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High-fat diet (HFD) induces low-grade chronic inflammation and insulin resistance. However, little is known about the mechanism underlying HFD-induced chronic inflammation in peripheral insulin-responsive tissues. Here, we show that colonic pro-inflammatory macrophages regulate insulin sensitivity under HFD conditions. To investigate the pathophysiological role of colonic macrophages, we generated macrophage-specific chemokine (C-C Motif) receptor 2 (Ccr2) knockout (M-Ccr2KO) and intestinal epithelial cell-specific tamoxifen-inducible Ccl2 knockout (Vil-Ccl2KO) mice. Both strains exhibited similar body weight to control under HFD. However, they exhibited decreased infiltration of colonic pro-inflammatory macrophages, decreased intestinal permeability, and inactivation of the colonic inflammasome. Interestingly, they showed significantly improved glucose tolerance and insulin sensitivity with decreased chronic inflammation of adipose tissue. Therefore, inhibition of pro-inflammatory macrophage infiltration prevents HFD-induced insulin resistance and could be a novel therapeutic approach for type 2 diabetes.

The canonical Wnt signaling pathway plays a major role in the regulation of embryogenesis and organogenesis, where signal strength-dependent cellular responses are of particular importance. To assess Wnt signal levels in individual cells, and to circumvent the integration site-dependent bias shown in previous Wnt reporter lines, we constructed a new Wnt signal reporter mouse line R26-WntVis. Heptameric TCF/LEF1 binding sequences were combined with a viral minimal promoter to confer a graded response to the reporter depending on Wnt signal strengths. The histone H2B-EGFP fusion protein was chosen as the fluorescent reporter to facilitate single-cell resolution analyses. This WntVis reporter gene was then inserted into the ROSA26 locus in an orientation opposite to that of the endogenous gene. The R26-WntVis allele was introduced into Wnt3a(-/-) and Wnt3a(vt/-) mutant mouse embryos and compared with wild-type embryos to assess its performance. The R26-WntVis reporter was activated in known Wnt-dependent tissues and responded in a graded fashion to signal intensity. This analysis also indicated that the major Wnt activity early in embryogenesis switched from Wnt3 to Wnt3a around E7.5. The R26-WntVis mouse line will be widely useful for the study of Wnt signal-dependent processes.

HEV LPA receptors differentially regulate lymphocyte recirculation, Naive lymphocytes continuously migrate from the blood into lymph nodes (LNs) via high endothelial venules (HEVs). To extravasate from the HEVs, lymphocytes undergo multiple adhesion steps, including tethering, rolling, firm adhesion and transmigration. We previously showed that autotaxin (ATX), an enzyme that generates lysophosphatidic acid (LPA), is highly expressed in HEVs, and that the ATX/LPA axis plays an important role in the lymphocyte transmigration across HEVs. However, the detailed mechanism underlying this axis’s involvement in lymphocyte transmigration has remained ill-defined. Here, we show that two LPA receptors, LPA4 and LPA6, are selectively expressed on HEV endothelial cells (ECs) and that LPA4 plays a major role in the lymphocyte transmigration across HEVs in mice. In the absence of LPA4 expression, lymphocytes accumulated heavily within the HEV EC layer, compared to wild-type (WT) mice. This accumulation was also observed in the absence of LPA6 expression, but it was less pronounced. Adoptive transfer experiments using WT lymphocytes revealed that the LPA4 deficiency in ECs specifically compromised the lymphocyte transmigration process, whereas the effect of LPA6 deficiency was not significant. These results indicate that the signals evoked in HEV ECs via the LPA4 and LPA6 differentially regulate lymphocyte extravasation from HEVs in the peripheral LNs.

Here we report a case of 57-year-old woman with renal angiomyolipoma associated with tuberous sclerosis complex involving inferior vena cava thrombus. We could perform less invasive nephrectomy with thrombectomy because everolimus administration reduced the inferior vena cava thrombus. To the best of our knowledge, this is the first report the use of everolimus before performing surgery to treat renal angiomyolipoma with inferior vena cava thrombus.

The Ovarian Carcinoma Immunoreactive Antigen Domain (OCIAD) containing proteins, Asrij/OCIAD1 and OCIAD2 are misexpressed in several human blood cell disorders. In mice, Asrij regulates bone marrow hematopoietic stem cell (BM HSC) quiescence and its depletion leads to a myeloproliferative disorder. Owing to the evolutionary relatedness, adjacent location and conserved role of the OCIAD1/2 proteins in regulating signaling, we expected a role for OCIAD2 in vertebrate hematopoiesis. In agreement with data mining results, we find OCIAD2 is expressed in wild type mouse CD150+ HSCs and various BM subpopulations, however its expression is lower and less heterogeneous compared to Asrij. To analyze in vivo function, we generated the first ociad2 knockout (KO, ociad2-/-) mice and show that they are viable, fertile and without overt abnormalities. However some organs atrophied with age. Flow cytometry analyses showed that OCIAD2 is dispensable for maintaining HSPCs. However, myeloid and B-cell frequencies were significantly increased, suggesting a functional role for OCIAD2 during lineage specification and maintenance. Further, transcript and protein analyses indicate that Asrij compensates for loss of OCIAD2 and vice versa. We suggest that the generic conserved requirement for Asrij in steady-state hematopoiesis is augmented by vertebrate duplication of OCIAD2.

Holocrine secretion is a specific mode of secretion involving secretion of entire cytoplasmic materials with remnants of dead cells, as observed in multicellular exocrine glands of reptiles, birds, and mammals. Here, we found that sebaceous glands in mice, representative of multicellular exocrine glands of mammals, exhibit a form of polarized stratified epithelium equipped with tight junctions (TJs), and found that holocrine secretion occurred outside the TJ barriers. Sebaceous glands share characteristics of stratified epithelia with interfollicular epidermis, including basal-layer-restricted cell proliferation, TJ barrier formation at a specific single layer of cells with apico-basolateral plasma membrane polarity, and cell death outside the TJ barrier. Knockout of claudin-1, a transmembrane adhesive protein in TJs, in mice caused leakage of the TJ barrier in sebaceous glands and incomplete degradation of the plasma membrane and nuclei during holocrine secretion. Claudin-1 knockout resulted in the accumulation of incompletely degenerated sebocytes in sebaceous ducts, suggesting that the TJ barrier was necessary for differentiation of holocrine secretion. The redefinition of sebaceous glands as TJ-forming stratified epithelia provides an important framework to understand the molecular mechanism of holocrine secretion.

