Timing of the G1/S transition in tobacco pollen vegetative cells as a primary step towards androgenesis in vitro

Key message

Mid-bicellular pollen vegetative cells in tobacco escape from G1 arrest and proceed to the G1/S transition towards androgenesis within 1 day under glutamine starvation conditions in vitro.

Abstract

In the Nicotiana tabacum pollen culture system, immature pollen grains at the mid-bicellular stage can mature in the presence of glutamine; however, if glutamine is absent, they deviate from their native cell fate in a few days. The glutamine-starved pollen grains cannot undergo maturation, even when supplied with glutamine later. Instead, they undergo cell division towards androgenesis slowly within 10 days in a medium containing appropriate nutrients. During the culture period, they ought to escape from G1 arrest to proceed into S phase as the primary step towards androgenesis. However, this event has not been experimentally confirmed. Here, we demonstrated that the pollen vegetative cells proceeded to the G1/S transition within approximately 15–36 h after the start of culture. These results were obtained by analyzing transgenic pollen possessing a fusion gene encoding nuclear-localizing GFP under the control of an E2F motif-containing promoter isolated from a gene encoding one of DNA replication licensing factors. Observations using a 5-ethynyl-2′-deoxyuridine DNA labeling and detection technique uncovered that the G1/S transition was soon followed by S phase. These hallmarks of vegetative cells undergoing dedifferentiation give us new insights into upstream events causing the G1/S transition and also provide a novel strategy to increase the frequency of the androgenic response in tobacco and other species, including recalcitrants.     

Detailed Identification of Fatty Acid Isomers Sheds Light on the Probable Precursors of Triacylglycerol Accumulation in Photoautotrophically Grown Chlamydomonas reinhardtii

Chlamydomonas reinhardtii is a model alga for studying triacylglycerol (TAG) accumulation in the photosynthetic production of biofuel. Previous studies were conducted under photoheterotrophic growth conditions in medium supplemented with acetate and/or ammonium. We wanted to demonstrate TAG accumulation under truly photoautotrophic conditions without reduced elements. We first reidentified all lipid components and fatty acids by mass spectrometry, because the currently used identification knowledge relies on data obtained in the 1980s. Accordingly, various isomers of fatty acids, which are potentially useful in tracing the flow of fatty acids leading to the accumulation of TAG, were detected. In strain CC1010 grown under photoautotrophic conditions, TAG accumulated to about 57.5 mol% of total lipids on a mole fatty acid basis after the transfer to nitrogen-deficient conditions. The content of monogalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol, and phosphatidylglycerol decreased drastically. The accumulated TAG contained 16:0 as the major acid and 16:4(4,7,10,13), 18:2(9,12), and 18:3(9,12,15), which are typically found in chloroplast lipids. Additionally, 18:1(11) and 18:3(5,9,12), which are specific to extrachloroplast lipids, were also abundant in the accumulated TAG. Photosynthesis and respiration slowed markedly after the shift to nitrogen-deficient conditions. These results suggest that fatty acids for the production of TAG were supplied not only from chloroplast lipids but also from other membranes within the cells, although the possibility of de novo synthesis cannot be excluded. Under nitrogen-replete conditions, supplementation with a high concentration of CO₂ promoted TAG production in the cells grown photoautotrophically, opening up the possibility to the continuous production of TAG using CO₂ produced by industry.     

Re-evaluation of liver stem/progenitor cells

Liver stem/progenitor cells (LPCs) are defined as cells that supply two types of liver epithelial cells, hepatocytes and cholangiocytes, during development, cellular turnover, and regeneration. Hepatoblasts, which are fetal LPCs derived from endoderm stem cells, robustly proliferate and differentiate into hepatocytes and cholangiocytes during fetal life. Between mid-gestation and the neonatal period, some cholangiocytes function as LPCs. Although LPCs in adult livers can be enriched in cells positive for cholangiocyte markers, their tissue localization and functions in cellular turnover remain obscure. On the other hand, it is well known that liver regeneration under conditions suppressing hepatocyte proliferation is supported by LPCs, though their origin has not been clearly identified. Recently many groups took advantage of new techniques including prospective isolation of LPCs by fluorescence-activated cell sorting and genetic lineage tracing to facilitate our understanding of epithelial supply in normal and injured livers. Those works suggest that, in normal livers, the turnover of hepatocytes mostly depends on duplication of hepatocytes. It is also demonstrated that liver epithelial cells as well as LPCs have great plasticity and flexible differentiation capability to respond to various types of injuries by protecting or repairing liver tissues.     

