Improving Agricultural Traits While Maintaining High Resistant Starch Content in Rice.

BACKGROUND: Resistant starch (RS) is beneficial for human health. Loss of starch branching enzyme IIb (BEIIb) increases the proportion of amylopectin long chains, which greatly elevates the RS content. Although high RS content cereals are desired, an increase in RS content is often accompanied by a decrease in seed weight. To further increase the RS content, genes encoding active-type starch synthase (SS) IIa, which elongates amylopectin branches, and high expression-type granule-bound SSI (GBSSI), which synthesizes amylose, were introduced into the be2b mutant rice. This attempt increased the RS content, but further improvement of agricultural traits was required because of a mixture of indica and japonica rice phonotype, such as different grain sizes, flowering times, and seed shattering traits. In the present study, the high RS lines were backcrossed with an elite rice cultivar, and the starch properties of the resultant high-yielding RS lines were analyzed. RESULTS: The seed weight of high RS lines was greatly improved after backcrossing, increasing up to 190% compared with the seed weight before backcrossing. Amylopectin structure, gelatinization temperature, and RS content of high RS lines showed almost no change after backcrossing. High RS lines contained longer amylopectin branch chains than the wild type, and lines with active-type SSIIa contained a higher proportion of long amylopectin chains compared with the lines with less active-SSIIa, and thus showed higher gelatinization temperature. Although the RS content of rice varied with the cooking method, those of high RS lines remained high after backcrossing. The RS contents of cooked rice of high RS lines were high (27-35%), whereas that of the elite parental rice was considerably low (< 0.7%). The RS contents of lines with active-type SSIIa and high-level GBSSI expression in be2b or be2b ss3a background were higher than those of lines with less-active SSIIa. CONCLUSIONS: The present study revealed that backcrossing high RS rice lines with elite rice cultivars could increase the seed weight, without compromising the RS content. It is likely that backcrossing introduced loci enhancing seed length and width as well as loci promoting early flowering for ensuring an optimum temperature during RS biosynthesis.

Impact of Meal Timing on Postprandial Interstitial Fluid Glucose Levels in Young Japanese Females.

Flash glucose monitoring (FGM) provides continuous and accessible measurement of the interstitial fluid glucose (ISFG) level and this system is useful for understanding blood glucose fluctuations. We examined differences in postprandial ISFG after the main mealtimes (breakfast, lunch, and dinner) in healthy young Japanese females. Nine healthy young females (aged 21.5±0.6 y old) were enrolled in this study. ISFG was continuously measured by FGM. Participants ate the same meal three times a day consecutively, thereby satisfying their daily energy requirements. Postprandial ISFG fluctuations were evaluated for 4 h after each meal. There were no significant differences in ISFG before the 3 main meals. The postprandial ISFG peak was the lowest after breakfast, increasing in the order of lunch and then dinner. The area under the curve of the 4-h postprandial ISFG was higher after lunch and dinner than after breakfast. The results of this study suggest that postprandial ISFG differ depending on mealtimes in young Japanese females.

Development of a Ligand Screening Tool Using Full-Length Human Peroxisome Proliferator-Activated Receptor-Expressing Cell Lines to Ameliorate Metabolic Syndrome.

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor superfamily and include three subtypes (PPARα, PPARδ, and PPARγ). They regulate gene expression in a ligand-dependent manner. PPARα plays an important role in lipid metabolism. PPARγ is involved in glucose metabolism and is a potential therapeutic target in Type 2 diabetes. PPARδ ligands are candidates for the treatment of metabolic disorders. Thus, the detection of PPAR ligands may facilitate the treatment of various diseases. In this study, to identify PPAR ligands, we engineered reporter cell lines that can be used to quantify PPARγ and PPARδ activity. We evaluated several known ligands using these reporter cell lines and confirmed that they are useful for PPAR ligand detection. Furthermore, we evaluated extracts of approximately 200 natural resources and found various extracts that enhance reporter gene activity. Finally, we identified a main alkaloid of the Evodia fruit, evodiamine, as a PPARγ activator using this screening tool. These results suggest that the established reporter cell lines may serve as a useful cell-based screening tool for finding PPAR ligands to ameliorate metabolic syndromes.

Discovery of peroxisome proliferator-activated receptor α (PPARα) activators with a ligand-screening system using a human PPARα-expressing cell line.

Peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that belongs to the superfamily of nuclear hormone receptors. PPARα is mainly expressed in the liver, where it activates fatty acid oxidation and lipoprotein metabolism and improves plasma lipid profiles. Therefore, PPARα activators are often used to treat patients with dyslipidemia. To discover additional PPARα activators as potential compounds for use in hypolipidemic drugs, here we established human hepatoblastoma cell lines with luciferase reporter expression from the promoters containing peroxisome proliferator-responsive elements (PPREs) and tetracycline-regulated expression of full-length human PPARα to quantify the effects of chemical ligands on PPARα activity. Using the established cell-based PPARα-activator screening system to screen a library of >12,000 chemical compounds, we identified several hit compounds with basic chemical skeletons different from those of known PPARα agonists. One of the hit compounds, a 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivative we termed compound 3, selectively up-regulated PPARα transcriptional activity, leading to PPARα target gene expression both in vitro and in vivo Of note, the half-maximal effective concentrations of the hit compounds were lower than that of the known PPARα ligand fenofibrate. Finally, fenofibrate or compound 3 treatment of high fructose-fed rats having elevated plasma triglyceride levels for 14 days indicated that compound 3 reduces plasma triglyceride levels with similar efficiency as fenofibrate. These observations raise the possibility that 1H-pyrazolo[3,4-b]pyridine-4-carboxylic acid derivatives might be effective drug candidates for selective targeting of PPARα to manage dyslipidemia.

Role of fusional convergence amplitude in postoperative phoria maintenance in children with intermittent exotropia.

PURPOSE: To examine the role of fusional convergence amplitude in postoperative phoria maintenance in childhood intermittent exotropia [X(T)]. METHODS: The medical records of 29 children aged 15 years or younger (mean age, 10.8 ± 2.4 years) and treated with monocular recession-resection for X(T) were reviewed retrospectively. The patients' fusional convergence amplitude (break point/total amplitudes), physiologic diplopia, and phoria maintenance (presence/absence of phoria maintenance and ability to maintain phoria) were assessed. The presence of phoria maintenance was confirmed by a cover test, and the ability to maintain phoria was quantified using the Bagolini red filter bar. Correlations of the amplitude size with the presence and ability of phoria maintenance were investigated. RESULTS: A significant correlation was seen between fusional amplitude (break point/total) and ability to maintain phoria at near and at far (break point: P < .05 at near/P < .01 at far; total: P < .05 at near/far). Neither the break point amplitude nor the total amplitude significantly differed between the patients with phoria maintenance and those without it (break point: P = .71 at near, P = .29 at far; total: P = .98 at near, P = .85 at far). Phoria maintenance correlated with the suppression of physiologic diplopia during phoria (P < .01). The deviation angle did not significantly correlate with fusional amplitude either at near (P = .58) or at far (P = .27). CONCLUSIONS: In childhood X(T), fusional amplitude plays a role in enforcing the patient's ability to maintain phoria. However, sufficient fusional amplitude does not guarantee fully functioning fusion if suppression is present during phoria.

Updated recommendation on molecular-targeted therapy for metastatic renal cell cancer.

Molecular-targeted therapy was recommended for the systemic therapy of renal cell cancer (RCC) in the RCC guidelines, but these guidelines do not address the order of administration of the multiple presently available agents. There are several aspects that remain unknown regarding the optimal administration order and combination of molecular-targeted drugs. Until the optimal treatment sequence is determined by clinical trials, treatment individualization is required for each patient based on patient and disease characteristics. We herein investigate 12 cases of RCC patients who received axitinib. Axitinib was used as the first-line drug in 4 cases, second-line in 5 cases, third-line in 1 case and as a fourth-line drug in 2 cases. Partial response (PR) was observed in 4 cases (30%) and stable disease in 4 cases (30%) during axitinib treatment, with an overall response rate of 60%. The duration of PR ranged from 6 to 19 months. Based on our cases, axitinib exhibited reasonable therapeutic efficacy as first- as well as second-line treatment. However, more cases are required to draw firm conclusions.

Influence of Test Distance on Stereoacuity in Intermittent Exotropia.

