Comparison of artificial tooth position in dentures fabricated by heat curing and additive manufacturing.

BACKGROUND: The purpose of this study was to compare the displacement of tooth arrangement in dentures fabricated by additive manufacturing (AM) and heat curing. METHODS: Three-dimensional (3D) scanning was performed for edentulous jaw models. After the teeth were arranged, 3D scanning for the wax denture was performed. Heat-cured dentures were fabricated with heat-cure polymer resin. Based on data obtained by subtracting the model data from wax denture data, AM dentures were fabricated from ultraviolet-cured acrylic resin. Accuracy was verified by superimposing heat-cured and AM dentures on the tooth region data from the wax dentures and measuring displacement of the tooth arrangement. RESULTS: In the maxillary dentures, the amount of tooth displacement for the heat-cured dentures and for the AM dentures ranged from -0.08 to +0.06 mm and from -0.25 to +0.06 mm respectively. A significant difference was observed between two dentures. In the mandibular dentures, the amount of tooth displacement for the heat-cured dentures and for the AM dentures ranged from -0.09 to +0.07 mm and from -0.03 to +0.07 mm respectively. No significant difference was observed between two dentures. CONCLUSIONS: The artificial teeth of the maxillary dentures fabricated by AM showed a greater displacement compared to those by heat curing.

Recurrent Spindle Cell Carcinoma Shows Features of Mesenchymal Stem Cells.

This study investigated a case of spindle cell carcinoma (SpCC) in tongue pathological lesions. The patient experienced a local recurrence and distant metastasis after surgical intervention. Although standard chemotherapy was administered, a granulomatous mass continued to develop. This aggressive growth led to survival of the tumor. Secondary debulking surgery was performed to improve the patient's quality of life at the request of the patient. Using a tissue sample derived from the secondary debulking surgery, we performed an analysis of the tumor's cell surface antigens, differentiation potential, metastatic ability, and inhibition potential by anticancer reagents. In vitro analysis revealed that the cell population grown under adherent culture conditions expressed the mesenchymal stem cell (MSC) markers CD73, CD90, and CD105. The cell line established from this SpCC contained colony-forming unit fibroblasts (CFU-Fs) and exhibited multipotent differentiation into several mesenchymal lineages, including bone, cartilage, and fat. The SpCC cells also displayed vigorous mobilization. These characteristics suggested that they had the differentiation potential of mesenchymal cells, especially MSCs, rather than that of epithelial cells. The surgical specimen analyzed in this study resisted the molecular target reagent cetuximab, which is an epidermal growth factor receptor inhibitor. This clinical insight revealed that chemotherapy-resistant SpCC cells have different characteristics compared to most other cancer cells, which are sensitive to cetuximab. Our cell death assay revealed that SpCC cell death was induced by the anticancer drug imatinib, which is known to inhibit protein tyrosine kinase activity of ABL, platelet-derived growth factor receptor α (PDGFRα), and KIT. Here, we report recurrent SpCC with characteristics of MSCs and potential for treatment with imatinib.

Generation of interspecies limited chimeric nephrons using a conditional nephron progenitor cell replacement system.

Animal fetuses and embryos may have applications in the generation of human organs. Progenitor cells may be an appropriate cell source for regenerative organs because of their safety and availability. However, regenerative organs derived from exogenous lineage progenitors in developing animal fetuses have not yet been obtained. Here, we established a combination system through which donor cells could be precisely injected into the nephrogenic zone and native nephron progenitor cells (NPCs) could be eliminated in a time- and tissue-specific manner. We successfully achieved removal of Six2+ NPCs within the nephrogenic niche and complete replacement of transplanted NPCs with donor cells. These NPCs developed into mature glomeruli and renal tubules, and blood flow was observed following transplantation in vivo. Furthermore, this artificial nephron could be obtained using NPCs from different species. Thus, this technique enables in vivo differentiation from progenitor cells into nephrons, providing insights into nephrogenesis and organ regeneration.

Analysis of induced pluripotent stem cells carrying 22q11.2 deletion.

Given the complexity and heterogeneity of the genomic architecture underlying schizophrenia, molecular analyses of these patients with defined and large effect-size genomic defects could provide valuable clues. We established human-induced pluripotent stem cells from two schizophrenia patients with the 22q11.2 deletion (two cell lines from each subject, total of four cell lines) and three controls (total of four cell lines). Neurosphere size, neural differentiation efficiency, neurite outgrowth, cellular migration and the neurogenic-to-gliogenic competence ratio were significantly reduced in patient-derived cells. As an underlying mechanism, we focused on the role of DGCR8, a key gene for microRNA (miRNA) processing and mapped in the deleted region. In mice, Dgcr8 hetero-knockout is known to show a similar phenotype of reduced neurosphere size (Ouchi et al., 2013). The miRNA profiling detected reduced expression levels of miRNAs belonging to miR-17/92 cluster and miR-106a/b in the patient-derived neurospheres. Those miRNAs are reported to target p38α, and conformingly the levels of p38α were upregulated in the patient-derived cells. p38α is known to drive gliogenic differentiation. The inhibition of p38 activity by SB203580 in patient-derived neurospheres partially restored neurogenic competence. Furthermore, we detected elevated expression of GFAP, a gliogenic (astrocyte) marker, in postmortem brains from schizophrenia patients without the 22q11.2 deletion, whereas inflammation markers (IL1B and IL6) remained unchanged. In contrast, a neuronal marker, MAP2 expressions were decreased in schizophrenia brains. These results suggest that a dysregulated balance of neurogenic-to-gliogenic competence may underlie neurodevelopmental disorders such as schizophrenia.

Neural changes in the primate brain correlated with the evolution of complex motor skills.

Complex motor skills of eventual benefit can be learned after considerable trial and error. What do structural brain changes that accompany such effortful long-term learning tell us about the mechanisms for developing innovative behavior? Using MRI, we monitored brain structure before, during and after four marmosets learnt to use a rake, over a long period of 10-13 months. Throughout learning, improvements in dexterity and visuo-motor co-ordination correlated with increased volume in the lateral extrastriate cortex. During late learning, when the most complex behavior was maintained by sustained motivation to acquire the skill, the volume of the nucleus accumbens increased. These findings reflect the motivational state required to learn, and show accelerated function in higher visual cortex that is consistent with neurocognitive divergence across a spectrum of primate species.

Purified Human Dental Pulp Stem Cells Promote Osteogenic Regeneration.

Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies.

IL-10 gene transfer upregulates arcuate POMC and ameliorates hyperphagia, obesity and diabetes by substituting for leptin.

BACKGROUND: Obesity and metabolic syndrome are the major risk factors for cardiovascular disease. Obesity is caused by increased food intake and/or decreased energy expenditure. Leptin potently inhibits food intake and promotes energy expenditure. These effects of leptin involve the activation of proopiomelanocortin (POMC) neurons in the hypothalamus arcuate nucleus (ARC). Disruption of leptin signaling in POMC neuron is considered one of the major causes for obesity. AIMS: The present study aimed to examine whether overexpression of interleukin-10 (IL-10) could substitute for the leptin action and ameliorate obesity in leptin-deficient Lep(ob/ob) mice. DESIGN: Adeno-associated virus (AAV) expressing murine IL-10 (AAV-mIL-10) was injected into the skeletal muscle to overexpress IL-10 in mice. These mice were subsequently subjected to analysis of body weight, food intake, glucose metabolism and underlying mechanisms. RESULTS: In Lep(ob/ob) mice, AAV-IL-10 ameliorated hyperphagia, obesity, glucose intolerance and insulin resistance, as well as attenuated tumor necrosis factor-α expression. The IL-10 treatment also improved glucose-induced insulin release. Furthermore, IL-10 treatment increased POMC mRNA expression in ARC and phosphorylation of signal transducer and activator of transcription-3 (STAT3) in ARC and white adipose tissue (WAT). In neuron-specific STAT3-null mice that exhibited obesity and hyperphagia, AAV-mIL-10 administration failed to affect food intake, body weight and phosphorylation of STAT3 in WAT. CONCLUSIONS: These results demonstrate that peripheral overexpression of IL-10 induces STAT3 phosphorylation in ARC POMC neurons, and thereby ameliorates hyperphagia and obesity caused by leptin deficiency. IL-10 gene transfer may provide an effective approach for preventing progression of metabolic syndrome due to leptin resistance.

Voxel-based morphometry of the marmoset brain: In vivo detection of volume loss in the substantia nigra of the MPTP-treated Parkinson's disease model.

Movement dysfunction in Parkinson's disease (PD) is caused by the degeneration of dopaminergic (DA) neurons in the substantia nigra (SN). Here, we established a method for voxel-based morphometry (VBM) and automatic tissue segmentation of the marmoset monkey brain using a 7-T animal scanner and applied the method to assess DA degeneration in a PD model, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated animals, with tyrosine-hydroxylase staining. The most significant decreases of local tissue volume were detected in the bilateral SN of MPTP-treated marmoset brains (-53.0% in right and -46.5% in left) and corresponded with the location of DA neurodegeneration found in histology (-65.4% in right). In addition to the SN, the decreases were also confirmed in the locus coeruleus, and lateral hypothalamus. VBM using 7-T MRI was effective in detecting volume loss in the SN of the PD-model marmoset. This study provides a potential basis for the application of VBM with ultra-high field MRI in the clinical diagnosis of PD. The developed method may also offer value in automatic whole-brain evaluation of structural changes for the marmoset monkey.

Clinical utility of neuronal cells directly converted from fibroblasts of patients for neuropsychiatric disorders: studies of lysosomal storage diseases and channelopathy.

Methodologies for generating functional neuronal cells directly from human fibroblasts [induced neuronal (iN) cells] have been recently developed, but the research so far has only focused on technical refinements or recapitulation of known pathological phenotypes. A critical question is whether this novel technology will contribute to elucidation of novel disease mechanisms or evaluation of therapeutic strategies. Here we have addressed this question by studying Tay-Sachs disease, a representative lysosomal storage disease, and Dravet syndrome, a form of severe myoclonic epilepsy in infancy, using human iN cells with feature of immature postmitotic glutamatergic neuronal cells. In Tay-Sachs disease, we have successfully characterized canonical neuronal pathology, massive accumulation of GM2 ganglioside, and demonstrated the suitability of this novel cell culture for future drug screening. In Dravet syndrome, we have identified a novel functional phenotype that was not suggested by studies of classical mouse models and human autopsied brains. Taken together, the present study demonstrates that human iN cells are useful for translational neuroscience research to explore novel disease mechanisms and evaluate therapeutic compounds. In the future, research using human iN cells with well-characterized genomic landscape can be integrated into multidisciplinary patient-oriented research on neuropsychiatric disorders to address novel disease mechanisms and evaluate therapeutic strategies.

Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients.

Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.

Fetal sulcation and gyrification in common marmosets (Callithrix jacchus) obtained by ex vivo magnetic resonance imaging.

The present study characterized fetal sulcation patterns and gyrification in the cerebrum of the New World monkey group, common marmosets, using a 3D T2-weighted high-resolution anatomical magnetic resonance imaging (MRI) sequence from the fixed brain at 7-tesla ex vivo. Fetal sulcation in the marmoset cerebrum began to indent the lateral fissure and hippocampal sulcus in gestational week (GW) 12, and then the following sulci emerged: the callosal and calcarine sulci on GW 15; the superior temporal sulcus on GW 17; and the circular and occipitotemporal sulci on GW 18. The degree of cortical convolution was evaluated quantitatively based on 2D MRI slices by the gyrification index (GI) and based on 3D MRI data by sulcation index (SI). Both the mean GI and SI increased from GW 16, and were closely correlated with the cortical volume and the cortical surface area during fetal periods (their correlation coefficients marked more than 0.95). After birth, both the mean GI and SI decreased slightly by 2years of age, whereas the cortical volume and surface area continuously increased. Notably, histological analysis showed that the outer subventricular zone (oSVZ) in non-sulcal regions was thicker than that in the presumptive calcarine sulcal region on GW 13, preceding the infolding of the calcarine sulcus. The present results showed definite sulcal infolding on the cerebral cortical surface of the marmosets, with similar pattern and sequence of their emergences to other higher-order primates such as macaques and humans. Differential expansion of the oSVZ may be involved in gyral convolution and sulcal infolding in the developing cerebrum.

Differential cellular localization of antioxidant enzymes in the trigeminal ganglion.

Because of its high oxygen demands, neural tissue is predisposed to oxidative stress. Here, our aim was to clarify the cellular localization of antioxidant enzymes in the trigeminal ganglion. We found that the transcriptional factor Sox10 is localized exclusively in satellite glial cells (SGCs) in the adult trigeminal ganglion. The use of transgenic mice that express the fluorescent protein Venus under the Sox10 promoter enabled us to distinguish between neurons and SGCs. Although both superoxide dismutases 1 and 2 were present in the neurons, only superoxide dismutase 1 was identified in SGCs. The enzymes relevant to hydrogen peroxide degradation displayed differential cellular localization, such that neurons were endowed with glutathione peroxidase 1 and thioredoxin 2, and catalase and thioredoxin 2 were present in SGCs. Our immunohistochemical finding showed that only SGCs were labeled by the oxidative damage marker 8-hydroxy-2'-deoxyguanosine, which indicates that the antioxidant systems of SGCs were less potent. The transient receptor potential vanilloid subfamily member 1 (TRPV1), the capsaicin receptor, is implicated in inflammatory hyperalgesia, and we demonstrated that topical capsaicin application causes short-lasting mechanical hyperalgesia in the face. Our cell-based assay revealed that TRPV1 agonist stimulation in the presence of TRPV1 overexpression caused reactive oxygen species-mediated caspase-3 activation. Moreover, capsaicin induced the cellular demise of primary TRPV1-positive trigeminal ganglion neurons in a dose-dependent manner, and this effect was inhibited by a free radical scavenger and a pancaspase inhibitor. This study delineates the localization of antioxidative stress-related enzymes in the trigeminal ganglion and reveals the importance of the pivotal role of reactive oxygen species in the TRPV1-mediated caspase-dependent cell death of trigeminal ganglion neurons. Therapeutic measures for antioxidative stress should be taken to prevent damage to trigeminal primary sensory neurons in inflammatory pain disorders.

Atlas of the developing brain of the marmoset monkey constructed using magnetic resonance histology.

The developmental anatomy of the brain is largely directed by neural-based cues. Despite this knowledge, the developmental trajectory of the primate brain has not yet been fully characterized. To realize this goal, the advance in noninvasive imaging methods and new brain atlases are essential. The common marmoset (Callithrix jacchus), a small New World primate, is widely used in neuroscience research. The recent introduction of transgenic techniques has enabled the marmoset to be used as a genetically modifiable primate model for brain development. Here, a magnetic resonance histology technique involving the use of ultra-high-resolution ex vivo magnetic resonance imaging (MRI) was performed to identify the developmental anatomy of the marmoset brain at different time points from gestational week 8 through to birth. The data allowed the generation of a multidimensional atlas of brain structures at different developmental stages. Furthermore, in utero MRI techniques were developed to noninvasively monitor brain development during the embryonic and fetal stages. The multidimensional atlas and the MRI tools developed herein are anticipated to further our understanding of the developing primate brain.

Effect of κ-opioid receptor agonist on the growth of non-small cell lung cancer (NSCLC) cells.

BACKGROUND: It is becoming increasingly recognised that opioids are responsible for tumour growth. However, the effects of opioids on tumour growth have been controversial. METHODS: The effects of κ-opioid receptor (KOR) agonist on the growth of non-small cell lung cancer (NSCLC) cells were assessed by a cell proliferation assay. Western blotting was performed to ascertain the mechanism by which treatment with KOR agonist suppresses tumour growth. RESULTS: Addition of the selective KOR agonist U50,488H to gefitinib-sensitive (HCC827) and gefitinib-resistant (H1975) NSCLC cells produced a concentration-dependent decrease in their growth. These effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. Furthermore, the growth-inhibitory effect of gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. With regard to the inhibition of tumour growth, the addition of U50, 488H to H1975 cells produced a concentration-dependent decrease in phosphorylated-glycogen synthase kinase 3ß (p-GSK3ß). CONCLUSION: The present results showed that stimulation of KOR reduces the growth of gefitinib-resistant NSCLC cells through the activation of GSK3ß.

Host-derived MMP-13 exhibits a protective role in lung metastasis of melanoma cells by local endostatin production.

BACKGROUND: Although matrix metalloproteinases (MMPs) are implicated in tumourigenesis and cancer progression, the role of MMP-13 in melanoma cell metastases is poorly understood. METHODS: Lung metastases of mouse melanoma B16BL6 cells were analysed in MMP-13 knockout (KO) and wild-type (WT) mice after intravenous injection. The mRNA and protein expression of MMP-13 in lung tissues was analysed by RT-PCR, real-time PCR, immunoblotting and immunohistochemistry. The expression of SDF-1α, CXCR4 and endostatin, and effects of endostatin to cultured melanoma cells and lung metastases were also studied. RESULTS: Lung metastases of B16BL6 cells were significantly higher by 2.5-5.7-fold in MMP-13 KO mice than in WT mice. The expression of MMP-13 in WT mouse lung tissue was stimulated on day 1 after intravenous injection of the melanoma cells and MMP-13 was immunolocalised to vascular endothelial cells in the lungs. Endostatin formation, but not degradation of SDF-1α, in the lung tissue was associated with reduced lung metastasis in WT mice. Endostatin significantly inhibited migration of B16BL6 cells in monolayer wounding assay and remarkably suppressed Matrigel invasion and transendothelial invasion of the cells. In addition, lung metastases of melanoma cells in MMP-13 KO mice were reduced by intraperitoneal administration of endostatin. CONCLUSION: Our results suggest that MMP-13 is overproduced by endothelial cells in the lungs with melanoma cells and has a protective role in lung metastasis by local generation of endostatin.

Population-averaged standard template brain atlas for the common marmoset (Callithrix jacchus).

Advanced magnetic resonance (MR) neuroimaging analysis techniques based on voxel-wise statistics, such as voxel-based morphometry (VBM) and functional MRI, are widely applied to cognitive brain research in both human subjects and in non-human primates. Recent developments in imaging have enabled the evaluation of smaller animal models with sufficient spatial resolution. The common marmoset (Callithrix jacchus), a small New World primate species, has been widely used in neuroscience research, to which voxel-wise statistics could be extended with a species-specific brain template. Here, we report, for the first time, a tissue-segmented, population-averaged standard template of the common marmoset brain. This template was created by using anatomical T(1)-weighted images from 22 adult marmosets with a high-resolution isotropic voxel size of (0.2 mm)(3) at 7-Tesla and DARTEL algorithm in SPM8. Whole brain templates are available at International Neuroinformatics Japan Node website, http://brainatlas.brain.riken.jp/marmoset/.

Reconsideration of insulin signals induced by improved laboratory animal diets, Japanese and American diets, in IRS-2 deficient mice.

Current Japanese and American diets and Japanese diet immediately after the War were converted to laboratory animal diets. As a result, current laboratory animal diet (CA-1, CLEA) unexpectedly resembled the diet of Japanese after the War. This is considered to result in an under-evaluation of diabetes research using laboratory animals at present. Therefore, changes in insulin signals caused by current Japanese and American diets were examined using IRS-2 deficient mice ( IRS2(-/-) mice) and mechanisms of aggravation of type 2 diabetes due to modern diets were examined. IRS2(-/-) mice at 6 weeks of age were divided into three groups: Japanese diet (Jd) group, American diet (Ad) group and CA-1 diet [regular diet (Rd)] group. Each diet was given to the dams from 7 days before delivery. When the IRS2(-/-) mice reached 6 weeks of age, the glucose tolerance test (GTT), insulin tolerance test (ITT) and organ sampling were performed. The sampled organs and white adipose tissue were used for analysis of RNA, enzyme activity and tissues. In GTT and ITT, the Ad group showed worse glucose tolerance and insulin resistance than the Rd group. Impaired glucose tolerance of the Jd group was the same as that of the Rd group, but insulin resistance was worse than in the Rd group. These results were caused an increase in fat accumulation and adipocytes in the peritoneal cavity by lipogenic enzyme activity in the liver and muscle, and the increase in TNFalpha of hypertrophic adipocyte origin further aggravated insulin resistance and the increase in resistin also aggravated the impaired glucose tolerance, leading to aggravation of type 2 diabetes. The Japanese and American diets given to the IRS2(-/-) mice, which we developed, showed abnormal findings in some IRS2(-/-) mice but inhibited excessive reactions of insulin signals as diets used in ordinary nutritional management.

The intestinal epithelium compensates for p53-mediated cell death and guarantees organismal survival.

Mdm2 is the major inhibitor of the p53 tumor suppressor. Loss of Mdm2 in mice or in specific tissues of the mouse always yields p53-dependent lethal phenotypes. However, the role of Mdm2 in tissues with high turnover capacity is unknown. We have engineered mice lacking Mdm2 in the intestinal epithelium using the Cre/LoxP system. Loss of Mdm2 (Mdm2(intDelta)) results in viable animals, but neonates display multiple intestinal abnormalities such as hyperplasia, enterocyte vacuolization, and inflammation. These defects correlate with a drastic increase in p53-dependent apoptosis in highly proliferative and differentiated cells. Unexpectedly, the observed phenotypes disappear with age. The tissue selects against Mdm2-null cells and increases its proliferative capacity. Additionally, the intestinal stem and progenitor cell populations are enriched leading to an increase in crypt fission events. Enhanced proliferation is achieved by activation of the canonical Wnt and EGFR-mediated Ras/MAPK pathways. While Mdm2 is a critical inhibitor of p53 in the intestinal epithelium, the tissue employs a series of processes that compensate for cell death.

In vivo imaging in humanized mice.

The radiological modalities that are currently utilized as critical components in clinical medicine have also been adapted to small-animal imaging, among which are ultrasound imaging, X-ray computerized tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT). Optical imaging techniques such as bioluminescence imaging (BLI) and fluorescence imaging (FLI) are approaches that are commonly used in small animals. Longitudinal surveys of living (i.e., nonsacrificed) animal models with these modalities provide some clues for the development of clinical applications. The techniques are absolutely essential for translational research. However, there are currently few tools available with sufficient spatial or temporal resolution ideal for all experimental studies. In this chapter, we provide a rationale and techniques for visualizing target cells in living small animals and an overview of the advantages and limitations of current imaging technology. Finally, we introduce a humanized mouse and a novel in vivo imaging system that we have developed. We also discuss real-time observations of reconstructs and clinical manifestations.

Stochastic approach to molecular interactions and computational theory of metabolic and genetic regulations.

The underlying molecular mechanisms of metabolic and genetic regulations are computationally identical and can be described by a finite state Markov process. We establish a common computational model for both regulations based on the stationary distribution of the Markov process with the aim of establishing a unified, quantitative model of general biological regulations. Various existing results regarding intracellular regulations are derived including the classical Michaelis-Menten equation and its generalization to more complex allosteric enzymes in a systematic way. The notion of probability flow is introduced to distinguish the equilibrium stationary distribution from the non-equilibrium one; it plays a crucial role in the analysis of stationary state equations. A graphical criterion to guarantee the existence of an equilibrium stationary distribution is derived, which turns out to be identical to the classical Wegscheider condition. Simple graphical methods to compute the equilibrium and non-equilibrium stationary distributions are derived based crucially on the probability flow, which dramatically simplifies the classical methods still used in enzymology.