Obesity is characterized by an expansion of white adipose tissue (WAT) mass, which mainly consists of adipocytes. During the commitment and differentiation of adipocytes, PPARγ functions as a key transcriptional factor for adipogenesis, and is associated with its suppressive coregulator, TAZ. Previous studies have shown the importance of TAZ in adipogenesis using an in vitro model; however, the understanding of its role in adipogenesis in vivo remains limited. Here, we report a unique obese mouse model that is associated with TAZ downregulation, which arose from the overexpression of Yap, a Taz paralog. YAP activation facilitated Hippo signaling feedback, which induced a compensatory reduction in YAP, subsequently neutralizing its functional activity. This feedback also induced TAZ suppression and exclusion from the nucleus. In Yap transgenic mice, TAZ downregulation in adipose stem cells activated PPARγ, leading to their differentiation into mature adipocytes and consequently increased adipose tissue. These results highlight the in vivo necessity of TAZ for adipocyte commitment and differentiation, which could provide insight into anti-obesity therapeutics.

Significance Each neuron forms a single axon and multiple dendrites, and this configuration is important for wiring the brain. How only a single axon extends from a neuron, however, remains unknown. This study demonstrates that CAMSAP3, a protein that binds the minus-end of microtubules, preferentially localizes along axons in hippocampal neurons. Remarkably, mutations of CAMSAP3 lead to production of multiple axons in these neurons. In attempts to uncover mechanisms underlying this abnormal axon extension, the authors found that CAMSAP3-anchored microtubules escape from acetylation, a process mediated by α-tubulin acetyltransferase-1, and depletion of this enzyme abolishes abnormal axon formation in CAMSAP3 mutants. These findings reveal that CAMSAP3 controls microtubule dynamics, preventing tubulin acetylation; this mechanism is required for single-axon formation., The molecular mechanisms that guide each neuron to become polarized, forming a single axon and multiple dendrites, remain unknown. Here we show that CAMSAP3 (calmodulin-regulated spectrin-associated protein 3), a protein that regulates the minus-end dynamics of microtubules, plays a key role in maintaining neuronal polarity. In mouse hippocampal neurons, CAMSAP3 was enriched in axons. Although axonal microtubules were generally acetylated, CAMSAP3 was preferentially localized along a less-acetylated fraction of the microtubules. CAMSAP3-mutated neurons often exhibited supernumerary axons, along with an increased number of neurites having nocodazole-resistant/acetylated microtubules compared with wild-type neurons. Analysis using cell lines showed that CAMSAP3 depletion promoted tubulin acetylation, and conversely, mild overexpression of CAMSAP3 inhibited it, suggesting that CAMSAP3 works to retain nonacetylated microtubules. In contrast, CAMSAP2, a protein related to CAMSAP3, was detected along all neurites, and its loss did not affect neuronal polarity, nor did it cause increased tubulin acetylation. Depletion of α-tubulin acetyltransferase-1 (αTAT1), the key enzyme for tubulin acetylation, abolished CAMSAP3 loss-dependent multiple-axon formation. These observations suggest that CAMSAP3 sustains a nonacetylated pool of microtubules in axons, interfering with the action of αTAT1, and this process is important to maintain neuronal polarity.

Grb2-associated regulator of Erk/MAPK (GAREM), is an adaptor protein related to the several cell growth factor receptor-signaling. The GAREM family has two subtypes, GAREM1 and GAREM2, both encoded in the human and mouse genome. Recent genome-wide research identified GAREM2 as a candidate of neurodegenerative diseases. Here, we use knockout (KO) mice to show the role of GAREM2, that is highly expressed in the brain. According to the comprehensive behavioral battery, they exhibited less anxiety both in elevated plus maze and open field tests, mildly increased social approaching behavior in the reciprocal social interaction test, and longer latency to immobility in the tail suspension test as compared to wild-type (WT). Additionally, the extension of neurites in the primary cultured neurons was suppressed in ones derived from GAREM2 KO mice. Furthermore, we also identified Intersectin, as a binding partner of GAREM2 in this study. Intersectin is also a multi-domain adaptor protein that regulates endocytosis and cell signaling, which can potentially alter the subcellular localization of GAREM2. The important molecules, such as the neurotrophin receptor and Erk family, that are involved in the signaling pathway of the neural cell growth in the mouse brain, have been reported to participate in emotional behavior. As GAREM plays a role in the cellular growth factor receptor signaling pathway, GAREM2 may have a common role related to the transduction of Erk signaling in the higher brain functions.

Significance IFN regulatory factor 3 (IRF3) is one of the most well-characterized transcription factors involved in the regulation of innate immune responses. Many studies have used conventional Irf3 -deficient mice to analyze the functions of IRF3 in immunity and other biological systems; however, these mice were found to carry another null mutation in the Bcl2l12 gene, which has confounded the analyses of these mice. For this study, we generated conditional Irf3 -deficient mice while leaving the Bcl2l12 gene intact. Conditional ablation of IRF3 in myeloid cells showed increased survival when challenged with endotoxin-induced shock, indicating a myeloid cell-specific role of IRF3. The IRF3 conditional knockout mouse may be a useful tool to identify cell type-specific functions of this transcription factor.

Rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) is a cytoskeletal protein that promotes directional cell migration and angiogenesis in vitro and is implicated in human carcinomas and coronary artery disease. To study the role of Rudhira during development in vivo, we generated the first knockout mouse for rudhira and show that Rudhira is essential for mouse development. Rudhira null embryos die at embryonic day (E) 9.5 accompanied by severe vascular patterning defects in embryonic and extra-embryonic tissues. To identify the molecular processes downstream of rudhira, we analyzed the transcriptome of intact knockout yolk sacs. Genome-wide transcriptome analysis showed that Rudhira functions in angiogenesis and its related processes such as cell adhesion, extracellular matrix organization, peptidase activity and TGFβ signaling. Since Rudhira is also expressed in endothelial cells (ECs), we further generated Tie2Cre-mediated endothelial knockout (CKO) of rudhira. CKO embryos survive to E11.5 and similar to the global knockout, display gross vascular patterning defects, showing that endothelial Rudhira is vital for development. Further, Rudhira knockdown ECs in culture fail to sprout in a spheroid-sprouting assay, strongly supporting its role in vascular patterning. Our study identifies an essential role for Rudhira in blood vessel remodeling and provides a mouse model for cardiovascular development.

