Derivation of Transgene-Free Human Induced Pluripotent Stem Cells from Human Peripheral T Cells in Defined Culture Conditions

Recently, induced pluripotent stem cells (iPSCs) were established as promising cell sources for revolutionary regenerative therapies. The initial culture system used for iPSC generation needed fetal calf serum in the culture medium and mouse embryonic fibroblast as a feeder layer, both of which could possibly transfer unknown exogenous antigens and pathogens into the iPSC population. Therefore, the development of culture systems designed to minimize such potential risks has become increasingly vital for future applications of iPSCs for clinical use. On another front, although donor cell types for generating iPSCs are wide-ranging, T cells have attracted attention as unique cell sources for iPSCs generation because T cell-derived iPSCs (TiPSCs) have a unique monoclonal T cell receptor genomic rearrangement that enables their differentiation into antigen-specific T cells, which can be applied to novel immunotherapies. In the present study, we generated transgene-free human TiPSCs using a combination of activated human T cells and Sendai virus under defined culture conditions. These TiPSCs expressed pluripotent markers by quantitative PCR and immunostaining, had a normal karyotype, and were capable of differentiating into cells from all three germ layers. This method of TiPSCs generation is more suitable for the therapeutic application of iPSC technology because it lowers the risks associated with the presence of undefined, animal-derived feeder cells and serum. Therefore this work will lead to establishment of safer iPSCs and extended clinical application.     

Photo-Induced P-Methylbenzylation of 1,3-Dimethylthymine and 1, 3-Dimethyluracil in the Presence of Trifluoroacetic Acid

Abstract

Photolysis of a solution of a pyrimidine (i. e., 1, 3-dimethylthymine and 1, 3-dimethyluracil) in p-xylene in the presence of trifluoroacetic acid afforded mainly the 5, 6-dihydropyrimidine derivative together with the 5-p-methylbenzylated product and the 6-isomer as well. It is suggested that the first two products result from the C⁶-protonated pyrimidine electron adduct (III), while the 6-isomer is derived from the O⁴-protonated isomer (II).     

Rapid Structural Changes and Acidification of Guard Cell Vacuoles during Stomatal Closure Require Phosphatidylinositol 3,5-Bisphosphate

Rapid stomatal closure is essential for water conservation in plants and is thus critical for survival under water deficiency. To close stomata rapidly, guard cells reduce their volume by converting a large central vacuole into a highly convoluted structure. However, the molecular mechanisms underlying this change are poorly understood. In this study, we used pH-indicator dyes to demonstrate that vacuolar convolution is accompanied by acidification of the vacuole in fava bean (Vicia faba) guard cells during abscisic acid (ABA)–induced stomatal closure. Vacuolar acidification is necessary for the rapid stomatal closure induced by ABA, since a double mutant of the vacuolar H⁺-ATPase vha-a2 vha-a3 and vacuolar H⁺-PPase mutant vhp1 showed delayed stomatal closure. Furthermore, we provide evidence for the critical role of phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] in changes in pH and morphology of the vacuole. Single and double Arabidopsis thaliana null mutants of phosphatidylinositol 3-phosphate 5-kinases (PI3P5Ks) exhibited slow stomatal closure upon ABA treatment compared with the wild type. Moreover, an inhibitor of PI3P5K reduced vacuolar acidification and convolution and delayed stomatal closure in response to ABA. Taken together, these results suggest that rapid ABA-induced stomatal closure requires PtdIns(3,5)P₂, which is essential for vacuolar acidification and convolution.     

Risk Literacy and Risk Perception Among Undergraduates in Mainland China: The Case of BSE

Risk response and cognitive characteristics of different groups are important aspects in risk research. In this article we discuss the general features of risk literacy and perception among undergraduates at Nanjing University and Huaiyin Institute of Technology in China in the case of bovine spongiform encephalopathy (BSE). The results show that: (1) there seem to be no relationships between respondents’ knowledge about BSE and their risk identification; (2) between all contrast samples, the greatest divergence appears in the judgment about probability of risk while the smallest one is the trust in science and technology to avoid BSE; (3) the judgment of hazard probability would be related to gender, family location, and the backgrounds of college and academic specialty. And only gender has impact on the affirmation of hazard with a significant level of 0.05. These findings provide insights into the comprehension of undergraduates’ risk literacy and perception, and can contribute to the improvement of risk communication and management in China.     

Cep57 and Cep57L1 maintain centriole engagement in interphase to ensure centriole duplication cycle

Centrioles duplicate in interphase only once per cell cycle. Newly formed centrioles remain associated with their mother centrioles. The two centrioles disengage at the end of mitosis, which licenses centriole duplication in the next cell cycle. Therefore, timely centriole disengagement is critical for the proper centriole duplication cycle. However, the mechanisms underlying centriole engagement during interphase are poorly understood. Here, we show that Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase. Codepletion of Cep57 and Cep57L1 induces precocious centriole disengagement in interphase without compromising cell cycle progression. The disengaged daughter centrioles convert into centrosomes during interphase in a Plk1-dependent manner. Furthermore, the centrioles reduplicate and the centriole number increases, which results in chromosome segregation errors. Overall, these findings demonstrate that the maintenance of centriole engagement by Cep57 and Cep57L1 during interphase is crucial for the tight control of centriole copy number and thus for proper chromosome segregation.     

