Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming

Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.     

Effects of excess light energy on excitation-energy dynamics in a pennate diatom Phaeodactylum tricornutum

Photosynthesis Research - Photosynthesis starts when a pigment in the photosynthetic antennae absorbs a photon. The electronic excitation energy is then transferred through the network of...     

Discriminating between Glaucoma and Normal Eyes Using Optical Coherence Tomography and the ‘Random Forests’ Classifier

Purpose

To diagnose glaucoma based on spectral domain optical coherence tomography (SD-OCT) measurements using the ‘Random Forests’ method.

Methods

SD-OCT was conducted in 126 eyes of 126 open angle glaucoma (OAG) patients and 84 eyes of 84 normal subjects. The Random Forests method was then applied to discriminate between glaucoma and normal eyes using 151 OCT parameters including thickness measurements of circumpapillary retinal nerve fiber layer (cpRNFL), the macular RNFL (mRNFL) and the ganglion cell layer-inner plexiform layer combined (GCIPL). The area under the receiver operating characteristic curve (AROC) was calculated using the Random Forests method adopting leave-one-out cross validation. For comparison, AROCs were calculated based on each one of the 151 OCT parameters.

Results

The AROC obtained with the Random Forests method was 98.5% [95% Confidence interval (CI): 97.1–99.9%], which was significantly larger than the AROCs derived from any single OCT parameter (maxima were: 92.8 [CI: 89.4–96.2] %, 94.3 [CI: 91.1–97.6] % and 91.8 [CI: 88.2–95.4] % for cpRNFL-, mRNFL- and GCIPL-related parameters, respectively; P<0.05, DeLong’s method with Holm’s correction for multiple comparisons). The partial AROC above specificity of 80%, for the Random Forests method was equal to 18.5 [CI: 16.8–19.6] %, which was also significantly larger than the AROCs of any single OCT parameter (P<0.05, Bootstrap method with Holm’s correction for multiple comparisons).

Conclusions

The Random Forests method, analyzing multiple SD-OCT parameters concurrently, significantly improves the diagnosis of glaucoma compared with using any single SD-OCT measurement.     

Micromechanics of human mitotic chromosomes

Eukaryote cells dramatically reorganize their long chromosomal DNAs to facilitate their physical segregation during mitosis. The internal organization of folded mitotic chromosomes remains a basic mystery of cell biology; its understanding would likely shed light on how chromosomes are separated from one another as well as into chromosome structure between cell divisions. We report biophysical experiments on single mitotic chromosomes from human cells, where we combine micromanipulation, nano-Newton-scale force measurement and biochemical treatments to study chromosome connectivity and topology. Results are in accord with previous experiments on amphibian chromosomes and support the 'chromatin network' model of mitotic chromosome structure. Prospects for studies of chromosome-organizing proteins using siRNA expression knockdowns, as well as for differential studies of chromosomes with and without mutations associated with genetic diseases, are also discussed.     

Overexpression of Bcl2 in osteoblasts inhibits osteoblast differentiation and induces osteocyte apoptosis

Bcl2 subfamily proteins, including Bcl2 and Bcl-X(L), inhibit apoptosis. As osteoblast apoptosis is in part responsible for osteoporosis in sex steroid deficiency, glucocorticoid excess, and aging, bone loss might be inhibited by the upregulation of Bcl2; however, the effects of Bcl2 overexpression on osteoblast differentiation and bone development and maintenance have not been fully investigated. To investigate these issues, we established two lines of osteoblast-specific BCL2 transgenic mice. In BCL2 transgenic mice, bone volume was increased at 6 weeks of age but not at 10 weeks of age compared with wild-type mice. The numbers of osteoblasts and osteocytes increased, but osteoid thickness and the bone formation rate were reduced in BCL2 transgenic mice with high expression at 10 weeks of age. The number of BrdU-positive cells was increased but that of TUNEL-positive cells was unaltered at 2 and 6 weeks of age. Osteoblast differentiation was inhibited, as shown by reduced Col1a1 and osteocalcin expression. Osteoblast differentiation of calvarial cells from BCL2 transgenic mice also fell in vitro. Overexpression of BCL2 in primary osteoblasts had no effect on osteoclastogenesis in co-culture with bone marrow cells. Unexpectedly, overexpression of BCL2 in osteoblasts eventually caused osteocyte apoptosis. Osteocytes, which had a reduced number of processes, gradually died with apoptotic structural alterations and the expression of apoptosis-related molecules, and dead osteocytes accumulated in cortical bone. These findings indicate that overexpression of BCL2 in osteoblasts inhibits osteoblast differentiation, reduces osteocyte processes, and causes osteocyte apoptosis.     

Requirement of insertion sequence IS1 for thermal adaptation of Pro-Tk-subtilisin from hyperthermophilic archaeon

Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis matures from Pro-Tk-subtilisin (Pro-TKS) upon autoprocessing and degradation of propeptide. Pro-TKS contains the insertion sequence (IS1) at the N-terminus of the mature domain as compared to bacterial pro-subtilisins. To analyze the role of IS1, the Pro-TKS derivative without IS1 (?IS1-Pro-TKS) and its active-site mutants (?IS1-Pro-S324A and ?IS1-Pro-S324C) were constructed and characterized. ?IS1-Pro-S324A and ?IS1-Pro-TKS represent an unautoprocessed and autoprocessed form of ?IS1-Pro-TKS, respectively. The CD and ANS fluorescence spectra of these proteins indicate that folding of ?IS1-Pro-TKS is not completed by binding of Ca²⁺ ions but is completed by the subsequent autoprocessing reaction. Thermal denaturation of these proteins analyzed by DSC and CD spectroscopy indicates that unautoprocessed ?IS1-Pro-TKS is less stable than autoprocessed ?IS1-Pro-TKS by 26.3?°C in T _m. The stability of autoprocessed ?IS1-Pro-TKS is comparable to that of Pro-TKS, which is slightly lower than that of unautoprocessed Pro-TKS. These results suggest that ?IS1-Pro-TKS is fully folded and greatly stabilized by autoprocessing. ?IS1-Pro-TKS more slowly matured to ?IS1-Tk-subtilisin than Pro-TKS did, due to a decrease in the autoprocessing rate. We propose that IS1 is required not only for hyperstabilization of Pro-TKS but also for its rapid maturation.     

Discovery and optimization of a novel series of GPR142 agonists for the treatment of type 2 diabetes mellitus

The discovery and initial optimization of a series of phenylalanine based agonists for GPR142 is described. The structure-activity-relationship around the major areas of the molecule was explored to give agonists 90 times more potent than the initial HTS hit in a human GPR142 inositol phosphate accumulation assay. Removal of CYP inhibition by exploration of the pyridine A-ring is also described.