The early post-implantation mouse embryo changes dramatically in both size and shape. These morphological changes are based on characteristic cellular behaviors, including cell growth and allocation. To perform clonal analysis, we established a Cre/loxP-based reporter mouse line, R26R-ManGeKyou, that enables clonal labeling with multiple colors. We also developed a novel ImageJ plugin, LP-Clonal, for quantitative measurement of the tilt angle of clonal cluster shape, enabling identification of the direction of cluster expansion. We carried out long-term and short-term lineage tracking. We also performed time-lapse imaging to characterize cellular behaviors using R26-PHA7-EGFP and R26R-EGFP. These images were subjected to quantitative image analyses. We found that the proximal visceral endoderm overlying the extra-embryonic ectoderm shows coherent cell growth in a proximal–anterior to distal–posterior direction. We also observed that directional cell migration is coupled with cell elongation in the anterior region. Our observations suggest that the behaviors of visceral endoderm cells vary between regions during peri-implantation stages.

Precise regulation of migration termination is crucial for the establishment of cortical laminar structure. However, little is known about how neurons terminate migration. Here we analyzed the role of Plexin A2 and A4 (PlxnA2/A4) receptors and Semaphorin 6A (Sema6A) ligand on this process, focusing on an interaction between migrating neurons and their substrates. In PlxnA2/A4 double-knockout as well as Sema6A mutant mice, a superficial layer neurons subset ectopically invades layer I. This abnormality manifests at a time when these neurons arrive at their destinations. PlxnA2/A4 proteins are abundant on their leading processes, while Sema6A mRNA is enriched in somata of radial glial cells. Cell-targeted gene expression and conditional knockouts confirm that these molecules are required in their respective cells. Collectively, these results suggest that the well-timed appearance of the repulsive signaling mediated by Sema6A-PlxnA2/A4 weakens the interaction between migrating neurons and radial glial cells, which leads to migration termination.

SummaryRett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Although emerging evidence suggests that MeCP2 deficiency is associated with dysregulation of mechanistic target of rapamycin (mTOR), which functions as a hub for various signaling pathways, the mechanism underlying this association and the molecular pathophysiology of RTT remain elusive. We show here that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs) as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that miR-199a positively controls mTOR signaling by targeting inhibitors for mTOR signaling. miR-199a and its targets have opposite effects on mTOR activity, ameliorating and inducing RTT neuronal phenotypes, respectively. Furthermore, genetic deletion of miR-199a-2 led to a reduction of mTOR activity in the brain and recapitulated numerous RTT phenotypes in mice. Together, these findings establish miR-199a as a critical downstream target of MeCP2 in RTT pathogenesis by linking MeCP2 with mTOR signaling.

Oa1 is the casual gene for ocular albinism-1 in humans. The gene product OA1, alternatively designated as GPR143, belongs to G-protein coupled receptors. It has been reported that OA1 is a specific receptor for 3, 4-dihydroxy- L-phenylalanine (DOPA) in retinal pigmental epithelium where DOPA facilitates the pigmentation via OA1 stimulation. We have recently shown that OA1 mediates DOPA-induced depressor response in rat nucleus tractus solitarii. However, the distribution and function of OA1 in other regions are largely unknown. We have generated oa1 knockout mice and examined OA1 expression in both neuronal and non-neuronal tissues by immunohistochemical analyses using anti-mouse OA1 monoclonal antibodies. In the telencephalon, OA1 was expressed in cerebral cortex and hippocampus. Predominant expression of OA1 was observed in the pyramidal neurons in these regions. OA1 was also expressed in habenular nucleus, hypothalamus, substantia nigra, and medulla oblongata. The expression of OA1 in the nucleus tractus solitarii of medulla oblongata may support the reduction of blood pressure by the microinjection of DOPA into this region. Outside of the nervous system, OA1 was expressed in heart, lung, liver, kidney and spleen. Abundant expression was observed in the renal tubules and the splenic capsules. These peripheral regions are innervated by numerous sympathetic nerve endings. In addition, substantia nigra contains a large population of dopaminergic neurons. Thus, the immunohistochemical analyses suggest that OA1 may modulate the monoaminergic functions in both peripheral and central nervous systems.

Epigenetic regulation is required to ensure the precise spatial and temporal pattern of gene expression that is necessary for embryonic development. Although the roles of some epigenetic modifications in embryonic development have been investigated in depth, the role of methylation at lysine 79 (H3K79me) is poorly understood. Dot1L, a unique methyltransferase for H3K79, forms complexes with distinct sets of co-factors. To further understand the role of H3K79me in embryogenesis, we generated a mouse knockout of Mllt10, the gene encoding Af10, one Dot1L complex co-factor. We find homozygous Mllt10 knockout mutants (Mllt10-KO) exhibit midline facial cleft. The midfacial defects of Mllt10-KO embryos correspond to hyperterolism and are associated with reduced proliferation of mesenchyme in developing nasal processes and adjacent tissue. We demonstrate that H3K79me level is significantly decreased in nasal processes of Mllt10-KO embryos. Importantly, we find that expression of AP2α, a gene critical for midfacial development, is directly regulated by Af10-dependent H3K79me, and expression AP2α is reduced specifically in nasal processes of Mllt10-KO embryos. Suppression of H3K79me completely mimicked the Mllt10-KO phenotype. Together these data are the first to demonstrate that Af10-dependent H3K79me is essential for development of nasal processes and adjacent tissues, and consequent midfacial formation.

NIPAL4 is one of the causative genes for autosomal recessive congenital ichthyosis. However, the role of NIPAL4 in skin barrier formation and the molecular mechanism of ichthyosis pathology caused by NIPAL4 mutations, have not yet been determined. Here, we found that Nipal4-knockout (KO) mice exhibited neonatal lethality due to skin barrier defects. Histological analyses showed several morphological abnormalities in the Nipal4-KO epidermis, including impairment of lipid multilayer structure formation, hyperkeratosis, immature keratohyalin granules, and developed heterochromatin structures. The levels of the skin barrier lipid acylceramide were decreased in Nipal4-KO mice. Expression of genes involved in skin barrier formation normally increases during keratinocyte differentiation, in which chromatin remodeling is involved. However, the induction of Krt1, Lor, Flg, Elovl1, and Dgat2 was impaired in Nipal4-KO mice. NIPAL4 is a putative Mg2+ transporter, and Mg2+ concentration in differentiated keratinocytes of Nipal4-KO mice was indeed lower than that of wild-type mice. Our results suggest that low Mg2+ concentration causes aberration in the proper chromatin remodeling process, which in turn leads to failure of differentiation-dependent gene induction in keratinocytes. Our findings provide insights into Mg2+-dependent regulation of gene expression and skin barrier formation during keratinocyte differentiation.