Modulation of the carbohydrate-binding specificity of two Xenopus proto-type galectins by site-directed mutagenesis

The galectin family is a representative soluble lectin group, which is responsible for the modulation of various cell functions. Although the carbohydrate-binding specificity of galectins has been well-studied, the relationship between protein structure and specificity remains to be elucidated. We previously reported the characteristics of a Xenopus laevis skin galectin, xgalectin-Va, which had diverged from galectin-1. The carbohydrate selectivity of xgalectin-Va was different from that of human galectin-1 and xgalectin-Ib (a Xenopus laevis galectin-1 homolog). In this study, we clarified the key residues for this selectivity by site-directed mutagenesis. Substitution of two amino acids of xgalectin-Va, Val56Gly/Lys76Arg, greatly enhanced the binding ability to N-acetyllactosamine and conferred significant T-cell growth inhibition activity, although the wild type had no activity. These two residues, Gly54 and Arg74 in galectin-1, would cooperatively contribute to the N-acetyllactosamine recognition. The loop region between the S4 and S5 β-strands was involved in the binding to the TF-antigen disaccharide. The loop substitution successfully changed the carbohydrate selectivity of xgalectin-Va and xgalectin-Ib.     

Eradication of the mongoose is crucial for the conservation of three endemic bird species in Yambaru,Okinawa Island,Japan

Okinawa Island, Japan, is a globally important biodiversity hotspot. Three endemic bird species, Okinawa rail (Hypotaenidia okinawae), Okinawa woodpecker (Dendrocopos noguchii), and Okinawa robin (Larvivora namiyei), are found only in the Yambaru region of the northern part of Okinawa Island. In order to conserve endemic species, it is important to determine the effect of alien species on endemic species. We conducted playback surveys four times every three years from 2007 to 2016 to evaluate the recent distribution of these three forest-dwelling bird species during the breeding season. Then, the association between the numbers of detections of these three species with the invasive mongoose density and the hardwood forest area was evaluated with a generalized additive mixed model (GAMM). The results showed that the distribution areas of these bird species have been recovering since the 2007 within the small Indian mongoose (Urva auropunctata) controlled area. The GAMM results showed that these bird species were abundant in areas with fewer small Indian mongooses and larger areas of hardwood forests. Thus, the mongoose had a negative impact not only on the flightless rails but also on the woodpeckers and the robins. In recent years, most of the old-growth forests have been designated as protected forests, and large-scale logging is no longer taking place in Yambaru. Eradication of the mongoose is particularly important for the conservation of these three endemic bird species.

     

The clinical significance of 5% change in vital capacity in patients with idiopathic pulmonary fibrosis: extended analysis of the pirfenidone trial

Background

The rate of decline in forced expiratory volume in 1 second (FEV₁) is representative of the natural history of COPD. Sparse information exists regarding the associations between the magnitude of annualised loss of FEV₁ with other endpoints.

Methods

Retrospective analysis of UPLIFT^® trial (four-year, randomized, double-blind, placebo-controlled trial of tiotropium 18 μg daily in chronic obstructive pulmonary disease [COPD], n = 5993). Decline of FEV₁ was analysed with random co-efficient regression. Patients were categorised according to quartiles based on the rate of decline (RoD) in post-bronchodilator FEV_1. The St George's Respiratory Questionnaire (SGRQ) total score, exacerbations and mortality were assessed within each quartile.

Results

Mean (standard error [SE]) post-bronchodilator FEV₁ increased in the first quartile (Q1) by 37 (1) mL/year. The other quartiles showed annualised declines in FEV₁ (mL/year) as follows: Q2 = 24 (1), Q3 = 59 (1) and Q4 = 125 (2). Age, gender, respiratory medication use at baseline and SGRQ did not distinguish groups. The patient subgroup with the largest RoD had less severe lung disease at baseline and contained a higher proportion of current smokers. The percentage of patients with ≥ 1 exacerbation showed a minimal difference from the lowest to the largest RoD, but exacerbation rates increased with increasing RoD. The highest proportion of patients with ≥ 1 hospitalised exacerbation was in Q4 (Q1 = 19.5% [tiotropium], 26% [control]; Q4 = 33.8% [tiotropium] and 33.1% [control]). Time to first exacerbation and hospitalised exacerbation was shorter with increasing RoD. Rate of decline in SGRQ increased in direct proportion to each quartile. The group with the largest RoD had the highest mortality.

Conclusion

Patients can be grouped into different RoD quartiles with the observation of different clinical outcomes indicating that specific (or more aggressive) approaches to management may be needed.

Trial Registration

ClinicalTrials.gov number, NCT00144339     

Time-lapse imaging reveals symmetric neurogenic cell division of GFAP-expressing progenitors for expansion of postnatal dentate granule neurons

Granule cells in the hippocampus, a region critical for memory and learning, are generated mainly during the early postnatal period but neurogenesis continues in adulthood. Postnatal neuronal production is carried out by primary progenitors that express glial fibrillary acidic protein (GFAP) and they are assumed to function as stem cells. A central question regarding postnatal dentate neurogenesis is how astrocyte-like progenitors produce neurons. To reveal cell division patterns and the process of neuronal differentiation of astrocyte-like neural progenitors, we performed time-lapse imaging in cultured hippocampal slices from early postnatal transgenic mice with mouse GFAP promoter-controlled enhanced green fluorescent protein (mGFAP-eGFP Tg mice) in combination with a retrovirus carrying a red fluorescent protein gene. Our results showed that the majority of GFAP-eGFP+ progenitor cells that express GFAP, Sox2 and nestin divided symmetrically to produce pairs of GFAP+ cells (45%) or pairs of neuron-committed cells (45%), whereas a minority divided asymmetrically to generate GFAP+ cells and neuron-committed cells (10%). The present results suggest that a substantial number of GFAP-expressing progenitors functions as transient amplifying progenitors, at least in an early postnatal dentate gyrus, although a small population appears to be stem cell-like progenitors. From the present data, we discuss possible cell division patterns of adult GFAP+ progenitors.