A physiologically based pharmacokinetic and pharmacodynamic model for disposition of FF-10832.

This study aimed to quantitatively clarify the critical factors responsible for the superior antitumor efficacy of a liposomal gemcitabine (2,2-difluorodeoxycytidine; dFdC) formulation, FF-10832, compared with dFdC. The underlying hypothesis is the different exposure of tumors to its active metabolite, dFdC triphosphate (dFdCTP), between the two formulations. Therefore, physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models for encapsulated and unencapsulated dFdC were constructed considering the tumor dFdCTP concentration as an index of antitumor activity. To estimate drug the parameters, the time profiles of encapsulated and unencapsulated dFdC in the blood and those of dFdC and dFdCTP in tumors were measured following the intravenous bolus administration of FF-10832 or dFdC. dFdC metabolism and transport in the liver S9 fraction and isolated hepatocytes, respectively, were experimentally determined. The tumor growth curve in a mouse xenograft model following the administration of FF10832 and dFdC was also used to construct the PD model. The sensitivity analysis of the PBPK/PD model revealed the critical factors affecting antitumor efficacy, which included the total and intratumor tissue uptake clearances for liposomal formulation and the cytidine deaminase and deoxycytidine deaminase activities in tumors. Thus, these parameters are potential biomarkers for predicting the efficacy of the liposomal formulation of dFdC.

Multi-omics approach reveals posttranscriptionally regulated genes are essential for human pluripotent stem cells.

The effects of transcription factors on the maintenance and differentiation of human-induced or embryonic pluripotent stem cells (iPSCs/ESCs) have been well studied. However, the importance of posttranscriptional regulatory mechanisms, which cause the quantitative dissociation of mRNA and protein expression, has not been explored in detail. Here, by combining transcriptome and proteome profiling, we identified 228 posttranscriptionally regulated genes with strict upregulation of the protein level in iPSCs/ESCs. Among them, we found 84 genes were vital for the survival of iPSCs and HDFs, including 20 genes that were specifically necessary for iPSC survival. These 20 proteins were upregulated only in iPSCs/ESCs and not in differentiated cells derived from the three germ layers. Although there are still unknown mechanisms that downregulate protein levels in HDFs, these results reveal that posttranscriptionally regulated genes have a crucial role in iPSC survival.

Evaluating totipotency using criteria of increasing stringency.

Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions.

Involvement of Claudin-11 in Disruption of Blood-Brain, -Spinal Cord, and -Arachnoid Barriers in Multiple Sclerosis.

It is important to understand the molecular mechanisms of barrier disruption in the central nervous system (CNS) of patients with multiple sclerosis (MS). The purpose of the present study was to clarify whether claudin-11 is involved in the disruption of two endothelial barriers (blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB)) and two epithelial barriers (blood-arachnoid barrier (BAB) and blood-CSF barrier (BCSFB)) in the CNS in MS. Immunohistochemical analysis revealed that, in both normal human and mouse, claudin-11 is co-localized with claudin-5 in the brain and spinal cord capillaries. The absolute protein expression level of claudin-11 was nearly equal to that of claudin-5 in rat brain capillaries, but was 2.81-fold greater in human brain capillaries. The protein expressions of claudin-11 were significantly downregulated in the brain and spinal cord capillaries of an MS patient and experimental autoimmune encephalomyelitis (EAE) mice. Specific downregulation of claudin-11 with siRNA significantly increased the transfer of membrane-impermeable FITC-dextran across human brain capillary endothelial cell (hCMEC/D3) monolayer. As for the epithelial barrier, claudin-11 protein expression was not decreased in choroid plexus epithelial cells forming the BCSFB in EAE mice, whereas it was decreased in brain and spinal cord meninges that form the BAB. Specific downregulation of claudin-11 with siRNA in a rat choroid plexus epithelial cell (TR-CSFB) monolayer significantly increased the permeability of FITC-dextran. In conclusion, our present findings indicate that claudin-11 expression at the BBB, BSCB, and BAB, but not the BCSFB, is downregulated in multiple sclerosis, impairing the functional integrity of these barriers.

Laparoscopic and Endoscopic Cooperative Surgery for Plexiform Angiomyxoid Myofibroblastic Tumor.

Plexiform angiomyxoid myofibroblastic tumor (PAMT) is a recently described distinctive gastric mesenchymal entity with a peculiar plexiform pattern, bland spindle cells and a myxoid stroma rich in arborizing blood vessels. In this study, we report a new case of this rare gastric tumor resected by laparoscopic and endoscopic cooperative surgery (LECS). A 39-year-old Japanese man was admitted with a gastric mass. Gastroscopy showed an elevated mass in the anterior wall of the gastric antrum. Endoscopic ultrasound examination revealed a focal hypoechoic lesion protruding into the lumen. A partial gastrectomy by LECS was performed, and the patient made an uneventful recovery and remains well 9 months later. The tumor in this case depicted all the typical histopathologic and immunochemical features of gastric PAMT (c-kit negative and smooth muscle actin-positive). Especially, it was characterized by multiple nodules protruding outward within the serosa. Therefore, it is important that the resection line is determined on the serosa to ensure the complete resection of these nodules together.

Screening of Human cDNA Library Reveals Two differentiation-Related Genes, HHEX and HLX, as Promoters of Early Phase Reprogramming toward Pluripotency.