Patients who undergo hemodialysis often suffer from cardiovascular disease (CVD), and evaluation of coronary artery calcification is extremely important. These evaluations are typically conducted using a noninvasive method including electron beam computed tomography (CT) or multi-detector CT, and the Agatston method to calculate the coronary artery calcification score (CACS). However, it is difficult to use for patients undergoing dialysis. Because patients undergoing dialysis is too strong in coronary artery calcification, and results become incorrect. Therefore, we were looking for a calcified evaluation place peculiar to a patients undergoing dialysis. We obtained pelvic artery calcification scores (PACS) using a 64-row multi-slice CT to assess the presence of calcification within a triangular space bordered by bordered by osseous structure. We used the Agatston method to calculate PACS. We compared male patients undergoing dialysis with male patients with normal renal function. Patients undergoing hemodialysis had a significantly higher incidence of pelvic artery calcification than normal controls (79.7% vs. 5.5%). In the dialysis group, CACS was 1660.2 (0-9056.1), and PACS was 48.8 (0-2943.1). We found a correlation between PACS and CACS and between PACS and dialysis period. We found penile artery calcification in male patients undergoing hemodialysis was more than normal controls, and it was possible to quantify PACS using the Agatston method. This study suggested the possibility that PACS became the vascular calcification evaluation method of the hemodialysis patient.

Complete recovery using interferon therapy in Japanese hepatitis C virus (HCV)-infected dialysis patients is difficult to achieve because70 % of the HCV genotypes observed in Japan are type 1. In 2016, new direct-acting antiviral drugs against HCV genotype 1 were reported to be effective and safe for HCV-infected hemodialysis patients. Although new direct antiviral therapy has become available, no large-scale studies evaluating the status of HCV infection in Japanese hemodialysis patients have been conducted since 2007. Therefore, we conducted a questionnaire survey to determine the current status of HCV infection in patients. Our results indicated that the HCV antibody prevalence was 5.02 %, and HCV RNA prevalence was 72.3 %. Genotype testing revealed that 62.1 % of patients had HCV genotype 1. New direct antiviral therapy may improve the survival of Japanese HCV-infected dialysis patients.

In addition to thymic stromal lymphopoietin and IL-33, IL-25 is known to induce TWe investigated the contribution of IL-25 to CHS using Il25CHS was evaluated by means of measurement of ear skin thickness in mice after fluorescein isothiocyanate painting. Skin dendritic cell (DC) migration, hapten-specific TIn contrast to thymic stromal lymphopoietin, we found that IL-25 was not essential for skin DC migration or hapten-specific TOur results identify a novel IL-25 inflammatory pathway involved in induction of T

Podocytes form the outer part of the glomerular filter, where they have to withstand enormous transcapillary filtration forces driving glomerular filtration. Detachment of podocytes from the glomerular basement membrane precedes most glomerular diseases. However, little is known about the regulation of podocyte adhesion in vivo. Thus, we systematically screened for podocyte-specific focal adhesome (FA) components, using genetic reporter models in combination with iTRAQ-based mass spectrometry. This approach led to the identification of FERM domain protein EPB41L5 as a highly enriched podocyte-specific FA component in vivo. Genetic deletion of Epb41l5 resulted in severe proteinuria, detachment of podocytes, and development of focal segmental glomerulosclerosis. Remarkably, by binding and recruiting the RhoGEF ARGHEF18 to the leading edge, EPB41L5 directly controls actomyosin contractility and subsequent maturation of focal adhesions, cell spreading, and migration. Furthermore, EPB41L5 controls matrix-dependent outside-in signaling by regulating the focal adhesome composition. Thus, by linking extracellular matrix sensing and signaling, focal adhesion maturation, and actomyosin activation EPB41L5 ensures the mechanical stability required for podocytes at the kidney filtration barrier. Finally, a diminution of EPB41L5-dependent signaling programs appears to be a common theme of podocyte disease, and therefore offers unexpected interventional therapeutic strategies to prevent podocyte loss and kidney disease progression.

PKA phosphorylates multiple molecules involved in calcium (Ca2+) handling in cardiac myocytes and is considered to be the predominant regulator of β-adrenergic receptor–mediated enhancement of cardiac contractility; however, recent identification of exchange protein activated by cAMP (EPAC), which is independently activated by cAMP, has challenged this paradigm. Mice lacking Epac1 (Epac1 KO) exhibited decreased cardiac contractility with reduced phospholamban (PLN) phosphorylation at serine-16, the major PKA-mediated phosphorylation site. In Epac1 KO mice, intracellular Ca2+ storage and the magnitude of Ca2+ movement were decreased; however, PKA expression remained unchanged, and activation of PKA with isoproterenol improved cardiac contractility. In contrast, direct activation of EPAC in cardiomyocytes led to increased PLN phosphorylation at serine-16, which was dependent on PLC and PKCε. Importantly, Epac1 deletion protected the heart from various stresses, while Epac2 deletion was not protective. Compared with WT mice, aortic banding induced a similar degree of cardiac hypertrophy in Epac1 KO; however, lack of Epac1 prevented subsequent cardiac dysfunction as a result of decreased cardiac myocyte apoptosis and fibrosis. Similarly, Epac1 KO animals showed resistance to isoproterenol- and aging-induced cardiomyopathy and attenuation of arrhythmogenic activity. These data support Epac1 as an important regulator of PKA-independent PLN phosphorylation and indicate that Epac1 regulates cardiac responsiveness to various stresses.

LMTK3 belongs to the LMTK family of protein kinases that are predominantly expressed in the brain. Physiological functions of LMTK3 and other members of the LMTK family in the CNS remain unknown. In this study, we performed a battery of behavioral analyses usingLmtk3−/−mice and showed that these mice exhibit abnormal behaviors, including pronounced locomotor hyperactivity, reduced anxiety behavior, and decreased depression-like behavior. Concurrently, the dopamine metabolite levels and dopamine turnover rate are increased in the striata ofLmtk3−/−mice compared with wild-type controls. In addition, using cultured primary neurons fromLmtk3−/−mice, we found that LMTK3 is involved in the endocytic trafficking ofN-methyl-d-aspartate receptors, a type of ionotropic glutamate receptor. Altered membrane traffic of the receptor inLmtk3−/−neurons may underlie behavioral abnormalities in the mutant animals. Together, our data suggest that LMTK3 plays an important role in regulating locomotor behavior in mice.

SummarySystems-level identification and analysis of cellular circuits in the brain will require the development of whole-brain imaging with single-cell resolution. To this end, we performed comprehensive chemical screening to develop a whole-brain clearing and imaging method, termed CUBIC (clear, unobstructed brain imaging cocktails and computational analysis). CUBIC is a simple and efficient method involving the immersion of brain samples in chemical mixtures containing aminoalcohols, which enables rapid whole-brain imaging with single-photon excitation microscopy. CUBIC is applicable to multicolor imaging of fluorescent proteins or immunostained samples in adult brains and is scalable from a primate brain to subcellular structures. We also developed a whole-brain cell-nuclear counterstaining protocol and a computational image analysis pipeline that, together with CUBIC reagents, enable the visualization and quantification of neural activities induced by environmental stimulation. CUBIC enables time-course expression profiling of whole adult brains with single-cell resolution.

Kitamura et al. identify NLRC4 as causing familial cold autoinflammatory syndrome using whole exome sequencing on a family with multiple affected family members. They identify a mutation in the NOD domain and show that the mutant protein increases Nlrc4 oligomerization and is associated with increased IL-1β. Transgenic mice with the same NLRC4 mutation are shown to develop a similar FCAS-like syndrome., Autoinflammatory syndromes cause sterile inflammation in the absence of any signs of autoimmune responses. Familial cold autoinflammatory syndrome (FCAS) is characterized by intermittent episodes of rash, arthralgia, and fever after exposure to cold stimuli. We have identified a missense mutation in the NLRC4 gene in patients with FCAS. NLRC4 has been known as a crucial sensor for several Gram-negative intracellular bacteria. The mutation in NLRC4 in FCAS patients promoted the formation of NLRC4-containing inflammasomes that cleave procaspase-1 and increase production of IL-1β. Transgenic mice that expressed mutant Nlrc4 under the invariant chain promoter developed dermatitis and arthritis. Inflammation within tissues depended on IL-1β–mediated production of IL-17A from neutrophils but not from T cells. Our findings reveal a previously unrecognized link between NLRC4 and a hereditary autoinflammatory disease and highlight the importance of NLRC4 not only in the innate immune response to bacterial infections but also in the genesis of inflammatory diseases.

The DISC1-interacting protein CAMDI has been suggested to promote radial migration through centrosome regulation. However, its physiological relevance is unclear. Here, we report the generation and characterization of CAMDI-deficient mice. CAMDI-deficient mice exhibit delayed radial migration with aberrant neural circuit formation and psychiatric behaviors including hyperactivity, repetitive behavior, and social abnormality typically observed in autism spectrum disorder patients. Analyses of direct targets of CAMDI identify HDAC6 whose α-tubulin deacetylase activity is inhibited by CAMDI at the centrosome. CAMDI deficiency increases HDAC6 activity, leading to unstable centrosomes with reduced γ-tubulin and acetylated α-tubulin levels. Most importantly, psychiatric behaviors as well as delayed migration are significantly rescued by treatment with Tubastatin A, a specific inhibitor of HDAC6. Our findings indicate that HDAC6 hyperactivation by CAMDI deletion causes psychiatric behaviors, at least in part, through delayed radial migration due to impaired centrosomes.

Summary: Altered DNA methylation plays an important role in the onset and progression of kidney disease. However, little is known about how the changes arise in disease states. Here, we report that KAT5-mediated DNA damage repair is essential for the maintenance of kidney podocytes and is associated with DNA methylation status. Podocyte-specific KAT5-knockout mice develop severe albuminuria with increased DNA double-strand breaks (DSBs), increased DNA methylation of the nephrin promoter region, and decreased nephrin expression. Podocyte KAT5 expression is decreased, whereas DNA DSBs and DNA methylation are increased in diabetic nephropathy; moreover, KAT5 restoration by gene transfer attenuates albuminuria. Furthermore, KAT5 decreases DNA DSBs and DNA methylation at the same nephrin promoter region, which indicates that KAT5-mediated DNA repair may be related to DNA methylation status. These results suggest a concept in which an environment of DNA damage repair, which occurs with decreased KAT5, may affect DNA methylation status. : Hishikawa et al. reveal that KAT5-mediated DNA repair is essential for podocyte maintenance and is related to changes in DNA methylation status. Decreased podocyte KAT5 expression may contribute to the pathophysiology of diabetic nephropathy, suggesting a therapeutic target. Keywords: podocyte, DNA damage repair, DNA methylation, diabetic nephropathy

Background In addition to thymic stromal lymphopoietin and IL-33, IL-25 is known to induce T H 2 cytokine production by various cell types, including T H 2 cells, T H 9 cells, invariant natural killer T cells, and group 2 innate lymphoid cells, involved in T H 2-type immune responses. Because both T H 2-type and T H 17-type cells/cytokines are crucial for contact hypersensitivity (CHS), IL-25 can contribute to this by enhancing T H 2-type immune responses. However, the precise role of IL-25 in the pathogenesis of fluorescein isothiocyanate–induced CHS is poorly understood. Objective We investigated the contribution of IL-25 to CHS using Il25 −/− mice. Methods CHS was evaluated by means of measurement of ear skin thickness in mice after fluorescein isothiocyanate painting. Skin dendritic cell (DC) migration, hapten-specific T H cell differentiation, and detection of IL-1β–producing cells were determined by using flow cytometry, ELISA, and immunohistochemistry, respectively. Results In contrast to thymic stromal lymphopoietin, we found that IL-25 was not essential for skin DC migration or hapten-specific T H cell differentiation in the sensitization phase of CHS. Unexpectedly, mast cell– and non–immune cell–derived IL-25 was important for hapten-specific T H 17 cell–mediated rather than T H 2 cell–mediated inflammation in the elicitation phase of CHS by enhancing T H 17-related, but not T H 2-related, cytokines in the skin. In particular, IL-1β produced by dermal DCs in response to IL-25 was crucial for hapten-specific T H 17 cell activation, contributing to induction of local inflammation in the elicitation phase of CHS. Conclusion Our results identify a novel IL-25 inflammatory pathway involved in induction of T H 17 cell–mediated, but not T H 2 cell–mediated, CHS. IL-25 neutralization can be a potential approach for treatment of CHS.

Global gene deletion studies in mice and humans have established the pivotal role of runt related transcription factor-2 (Runx2) in both intramembranous and endochondral ossification processes during skeletogenesis. In this study, we for the first time generated mice carrying a conditional Runx2 allele with exon 4, which encodes the Runt domain, flanked by loxP sites. These mice were crossed with α1(I)-collagen-Cre or α1(II)-collagen-Cre transgenic mice to obtain osteoblast-specific or chondrocyte-specific Runx2 deficient mice, respectively. As seen in Runx2(-/-) mice, perinatal lethality was observed in α1(II)-Cre;Runx2(flox/flox) mice, but this was not the case in animals in which α1(I)-collagen-Cre was used to delete Runx2. When using double-staining with Alizarin red for mineralized matrix and Alcian blue for cartilaginous matrix, we observed previously that mineralization was totally absent at embryonic day 15.5 (E15.5) throughout the body in Runx2(-/-) mice, but was found in areas undergoing intramembranous ossification such as skull and clavicles in α1(II)-Cre;Runx2(flox/flox) mice. In newborn α1(II)-Cre;Runx2(flox/flox) mice, mineralization impairment was restricted to skeletal areas undergoing endochondral ossification including long bones and vertebrae. In contrast, no apparent skeletal abnormalities were seen in mutant embryo, newborn, and 3-week-old to 6-week old-mice in which Runx2 had been deleted with the α1(I)-collagen-Cre driver. These results suggest that Runx2 is absolutely required for endochondral ossification during embryonic and postnatal skeletogenesis, but that disrupting its expression in already committed osteoblasts as achieved here with the α1(I)-collagen-Cre driver does not affect overtly intramembranous and endochondral ossification. The Runx2 floxed allele established here is undoubtedly useful for investigating the role of Runx2 in particular cells.

During injury to the central nervous system (CNS), astrocytes and microglia proliferate and migrate around the lesion sites. Recently, it has been reported that one of the water channels, aquaporin-4 (AQP4) is seemed to have a role in astroglial migration and glial scar formation caused by brain injury, although its molecular mechanism is largely unknown. In the present study, we examined the expression profiles in wild-type (WT) and AQP4-deficient (AQP4/KO) mice after a stab wound to the cerebral cortex. Three days after the stab wound, AQP4 expression was observed in activated microglia around the lesion site as well as in astrocytes. A microarray analysis revealed that 444 genes around the lesion site were upregulated 3 days after the wounding in WT mice. Surprisingly, most of these up-regulations were significantly attenuated in AQP4/KO mice. Real-time RT-PCR and immunofluorescence showed that osteopontin (OPN) expression around the lesion site was much lower in AQP4/KO mice than in WT mice. Moreover, the up-regulation of pro-inflammatory cytokines was significantly attenuated in AQP4/KO mice. Taken together, these results suggest that AQP4 plays an important role in immunological function in concert with OPN under pathological conditions in the CNS.

SummaryG-protein-coupled receptors (GPCRs) are known to possess two different conformations, active and inactive, and they spontaneously alternate between the two in the absence of ligands. Here, we analyzed the agonist-independent GPCR activity for its possible role in receptor-instructed axonal projection. We generated transgenic mice expressing activity mutants of the β2-adrenergic receptor, a well-characterized GPCR with the highest homology to odorant receptors (ORs). We found that mutants with altered agonist-independent activity changed the transcription levels of axon-targeting molecules—e.g., Neuropilin-1 and Plexin-A1—but not of glomerular segregation molecules—e.g., Kirrel2 and Kirrel3—thus causing shifts in glomerular locations along the anterior-posterior (A-P) axis. Knockout and in vitro experiments demonstrated that Gs, but not Golf, is responsible for mediating the agonist-independent GPCR activity. We conclude that the equilibrium of conformational transitions set by each OR is the major determinant of expression levels of A-P-targeting molecules.

Background & Aims The cyclic adenosine monophosphate (cAMP) and Ca 2+ signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. Methods We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6 −/− mice) and inositol 1,4,5-triphosphate (InsP 3 ) receptor-binding protein released with InsP 3 (Irbit −/− ) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl – current. Protein interactions were determined by immunoprecipitation analyses. Results Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca 2+ and cAMP. In resting cells, Irbit was sequestered by InsP 3 receptors (IP 3 Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP 3 Rs, which increased their affinity for InsP 3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP 3 , caused dissociation of Irbit from IP 3 Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit −/− or Slc26a6 −/− mice, or in salivary gland ducts expressing mutant forms of IP 3 Rs that could not undergo protein kinase A–mediated phosphorylation. Conclusions Irbit promotes synergy between the Ca 2+ and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjogren syndrome.

The use of live imaging approaches to examine and understand the dynamic processes that take place during mouse development has become widespread. Several groups have reported their success in generating different reporter mouse lines that express a variety of fluorescent markers for imaging. However, there is currently no established database of the reporter mouse lines available for live imaging, such as the Cre transgenic lines (Cre-X-Mice). Researchers therefore often have difficulties in determining which reporter mouse line meets their research purposes. In this review, we summarize some of the reporter mouse lines that have been generated for live imaging studies, and discuss their characteristics.

In the node of mouse embryo, rotational movements of cilia generate an external liquid flow known as nodal flow, which determines left-right asymmetric gene expression. How nodal flow is converted into asymmetric gene expression is still controversial, but the increase of Ca(2+) levels in endodermal cells to the left of the node has been proposed to play a role. However, Ca(2+) signals inside the node itself have not yet been described. By our optimized Ca(2+) imaging method, we were able to observe dynamic Ca(2+) signals in the node in live mouse embryos. Pharmacological disruption of Ca(2+) signals did not affect ciliary movements or nodal flow, but did alter the expression patterns of the Nodal and Cerl-2 genes. Quantitative analyses of Ca(2+) signal frequencies and distributions showed that during left-right axis establishment, formerly symmetric Ca(2+) signals became biased to the left side. In iv/iv mutant embryos that showed randomized laterality due to ciliary immotility, Ca(2+) signals were found to be variously left-sided, right-sided, or bilateral, and thus symmetric on average. In Pkd2 mutant embryos, which lacked polycystin-2, a Ca(2+)-permeable cation channel necessary for left-right axis formation, the Ca(2+) signal frequency was lower than in wild-type embryos. Our data support a model in which dynamic Ca(2+) signals in the node are involved in left-right patterning.

SummaryMast cells (MCs) are key effector cells in allergic reactions. However, the inhibitory mechanism that prevents excessive activation of MCs remains elusive. Here we show that leukocyte mono-immunoglobulin-like receptor 3 (LMIR3; also called CD300f) is a negative regulator of MC activation in vivo. LMIR3 deficiency exacerbated MC-dependent allergic responses in mice, including anaphylaxis, airway inflammation, and dermatitis. Both physical binding and functional reporter assays via an extracellular domain of LMIR3 showed that several extracellular lipids (including ceramide) and lipoproteins were possible ligands for LMIR3. Importantly, MCs were frequently surrounded by extracellular ceramide in vivo. Upon engagement of high-affinity immunoglobulin E receptor, extracellular ceramide-LMIR3 binding inhibited MC activation via immunoreceptor tyrosine-based inhibitory and switch motifs of LMIR3. Moreover, pretreatment with LMIR3-Fc fusion protein or antibody against either ceramide or LMIR3 interfered with this binding in vivo, thereby exacerbating passive cutaneous anaphylaxis. Thus, the interaction between extracellular ceramide and LMIR3 suppressed MC-dependent allergic responses.

After implantation, pluripotent epiblasts are converted to embryonic ectoderm through cell-cell interactions that significantly change the transcriptional and epigenetic networks. An entree to understanding this vital developmental transition is the t(w5) mutation of the mouse t complex. This mutation produces highly specific defects in the embryonic ectoderm before gastrulation, leading to death of the embryonic ectoderm. Using a positional cloning approach, we have now identified the mutated gene, completing a decades-long search. The gene, vacuolar protein sorting 52 (Vps52), is a mouse homolog of yeast VPS52 that is involved in the retrograde trafficking of endosomes. Our data suggest that Vps52 acts in extraembryonic tissues to support the growth and differentiation of embryonic ectoderm via cell-cell interactions. It is also required in the formation of embryonic structures at a later stage of development, revealing hitherto unknown functions of Vps52 in the development of a multicellular organism.

Osteoblasts secrete matrix vesicles and proteins to bone surfaces, but the molecular mechanisms of this secretion system remain unclear. The present findings reveal the roles of important genes in osteoblasts involved in regulation of extracellular matrix secretion. We especially focused on "soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor" (SNARE) genes and identified notable Syntaxin 4a (Stx4a) expression on the basolateral side of the plasma membrane of osteoblasts. Furthermore, Stx4a overexpression was found to increase mineralization by osteoblastic cells, whereas Stx4a knockdown reduced levels of mineralization. Also, BMP-4 and IGF-1 induced the localization of Stx4a to the basolateral side of the cells. To examine the function of Stx4a in osteoblasts, we generated osteoblast-specific Stx4a conditional knockout mice, which demonstrated an osteopenic phenotype due to reduced matrix secretion. Bone mineral density, shown by peripheral quantitative computed tomography (pQCT), was reduced in the femur metaphyseal and diaphyseal regions of Stx4a osteoblast-specific deficient mice, whereas bone parameters, shown by micro-computed tomography (μCT) and bone histomorphometric analysis, were also decreased in trabecular bone. In addition, primary calvarial cells from those mice showed decreased mineralization and lower secretion of matrix vesicles. Our findings indicate that Stx4a plays a critical role in bone matrix production by osteoblasts. © 2016 American Society for Bone and Mineral Research.

The long noncoding RNA Gomafu/MIAT/Rncr2 is thought to function in retinal cell specification, stem cell differentiation and the control of alternative splicing. To further investigate physiological functions of Gomafu, we created mouse knockout (KO) model that completely lacks the Gomafu gene. The KO mice did not exhibit any developmental deficits. However, behavioral tests revealed that the KO mice are hyperactive. This hyperactive behavior was enhanced when the KO mice were treated with the psychostimulant methamphetamine, which was associated with an increase in dopamine release in the nucleus accumbens. RNA sequencing analyses identified a small number of genes affected by the deficiency of Gomafu, a subset of which are known to have important neurobiological functions. These observations suggest that Gomafu modifies mouse behavior thorough a mild modulation of gene expression and/or alternative splicing of target genes.

Vascular development involvesde novoformation of a capillary plexus, which is then pruned and remodeled by angiogenic events. Cytoskeletal remodeling and directional endothelial migration are essential for developmental and pathological angiogenesis. Smad-dependent TGFβ signaling controls vascular patterning and is negatively regulated by microtubules. Here we show that a positive regulator of TGFβ signaling is essential for developmental vascular patterning and microtubule stability. Rudhira/BCAS3 is known to bind microtubules and to play a nodal role in cytoskeletal remodeling and directional endothelial cell (EC) migration invitro. We demonstrate that the molecular and cellular function of Rudhira is deployed at critical steps in vascular patterning. We generated the first floxed mice forrudhiraand find that global or endothelial knockout ofrudhiraresults in mid-gestation lethality due to aberrant embryonic and extra-embryonic vessel patterning and defective cardiac morphogenesis.Rudhiranull yolk sac ECs show random and retarded migration. Yolk sac transcriptome analysis revealed key mediators of angiogenic processes and TGFβ receptor signaling were perturbed inrudhiranull mutants. Molecular and biochemical analyses showed thatrudhiradepletion reduced microtubule stability but increased expression of pathway inhibitors leading to high levels of SMAD2/3 ubiquitination and reduced activation. These effects were not rescued by exogenous TGFβ. However, TGFβ treatment of wild type ECs increased Rudhira expression. Further, exogenous Rudhira, which promotes directional cell migration, caused increased SMAD2/3 nuclear translocation and reduced inhibitor levels. Therefore, we propose that Rudhira and TGFβ signaling are mutually dependent. Rudhira has a dual function in promoting TGFβ signaling, possibly by sequestering microtubules and simultaneously preventing SMAD2/3 ubiquitination to permit EC migration and vascular patterning. TGFβ signaling and aberrant human Rudhira (Breast Cancer Amplified Sequence 3, BCAS3) expression are both associated with tumour metastasis. Our study identifies a cytoskeletal, cell type-specific modulator of TGFβ signaling important in development and cancer.

