucleotide Polymorphism that Accompanies a Missense Mutation (Gln488His) Impedes the Dimerization of Hsp90.

A single nucleotide polymorphism (SNP) that causes a missense mutation of highly conserved Gln488 to His of the alpha isoform of the 90-kDa heat shock protein (Hsp90alpha) molecular chaperone is observed in Caucasians. The mutated Hsp90alpha severely reduced the growth of yeast cells. To investigate this molecular mechanism, we examined the domain-domain interactions of human Hsp90alpha by using bacterial 2-hybrid system. Hsp90alpha was expressed as a full-length form, N-terminal domain (residues 1-400), or middle (residues 401-617) plus C-terminal (residues 618-732) domains (MC domain/amino acids 401-732). The Gln488His substitution in MC domain did not affect the intra-molecular interaction with N-terminal domain, whereas the dimeric interaction-mediated by the inter-molecular interaction between MC domains was decreased to 32%. Gln488Ala caused a similar change, whereas Gln488Thr, which exceptionally occurs in mitochondrial Hsp90 paralog, fully maintained the dimeric interaction. Therefore, the SNP causing Gln488His mutation could abrogate the Hsp90 function due to reduced dimerization

Effects of fermented brown rice on the intestinal environments in healthy adult

Purpose : The aim of this study is to investigate the prebiotic effects of brown rice fermented by Aspergillus oryzae (FBRA) on the intestinal environment in vitro and in healthy adults. Methods : Fresh fecal slurries from six healthy adults were incubated with FBRA to confirm prebiotic potentials of FBRA. Another thirty-six healthy adults were randomly allocated to 2 groups for the clinical study. Subjects consumed 21.0 g/day of either FBRA or control food for 2 weeks, followed by a 12-week intermission and then 2-week ingestion vice versa. Main outcome measures were bifidobacterial numbers and organic acid concentration in feces. Sub outcome measures were fecal microbiota, fecal environments and bowel function. Results : Incubation of fecal slurries with FBRA in vitro resulted in increased organic acids with individual-specific patterns. Bifidobacterial numbers were increased during incubation. In the clinical study, all participants safely completed this study. FBRA had little effect on fecal number of bifidobacteria, concentrations of organic acids or putrefactive metabolites, fecal pH, or fecal microbiota. Conclusion : FBRA has the potentials as a prebiotic, however, we could not detect its effects on the intestinal environment in vivo. The results in a clinical study indicated that FBRA could be safely used for healthy adults

Live imaging of center of calcification formation during septum development in primary polyps of Acropora digitifera

Recent studies have revealed that stony corals create their extracellular skeletons via biologically controlled calcification, in which amorphous calcium carbonate (ACC), regarded as precursors of aragonite crystals, have been observed at nanoscale using electron microscopy. However, the exact mechanism by which ACC is generated, and how it contributes to skeletal growth in coral calcifying tissue, remains enigmatic. The septal skeleton of an individual polyp is composed of radially aligned plates extending upward from the aboral calcifying tissue. This structure includes microstructure known as the centers of calcification (CoC). However, despite its importance, direct in vivo observation of septal growth has not been reported. Observations under transmitted illumination using polarized light microscopy on calcifying tissue of young Acropora digitifera revealed small crystals, a few micrometers in size, that accompany subtle movements and that emerge exclusively on the inner wall of the pocket in extracellular calcifying fluid (ECF). Crystal growth initiated from small, scattered crystals on a glass plate resembles this phenomenon observed in coral skeletons. Time-lapse photographs of 12 individuals in early primary polyp settlement revealed this process in three individuals, documenting 13 of these crystal events. This phenomenon occurred solely at the bases of subsequently formed septa. These crystals differ notably from fusiform crystals and from dumbbell-like or rod-like crystals growing individually. Upright two-photon microscopy captured movement of sub-micron-sized fluorescent calcein-accumulating particles, emphasizing their presence on the surface of the growing fronts of septa. Methodological advances that facilitate comprehensive in vivo observation of sub-micron-sized structures, calcein-accumulating particles to the skeleton, are needed to develop a more detailed understanding of coral skeletal growth

E-selectin as a prognostic factor of patients hospitalized due to acute inflammatory respiratory diseases

When examining patients with acute inflammatory respiratory diseases, it is difficult to distinguish between infectious pneumonia and interstitial pneumonia and predict patient prognosis at the beginning of treatment. In this study, we assessed whether endothelial selectin (E-selectin) predicts the outcome of patients with acute inflammatory respiratory diseases. We measured E-selectin serum levels in 101 patients who were admitted to our respiratory care unit between January 2013 and December 2013 because of acute inflammatory respiratory diseases that were eventually diagnosed as interstitial pneumonia (n = 38) and lower respiratory tract infection (n = 63). Seven of these patients (n = 101) died. The pneumonia severity score was significantly higher and the oxygen saturation of arterial blood measured by pulse oximeter (SpO2)/fraction of inspiratory oxygen (FiO2) was significantly lower in the deceased patients than in the surviving patients. There were significantly fewer peripheral lymphocytes and significantly higher E-selectin serum levels in the deceased patients than in the surviving patients. In the multiple logistic regression analysis, the E-selectin serum levels and SpO2/FiO2 ratio were independent predictive factors of prognosis. The risk of death during acute respiratory disease was determined using a receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) was 0.871 as calculated from the ES, and the cutoff value was 6453.04 pg/ml, with a sensitivity of 1.00 and a specificity of 0.72 (p = 0.0027). E-selectin may be a useful biomarker for predicting the prognosis of patients with acute inflammatory respiratory diseases

MafB silencing in macrophages does not influence the initiation and growth of lung cancer induced by urethane

An increased number of tumor-associated macrophages (TAMs) that exhibit the M2 macrophage phenotype is related to poorer prognosis in cancer patients. MafB is a transcription factor regulating the differentiation of macrophages. However, involvement of MafB for the development of TAMs is unknown. This study was designed to investigate the role of MafB in a murine urethane-induced lung cancer model. Urethane was injected intraperitoneally into wild-type and dominant-negative MafB transgenic mice. Twenty-four weeks later, mice were sacrificed and their lungs removed for pathological analysis. The numbers and mean areas of lung cancer were evaluated. In addition, the numbers of Mac-3-positive macrophages were evaluated in each tumor. The numbers and mean areas of lung cancer induced by urethane administration were not significantly different between wild-type and dominant-negative MafB transgenic mice. The numbers of TAMs in lung cancer tissue were not significantly different between the two groups. MafB silencing using dominant-negative MafB did not influence the initiation and growth of lung cancer in mice exposed to urethane. These data suggest that MafB may not be related to the development of TAMs

A Novel Function of Noc2 in Agonist-Induced Intracellular Ca2+ Increase during Zymogen-Granule Exocytosis in Pancreatic Acinar Cells

Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca2+-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 µM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca2+]i-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca2+]i increases

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis

Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7- amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly- Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the kcat/Km figure (194 mM21?sec21) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 mM21?sec21). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCAhydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule