Graphic and movie illustrations of human prenatal development and their application to embryological education based on the human embryo specimens in the Kyoto collection.

Morphogenesis in the developing embryo takes place in three dimensions, and in addition, the dimension of time is another important factor in development. Therefore, the presentation of sequential morphological changes occurring in the embryo (4D visualization) is essential for understanding the complex morphogenetic events and the underlying mechanisms. Until recently, 3D visualization of embryonic structures was possible only by reconstruction from serial histological sections, which was tedious and time-consuming. During the past two decades, 3D imaging techniques have made significant advances thanks to the progress in imaging and computer technologies, computer graphics, and other related techniques. Such novel tools have enabled precise visualization of the 3D topology of embryonic structures and to demonstrate spatiotemporal 4D sequences of organogenesis. Here, we describe a project in which staged human embryos are imaged by the magnetic resonance (MR) microscope, and 3D images of embryos and their organs at each developmental stage were reconstructed based on the MR data, with the aid of computer graphics techniques. On the basis of the 3D models of staged human embryos, we constructed a data set of 3D images of human embryos and made movies to illustrate the sequential process of human morphogenesis. Furthermore, a computer-based self-learning program of human embryology is being developed for educational purposes, using the photographs, histological sections, MR images, and 3D models of staged human embryos.     

Deletion of the CaBIG1 gene reduces beta-1,6-glucan synthesis, filamentation, adhesion, and virulence in Candida albicans

The human fungal pathogen Candida albicans is able to change its shape in response to various environmental signals. We analyzed the C. albicans BIG1 homolog, which might be involved in beta-1,6-glucan biosynthesis in Saccharomyces cerevisiae. C. albicans BIG1 is a functional homolog of an S. cerevisiae BIG1 gene, because the slow growth of an S. cerevisiae big1 mutant was restored by introduction of C. albicans BIG1. CaBig1p was expressed constitutively in both the yeast and hyphal forms. A specific localization of CaBig1p at the endoplasmic reticulum or plasma membrane similar to the subcellular localization of S. cerevisiae Big1p was observed in yeast form. The content of beta-1,6-glucan in the cell wall was decreased in the Cabig1Delta strain in comparison with the wild-type or reconstituted strain. The C. albicans BIG1 disruptant showed reduced filamentation on a solid agar medium and in a liquid medium. The Cabig1Delta mutant showed markedly attenuated virulence in a mouse model of systemic candidiasis. Adherence to human epithelial HeLa cells and fungal burden in kidneys of infected mice were reduced in the Cabig1Delta mutant. Deletion of CaBIG1 abolished hyphal growth and invasiveness in the kidneys of infected mice. Our results indicate that adhesion failure and morphological abnormality contribute to the attenuated virulence of the Cabig1Delta mutant.     

Spatiotemporal organization of frog respiratory neurons visualized on the ventral medullary surface

We visualized the spatiotemporal activity of respiratory-related neurons in the frog using the isolated brainstem spinal cord preparation. We recorded optical signals from the ventral surface of the medulla using a voltage-sensitive dye, and calculated cross-correlations with the integrated respiratory activity of the trigeminal nerve. Lung burst-related depolarizing optical signals were observed bilaterally as longitudinal columns in the ventrolateral medulla between the levels of trigeminal and hypoglossal rootlets, mostly caudal to the vagal rootlet. However, we could not differentiate between neurons involved in rhythm generation and motoneurons. The dye weakened the buccal rhythm and slowed the lung rhythm, which might have influenced the results. Extracellular recording of respiratory neurons verified the optically identified area. Strychnine disrupted the spatiotemporal organization of optical signals, although trigeminal periodic bursts persisted. We conclude that the pattern generator but not the rhythm generator of lung burst in the frog involves glycinergic mechanisms and lies as longitudinal columns in the reticular formation of the ventrolateral medulla.     

Type XVII Collagen is a Key Player in Tooth Enamel Formation

Inherited tooth enamel hypoplasia occurs due to mutations in genes that encode major enamel components. Enamel hypoplasia also has been reported in junctional epidermolysis bullosa, caused by mutations in the genes that encode type XVII collagen (COL17), a component of the epithelial-mesenchymal junction. To elucidate the pathological mechanisms of the enamel hypoplasia that arise from the deficiency of epithelial-mesenchymal junction molecules, such as COL17, we investigated tooth formation in our recently established Col17^−/− and Col17 rescued mice. Compared with wild-type mice, the incisors of the Col17^−/− mice exhibited reduced yellow pigmentation, diminished iron deposition, delayed calcification, and markedly irregular enamel prisms, indicating the presence of enamel hypoplasia. The molars of the Col17^−/− mice demonstrated advanced occlusal wear. These abnormalities were corrected in the Col17 rescued humanized mice. Thus, the Col17^−/− mice clearly reproduced the enamel hypoplasia in human patients with junctional epidermolysis bullosa. We were able to investigate tooth formation in the Col17^−/− mice because the Col17^−/− genotype is not lethal. Col17^−/− mouse incisors had poorly differentiated ameloblasts that lacked enamel protein-secreting Tomes’ processes and reduced mRNA expression of amelogenin, ameloblastin, and of other enamel genes. These findings indicated that COL17 regulates ameloblast differentiation and is essential for normal formation of Tomes’ processes. In conclusion, COL17 deficiency disrupts the epithelial-mesenchymal interactions, leading to both defective ameloblast differentiation and enamel malformation.     

Stretching versus transitory icing: which is the more effective treatment for attenuating muscle fatigue after repeated manual labor?

Purpose

Effective recovery from muscle fatigue, especially during rest intervals between periods of high-intensity activity, is important to ensure optimal subsequent performance. Stretching and icing are two types of treatment used for muscle recovery in such situations. However, their effectiveness remains unclear because of a lack of adequate evidence and/or discrepant results of previous studies. We performed a study to elucidate the effects of stretching and icing on muscle fatigue in subjects performing alternating muscle contraction and rest.

Methods

Sixteen healthy male subjects aged 21–27 years were evaluated. Each subject performed repeated isometric muscle contraction exercises that involved lifting and holding a dumbbell to induce muscle fatigue. Four treatments were performed during the rest periods between isometric muscle contraction: static stretching, ballistic stretching, no stretching, or icing. Electromyography and relative muscle oxygen saturation measurements were performed during the exercises. Muscle fatigue was indirectly estimated by the decline in the median frequency of the electromyographic signal.

Results

Stretching between alternate isometric muscle contraction exercises resulted in a significantly lower median frequency of the electromyographic signal than did no stretching. There was no significant difference in the change in the median frequency between static and ballistic stretching. Conversely, icing between alternate exercises did not decrease the median frequency.

Conclusions

Stretching, whether static or ballistic, is not beneficial for recovery from muscle fatigue and may actually inhibit recovery. Icing may more effectively induce such recovery and thus may be a better choice between the two treatment techniques.     

Determinants of Local Progression After Computed Tomography-Guided Percutaneous Radiofrequency Ablation for Unresectable Lung Tumors: 9-Year Experience in a Single Institution

The purpose of this study was to retrospectively determine the local control rate and contributing factors to local progression after computed tomography (CT)-guided radiofrequency ablation (RFA) for unresectable lung tumor. This study included 138 lung tumors in 72 patients (56 men and 16 women; age 70.0 ± 11.6 years (range 31–94); mean tumor size 2.1 ± 1.2 cm [range 0.2–9]) who underwent lung RFA between June 2000 and May 2009. Mean follow-up periods for patients and tumors were 14 and 12 months, respectively. The local progression-free rate and survival rate were calculated to determine the contributing factors to local progression. During follow-up, 44 of 138 (32%) lung tumors showed local progression. The 1-, 2-, 3-, and 5-year overall local control rates were 61, 57, 57, and 38%, respectively. The risk factors for local progression were age (≥70 years), tumor size (≥2 cm), sex (male), and no achievement of roll-off during RFA (P < 0.05). Multivariate analysis identified tumor size ≥2 cm as the only independent factor for local progression (P = 0.003). For tumors <2 cm, 17 of 68 (25%) showed local progression, and the 1-, 2-, and 3-year overall local control rates were 77, 73, and 73%, respectively. Multivariate analysis identified that age ≥70 years was an independent determinant of local progression for tumors <2 cm in diameter (P = 0.011). The present study showed that 32% of lung tumors developed local progression after CT-guided RFA. The significant risk factor for local progression after RFA for lung tumors was tumor size ≥2 cm.     

Positive modulation of long-term potentiation at hippocampal CA1 synapses by low micromolar concentrations of zinc

The role of zinc, an endogenous N-methyl-d-aspartate (NMDA) receptor antagonist, in long-term potentiation (LTP) at hippocampal CA1 synapses is poorly understood. In the present study, the effect of exogenous zinc and zinc chelators on CA1 LTP was examined by using hippocampal slices from rats. CA1 LTP after tetanic stimulation (100 Hz, 1 s) was potentiated in the presence of 5 microM ZnCl(2), but not in the presence of 30 microM. In varying the frequency (10-100 Hz, 1 s), zinc (5 microM) caused a significant shift of the frequency-response curve and lowered the threshold in LTP induction. The present study is the first to demonstrate that CA1 LTP is potentiated by low micromolar concentrations of zinc. Endogenous zinc is likely to reach low micromolar concentrations in the extracellular compartment in CA1 LTP induction. On the other hand, zinc has no effect on field excitatory postsynaptic potentials (fEPSPs) after tetanic stimulation in the presence of 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, in which LTP was abolished, indicating that NMDA receptor activation is necessary for the potentiation of CA1 LTP by zinc. The pretreatment with ZnAF-2DA, a membrane-permeable zinc chelator, which was used to block the increase in intracellular Zn(2+), inhibited LTP and also LTP potentiated by zinc. It is likely that Zn(2+) taken up during LTP induction potentiates CA1 LTP via NMDA receptor activation.     

Proteomic and histopathological characterization of the interface between oral squamous cell carcinoma invasion fronts and non-cancerous epithelia

Oral squamous cell carcinomas (SCCs) are frequently associated with pre-invasive lesions including carcinoma in-situ (CIS), and CISs further form lateral interfaces against surrounding normal or dysplastic epithelia (ND). At the interface where keratin (K) 17 positive (+) SCC/CIS cells are in contact with K13 + ND cells, “cell competition” must be evoked between two such different cell types. Thus, the aim of this study was to characterize the histopathology of the SCC/CIS-ND interface and to determine protein profiles around the interface by proteomics. A total of 112 lateral interfaces were collected from 55 CIS and 57 SCC foci, and they were investigated by immunohistochemistry and liquid chromatography-tandem mass spectrometry. The interfaces were morphologically classified into three types: vertical, oblique, and convex. There were several cellular changes characteristic to the interface, including apoptosis and hyaline bodies, which were more emphasized in SCC/CIS sides. The results suggested that ND cells were winners of cell competition against SCC/CIS cells. Then, the interfaces were divided into four vertical segments, and each segment was separately laser-microdissected from tissue sections with immunostaining for K13 or K17; the four segments included SCC/CIS away from (#1) or adjacent to (#2) the interface, and ND adjacent to (#3) or away from (#4) the interface. Proteome analyses revealed approximately 4000 proteins from SCC/CIS sides [#1 and #2] and 2800 proteins from ND sides [#3 and #4]. We quantitatively selected the top 25 proteins including ladinin-1 or interleukin-1 receptor antagonist protein, which were most contrastively increased or decreased in SCC/CIS or ND sides, respectively, and their specific immunohistochemical expression modes were confirmed in tissue sections as well as in cultured SCC cells. These molecules should be involved in the cellular crosstalk toward cell competition at the lateral interface of oral SCC/CIS and would be new candidates for histopathological distinction of oral malignancies.     

Determination of bisphenol A concentrations in human biological fluids reveals significant early prenatal exposure

BACKGROUND: There is broad human exposure to bisphenol A (BPA), an estrogenic endocrine-disrupting chemical widely used for the production of plastic products. BPA is reported to affect preimplantation embryos or fetuses and alter their postnatal development at doses typically found in the environment. We measured contamination of BPA in various kinds of human biological fluids by a novel enzyme-linked immunosorbent assay. METHODS: Blood samples were obtained from healthy premenopausal women, women with early and full-term pregnancy, and umbilical cord at full-term delivery. Ovarian follicular fluids obtained during IVF procedures and amniotic fluids obtained at mid-term and full-term pregnancy were also subject to BPA measurements. RESULTS: BPA was present in serum and follicular fluid at approximately 1-2 ng/ml, as well as in fetal serum and full-term amniotic fluid, confirming passage through the placenta. Surprisingly, an approximately 5-fold higher concentration, 8.3 +/- 8.7 ng/ml, was revealed in amniotic fluid at 15-18 weeks gestation, compared with other fluids. CONCLUSION: These results suggest accumulation of BPA in early fetuses and significant exposure during the prenatal period, which must be considered in evaluating the potential for human exposure to endocrine-disrupting chemicals.