sFRP4 is an extracellular Wnt antagonist that fine-tunes its signal activity by direct binding to Wnts. Bone fragility under oxidative stress by diabetes and aging is partly related to the suppression of the Wnt signal through upregulated sFRP4. Here, to explore the functions of sFRP4 as a balancer molecule in bone development and remodeling, we analyzed the sFRP4 knock-in mouse strain. X-gal and immunohistochemically stained signals in sFRP4-LacZ heterozygous mice were detectable in restricted areas, mostly in osteoblasts and osteoclasts, of the femoral diaphysis after neonatal and postnatal stages. Histological and μCT analyses showed increased trabecular bone mass with alteration of the Wnt signal and osteogenic activity in sFRP4 mutants; this augmented the effect of the buildup of trabecular bone during the ageing period. Our results indicate that sFRP4 plays a critical role in bone development and remodeling by regulating osteoblasts and osteoclasts and that its functional loss prevents age-related bone loss in the trabecular bone area. These findings imply that sFRP4 functions as a key potential endogenous balancer of the Wnt signaling pathway by efficiently having direct influence on both bone formation and bone absorption during skeletal bone development and maintenance through remodeling.

// Toshiharu Sakurai 1 , Hiroaki Higashitsuji 2 , Hiroshi Kashida 1 , Tomohiro Watanabe 1 , Yoriaki Komeda 1 , Tomoyuki Nagai 1 , Satoru Hagiwara 1 , Masayuki Kitano 1 , Naoshi Nishida 1 , Takaya Abe 3 , Hiroshi Kiyonari 3, 4 , Katsuhiko Itoh 2 , Jun Fujita 2 , Masatoshi Kudo 1 1 Department of Gastroenterology and Hepatology, Kindai University Faculty of Medicine, Osaka-Sayama, Osaka, Japan 2 Department of Clinical Molecular Biology, Kyoto University, Kyoto, Japan 3 Genetic Engineering Team, RIKEN Center for Life Science Technologies, Kobe, Japan 4 Animal Resource Development Unit, RIKEN Center for Life Science Technologies, Kobe, Japan Correspondence to: Toshiharu Sakurai, email: sakurai@med.kindai.ac.jp Keywords: IBD, TNF, Bmi1, IL-17, treatment resistance Received: April 20, 2016 Accepted: December 31, 2016 Published: February 01, 2017 ABSTRACT Although long-standing colonic inflammation due to refractory inflammatory bowel disease (IBD) promotes the development of colitis-associated cancer (CAC), the molecular mechanisms accounting for the development of CAC remains largely unknown. In this study, we investigated the role of gankyrin in the development of CAC since gankyrin is overexpressed in sporadic colorectal cancers. We analyzed gene expression of colon tissues obtained from 344 patients with IBD and CAC and found that expression of gankyrin was much higher in colonic mucosa of patients with refractory IBD than in those with IBD in remission. Expression of gankyrin was upregulated in inflammatory cells as well as tumor cells in colonic mucosa of patients with CAC. Over-expressing studies utilizing tagged ganlyrin-cDNA identified physical interaction between ganlyrin and Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1). Importantly, the interaction between ganlyrin and SHP-1 leads to inhibition of STAT3 activation and to enhancement of TNF-α and IL-17 in inflammatory cells. To further address the role of gankyrin in the development of CAC, we created mice with intestinal epithelial cell-specific gankyrin ablation ( Vil-Cre;Gankyrin f/f ) and deletion of gankyrin in myeloid and epithelial cells ( Mx1-Cre;Gankyrin f/f ). Gankyrin deficiency in myeloid cells, but not in epithelial cells, reduced the activity of mitogen activated protein kinase and the expression of stem cell markers, leading to attenuated tumorigenic potential. These findings provide important insights into the pathogenesis of CAC and suggest that gankyrin is a promising target for developing therapeutic and preventive strategies against CAC.

INTRODUCTION AND OBJECTIVES: To compare the rate of chemo-sensitivity in neoadjuvant chemotherapy for muscle invasive bladder cancer between historical control and this interventional prospective study by using our M-VAC and CaG prediction systems. METHODS: Muscle invasive bladder cancer patients (Stage II-III) were engaged in this study. They were assigned to M-VAC, CaG, operation or radiation therapy according to the result of the prediction system which we had already established. According to their responses to the neoadjuvant chemotherapy, we categorized the patients into two groups: 0responders0 who achieved significant tumor shrinking (>60%), and 0non-responders0 ( 60%). Patients were evaluated either 0accurate0 or 0 inaccurate0 according to a tumor shrinkage rate cut off line of a 60% decrease after chemotherapies. The primary endpoint of this study was comparison with the historical controls in terms of the rate of chemo-sensitivity and efficacy. The secondary endpoint was down staging ( pT1) and the overall survival. RESULTS: From March 2011 to July 2013, an intention-to-treat 35 cancer samples from patients were entered in this study. Seven patients M-VAC, 22 CaG, 2 operation and 4 radiation therapy were received respectively. The predicted responder rate for M-VAC was 42.9% (15/35) and for CaG was 57.1% (20/35). Moreover, 74.3% (26/35) of all patients could be expected to respond to both or either of these two regimens by applying our two prediction systems. While, in previous historical control studies (M-VAC 39, CaG 37, total 76 cases), the total predicted response rate of the 76 cases was 57.9% (44/76) (p1⁄40.09). And in the predicted 0responder0 cases, the response rate of M-VAC neoadjuvant chemotherapy was 85.7% (6/7) and CaG was 89.5% (17/19). Therefore, the combined response rate in this group was 88.5% (23/26). In the historical control cases, the observed response rate of M-VAC/CaG therapy was 59.0% (23/39)/54.1% (20/37), and combined observed response rate was 56.6% (43/76) (p1⁄40.004). Down staging and overall survival between this prospective study and the historical control were not significant statistical differences, however in overall survival this prospective study was better tendency than that of the historical control (p1⁄40.233). CONCLUSIONS: Combining the M-VAC and CaG prediction systems would improve the chemo-sensitivity for muscle invasive bladder cancers.