Design of a New Energy‐Harvesting Electrochromic Window Based on an Organic Polymeric Dye,a Cobalt Couple,and PProDOT‐Me2

A new design for an energy‐harvesting electrochromic window (EH‐ECW) based on the fusion of two technologies, organic electrochromic windows and dye‐sensitized solar cells (DSSCs), is presented. Unlike other power‐generating smart windows, such as photoelectrochromic devices that are passive and only contain two states (i.e., a closed‐circuit colored state and an open‐circuit bleaching state), EH‐ECW allows active tuning of the transmittance by varying the applied potential and it functions as a photovoltaic cell based on a DSSC. The resulting device demonstrates a fast switching rate of 1 s in both the bleaching and coloring processes through the use of an electrochromic polymer as a counter electrode layer. To increase the transmittance of the device, a cobalt redox couple and a light‐colored, yet efficient, organic dye are used. The organic dye contains a polymeric structure that contributes to the high cyclic stability. The device exhibits a power conversion efficiency (PCE) of 4.5% (100 mW cm^‐2) under AM 1.5 irradiation, a change in transmittance of 34% upon applied potential, and shows only 3% degradation in the PCE after 5000 cycles.     

Gastrointestinal specific anxiety in irritable bowel syndrome: validation of the Japanese version of the visceral sensitivity index for university students

Objective

The visceral sensitivity index (VSI) is a useful self-report measure of the gastrointestinal symptom-specific anxiety (GSA) of patients with irritable bowel syndrome (IBS). Previous research has shown that worsening GSA in IBS patients is related to the severity of GI symptoms, suggesting that GSA is an important endpoint for intervention. However, there is currently no Japanese version of the VSI. We therefore translated the VSI into Japanese (VSI-J) and verified its reliability and validity.

Material and methods

Participants were 349 university students aged 18 and 19 years and recruited from an academic class. We analyzed data from the VSI-J, Anxiety Sensitivity Index (ASI), Hospital Anxiety and Depression scale (HAD), and Irritable Bowel Syndrome Severity Index (IBS-SI). The internal consistency, stability, and factor structure of the VSI-J and its associations with anxiety, depression and severity measures were investigated.

Results

The factor structure of the VSI-J is unidimensional and similar to that of the original VSI (Cronbach’s α?=?0.93). Construct validity was demonstrated by significant correlations with ASI (r?=?0.43, p?<?0.0001), HAD-ANX (r?=?0.19, p?=?0.0003), and IBS-SI scores (r?=?0.45, p?<?0.0001). Furthermore, the VSI-J was a significant predictor of severity scores on the IBS-SI and demonstrated good discriminant (p?<?0.0001) and incremental (p?<?0.0001) validity.

Conclusion

These findings suggest that the VSI-J is a reliable and valid measure of visceral sensitivity.

     

Effect of codon adaptation on codon-level and gene-level translation efficiency in vivo

Background

There is a significant difference between synonymous codon usage in many organisms, and it is known that codons used more frequently generally showed efficient decoding rate. At the gene level, however, there are conflicting reports on the existence of a correlation between codon adaptation and translation efficiency, even in the same organism.

Results

To resolve this issue, we cultured Escherichia coli under conditions designed to maintain constant levels of mRNA and protein and subjected the cells to ribosome profiling (RP) and mRNA-seq analyses. We showed that the RP results correlated more closely with protein levels generated under similar culture conditions than with the mRNA abundance from the mRNA-seq. Our result indicated that RP/mRNA ratio could be used as a measure of translation efficiency at gene level. On the other hand, the RP data showed that codon-specific ribosome density at the decoding site negatively correlated with codon usage, consistent with the hypothesis that preferred codons display lower ribosome densities due to their faster decoding rate. However, highly codon-adapted genes showed higher ribosome densities at the gene level, indicating that the efficiency of translation initiation, rather than higher elongation efficiency of preferred codons, exerted a greater effect on ribosome density and thus translation efficiency.

Conclusions

These findings indicate that evolutionary pressure on highly expressed genes influenced both codon bias and translation initiation efficiency and therefore explains contradictory findings that codon usage bias correlates with translation efficiency of native genes, but not with the artificially created gene pool, which was not subjected to evolution pressure.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1115) contains supplementary material, which is available to authorized users.     

A Proton Beam Therapy System Dedicated to Spot-Scanning Increases Accuracy with Moving Tumors by Real-Time Imaging and Gating and Reduces Equipment Size

Purpose

A proton beam therapy (PBT) system has been designed which dedicates to spot-scanning and has a gating function employing the fluoroscopy-based real-time-imaging of internal fiducial markers near tumors. The dose distribution and treatment time of the newly designed real-time-image gated, spot-scanning proton beam therapy (RGPT) were compared with free-breathing spot-scanning proton beam therapy (FBPT) in a simulation.

Materials and Methods

In-house simulation tools and treatment planning system VQA (Hitachi, Ltd., Japan) were used for estimating the dose distribution and treatment time. Simulations were performed for 48 motion parameters (including 8 respiratory patterns and 6 initial breathing timings) on CT data from two patients, A and B, with hepatocellular carcinoma and with clinical target volumes 14.6 cc and 63.1 cc. The respiratory patterns were derived from the actual trajectory of internal fiducial markers taken in X-ray real-time tumor-tracking radiotherapy (RTRT).

Results

With FBPT, 9/48 motion parameters achieved the criteria of successful delivery for patient A and 0/48 for B. With RGPT 48/48 and 42/48 achieved the criteria. Compared with FBPT, the mean liver dose was smaller with RGPT with statistical significance (p<0.001); it decreased from 27% to 13% and 28% to 23% of the prescribed doses for patients A and B, respectively. The relative lengthening of treatment time to administer 3 Gy (RBE) was estimated to be 1.22 (RGPT/FBPT: 138 s/113 s) and 1.72 (207 s/120 s) for patients A and B, respectively.

Conclusions

This simulation study demonstrated that the RGPT was able to improve the dose distribution markedly for moving tumors without very large treatment time extension. The proton beam therapy system dedicated to spot-scanning with a gating function for real-time imaging increases accuracy with moving tumors and reduces the physical size, and subsequently the cost of the equipment as well as of the building housing the equipment.     

Overtime Work and Prevalence of Diabetes in Japanese Employees: Japan Epidemiology Collaboration on Occupational Health Study

Objective

Epidemiologic evidence on long working hour and diabetes has been conflicting. We examined the association between overtime work and prevalence of diabetes among Japanese workers.

Methods

The subjects were 40,861 employees (35,170 men and 5,691 women), aged 16 to 83 years, of 4 companies in Japan. Hours of overtime were assessed using self-reported questionnaires. Diabetes was defined as a fasting plasma glucose ≥126 mg/dl (7.0 mmol/l), hemoglobin A1c ≥6.5% (48 mmol/mol), or current use of anti-diabetic drug. Multiple logistic regression analysis was used to calculate odds ratio of diabetes for each category of overtime.

Results

After adjustment for age, sex, company, smoking, and BMI, there was a suggestion of U-shaped relationship between overtime work and prevalence of diabetes (P for quadratic trend = 0.07). Compared with those who worked <45 hours of overtime per month, the adjusted odds ratios (95% confidence interval) of diabetes were 0.86 (0.77–0.94), 0.69 (0.53–0.89), and 1.03 (0.72–1.46) for those who worked 45–79, 80–99, and ≥100 hours of overtime per month, respectively. In one company (n = 33,807), where other potential confounders including shift work, job position, type of department, alcohol consumption, sleep duration, leisure time physical activity, and family history of diabetes was additionally adjusted for, similar result was obtained (P for quadratic trend = 0.05).

Conclusions

Long hours of overtime work may not be associated with increased prevalence of diabetes among Japanese workers.     

Multivariable Logistic Regression Model: A Novel Mathematical Model that Predicts Visual Field Sensitivity from Macular Ganglion Cell Complex Thickness in Glaucoma

Purpose

To design a mathematical model that can predict the relationship between the ganglion cell complex (GCC) thickness and visual field sensitivity (VFS) in glaucoma patients.

Design

Retrospective cross-sectional case series.

Method

Within 3 months from VFS measurements by the Humphrey field analyzer 10-2 program, 83 eyes underwent macular GCC thickness measurements by spectral-domain optical coherence tomography (SD-OCT). Data were used to construct a multiple logistic model that depicted the relationship between the explanatory variables (GCC thickness, age, sex, and spherical equivalent of refractive errors) determined by a regression analysis and the mean VFS corresponding to the SD-OCT scanned area. Analyses were performed in half or 8 segmented local areas as well as in whole scanned areas. A simple logistic model that included GCC thickness as the single explanatory variable was also constructed. The ability of the logistic models to depict the real GCC thickness/VFS in SAP distribution was analyzed by the χ² test of goodness-of-fit. The significance of the model effect was analyzed by analysis of variance (ANOVA).

Results

Scatter plots between the GCC thickness and the mean VFS showed sigmoid curves. The χ² test of goodness-of-fit revealed that the multiple logistic models showed a good fit for the real GCC thickness/VFS distribution in all areas except the nasal-inferior-outer area. ANOVA revealed that all of the multiple logistic models significantly predicted the VFS based on the explanatory variables. Although simple logistic models also exhibited significant VFS predictability based on the GCC thickness, the model effect was less than that observed for the multiple logistic models.

Conclusions

The currently proposed logistic models are useful methods for depicting relationships between the explanatory variables, including the GCC thickness, and the mean VFS in glaucoma patients.