Gene screenings have identified a number of reprogramming factors that induce pluripotency from somatic cells. However, the screening methods have mostly considered only factors that maintain pluripotency in embryonic stem cells, ignoring a potentially long list of other contributing factors involved. To expand the search, we developed a new screening method that examined 2,008 human genes in the generation of human induced pluripotent stem cells (iPSCs), including not only pluripotent genes but also differentiation-related genes that suppress pluripotency. We found the top 100 genes that increased reprogramming efficiency and discovered they contained many differentiation-related genes and homeobox genes. We selected two, HHEX and HLX, for further analysis. These genes enhanced the appearance of premature reprograming cells in the early phase of human iPSC induction, but had inhibitory effect on the late phase. In addition, when expressed in human iPSCs, HHEX and HLX interfered with the pluripotent state, indicating inverse effects on somatic reprograming and pluripotent maintenance. These results demonstrate that our screening is useful for identifying differentiation-related genes in somatic reprograming. Stem Cells 2016;34:2661-2669.

OVOL2 Maintains the Transcriptional Program of Human Corneal Epithelium by Suppressing Epithelial-to-Mesenchymal Transition.

In development, embryonic ectoderm differentiates into neuroectoderm and surface ectoderm using poorly understood mechanisms. Here, we show that the transcription factor OVOL2 maintains the transcriptional program of human corneal epithelium cells (CECs), a derivative of the surface ectoderm, and that OVOL2 may regulate the differential transcriptional programs of the two lineages. A functional screen identified OVOL2 as a repressor of mesenchymal genes to maintain CECs. Transduction of OVOL2 with several other transcription factors induced the transcriptional program of CECs in fibroblasts. Moreover, neuroectoderm derivatives were found to express mesenchymal genes, and OVOL2 alone could induce the transcriptional program of CECs in neural progenitors by repressing these genes while activating epithelial genes. Our data suggest that the difference between the transcriptional programs of some neuroectoderm- and surface ectoderm-derivative cells may be regulated in part by a reciprocally repressive mechanism between epithelial and mesenchymal genes, as seen in epithelial-to-mesenchymal transition.

[Effect of Viewing a Long Stereoscopic Film on Equilibrium Function and Autonomic Nervous System].

OBJECTIVES: Owing to the recent rapid advancements in image processing and three-dimensional (3-D) technologies, stereoscopic images can now be viewed on television as well as in theaters and on gaming consoles among others. However, with these advancements, there have also been reports on motion sickness and asthenopia induced by viewing stereoscopic films. Human equilibrium function deteriorates when viewing stereoscopic films, which may lead to motion sickness; however, the exact cause of such motion sickness remains unknown. Therefore, as part of hygiene research that contributes to society, it is important to consider the safety of viewing virtual 3D contents. METHODS: In this study, we investigated the effects of viewing 2-D/3-D video clips on the human body by stabilometry, electrogastrography (EGG), and subjective assessments. Seven subjects aged 22 to 24 viewed 2-D/3-D video clips for 60 min. RESULTS: A comparison of time series data obtained at rest shows a significant change in the EGG patterns 20 min after the start of viewing the video clips. Furthermore, sway values while viewing the 3-D video clips were considerably higher than those while viewing the 2-D video clips 60 min after the start of viewing. CONCLUSIONS: These findings show that the autonomic nervous system is affected first by long-term viewing of stereoscopic films, and the equilibrium function deteriorates gradually over the course of the exposure.

An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.

The generation of induced pluripotent stem cells (iPSCs) provides the opportunity to use patient-specific somatic cells, which are a valuable source for disease modeling and drug discovery. To promote research involving these cells, it is important to make iPSCs from easily accessible and less invasive tissues, like blood. We have recently reported the efficient generation of human iPSCs from adult fibroblasts using a combination of plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53. We herein report a modified protocol enabling efficient iPSC induction from CD34+ cord blood cells and from peripheral blood isolated from healthy donors using these plasmid vectors. The original plasmid mixture could induce iPSCs; however, the efficiency was low. The addition of EBNA1, an essential factor for episomal amplification of the vectors, by an extra plasmid greatly increased the efficiency of iPSC induction, especially when the induction was performed from αßT cells. This improvement enabled the establishment of blood-derived iPSCs from seven healthy donors ranging in age from their 20s to their 60s. This induction method will be useful for the derivation of patient-specific integration-free iPSCs and would also be applicable to the generation of clinical-grade iPSCs in the future.

ECAT11/L1td1 is enriched in ESCs and rapidly activated during iPSC generation, but it is dispensable for the maintenance and induction of pluripotency.

The principal factors that lead to proliferation and pluripotency in embryonic stem cells (ESCs) have been vigorously investigated. However, the global network of factors and their full signaling cascade is still unclear. In this study, we found that ECAT11 (L1td1) is one of the ESC-associated transcripts harboring a truncated fragment of ORF-1, a component of the L1 retrotransposable element. We generated an ECAT11 knock-in mouse by replacing its coding region with green fluorescent protein. In the early stage of development, the fluorescence was observed at the inner cell mass of blastocysts and epiblasts. Despite this specific expression, ECAT11-null mice grow normally and are fertile. In addition, ECAT11 was dispensable for both the proliferation and pluripotency of ESCs.We found rapid and robust activation of ECAT11 in fibroblasts after the forced expression of transcription factors that can give rise pluripotency in somatic cells. However, iPS cells could be established from ECAT11-null fibroblasts. Our data demonstrate the dispensability of ECAT11/L1td1 in pluripotency, despite its specific expression.