AIMS: To investigate influence of test distance on stereoacuity in intermittent exotropia (X[T]) using the same test conditions for both near and far distances. METHODS: Subjects were 38 consecutive patients with X(T). All the patients were between ages 6 and 15 years and had decimal visual acuity of 1.0 or better. Another inclusion criterion was presence of phoric condition at near and far distances. Stereoacuity was measured at a near distance of 40 cm and at a far distance of 5 m. The following test conditions were used for both test distances: separation of the two eyes using polarized glasses, and a target with a random dot pattern. All the stereograms had the same subtended angle of 2.5º, and binocular disparity of 480, 240, 120, and 60 arcsec. We used two stereogram types with crossed and uncrossed disparities. RESULTS: Far stereoacuity of 38 subjects measured with the crossed disparity was significantly worse than near stereoacuity (P<0.05, Wilcoxon signed-ranks test), although 30 (78.9%) of the 38 subjects showed no differences in stereopsis between the near and far distances. Far stereoacuity of 38 cases measured with the uncrossed disparity was significantly worse than at near (P<0.05, Wilcoxon signed-ranks test), although 20 (52.6%) of the 38 subjects showed no differences between stereoacuity at near and far. In comparison of stereoacuity with crossed disparity and uncrossed disparity, stereoacuity with crossed disparity was significantly better than that with uncrossed disparity both at near and far (P<0.05, Wilcoxon signed-ranks test). CONCLUSIONS: Stereoacuity in X(T) was different according to test distance when measured controlling subtended angle of stereogram at both distances. Far stereoacuity was significantly worse than near stereoacuity when measured using test targets with both crossed and uncrossed disparities. Additionally, stereoacuity measured with crossed disparity was better than that with uncrossed disparity at both distances.

Correction: Effects of a Closed Space Environment on Gene Expression in Hair Follicles of Astronauts in the International Space Station.

[This corrects the article DOI: 10.1371/journal.pone.0150801.].

Effects of a Closed Space Environment on Gene Expression in Hair Follicles of Astronauts in the International Space Station.

Adaptation to the space environment can sometimes pose physiological problems to International Space Station (ISS) astronauts after their return to earth. Therefore, it is important to develop healthcare technologies for astronauts. In this study, we examined the feasibility of using hair follicles, a readily obtained sample, to assess gene expression changes in response to spaceflight adaptation. In order to investigate the gene expression changes in human hair follicles during spaceflight, hair follicles of 10 astronauts were analyzed by microarray and real time qPCR analyses. We found that spaceflight alters human hair follicle gene expression. The degree of changes in gene expression was found to vary among individuals. In some astronauts, genes related to hair growth such as FGF18, ANGPTL7 and COMP were upregulated during flight, suggesting that spaceflight inhibits cell proliferation in hair follicles.

Expression, purification and characterization of the recombinant cysteine-rich antimicrobial peptide snakin-1 in Pichia pastoris.

Snakin-1 (SN-1) is a small cysteine-rich plant antimicrobial peptide with broad spectrum antimicrobial activity which was isolated from potato (Solanum tuberosum). Here, we carried out the expression of a recombinant SN-1 in the methylotrophic yeast Pichia pastoris, along with its purification and characterization. A DNA fragment encoding the mature SN-1 was cloned into pPIC9 vector and introduced into P. pastoris. A large amount of pure recombinant SN-1 (approximately 40 mg/1L culture) was obtained from a fed-batch fermentation culture after purification with a cation exchange column followed by RP-HPLC. The identity of the recombinant SN-1 was verified by MALDI-TOF MS, CD and (1)H NMR experiments. All these data strongly indicated that the recombinant SN-1 peptide had a folding with six disulfide bonds that was identical to the native SN-1. Our findings showed that SN-1 exhibited strong antimicrobial activity against test microorganisms and produced very weak hemolysis of mammalian erythrocytes. The mechanism of its antimicrobial action against Escherichia coli was investigated by both outer membrane permeability assay and cytoplasmic membrane depolarization assay. These assays demonstrated that SN-1 is a membrane-active antimicrobial peptide which can disrupt both outer and cytoplasmic membrane integrity. This is the first report on the recombinant expression and purification of a fully active SN-1 in P. pastoris.

Regulation of the human PDZK1 expression by peroxisome proliferator-activated receptor alpha.

Although PDZK1 is a well-known adaptor protein, the mechanisms for its role in transcriptional regulation are largely unknown. The peroxisome proliferator-activated receptor alpha (PPARalpha) is a ligand-activated transcription factor that plays an important role in the regulation of lipid homeostasis. Previously, we established a tetracycline-regulated human cell line that can be induced to express PPARalpha and identified candidate target genes, one of which was PDZK1. In this study, we cloned and characterized the promoter region of the human pdzk1 gene and determined the PPAR response element. Finally, we demonstrate that endogenous PPARalpha regulates PDZK1 expression.

Analysis of PPAR alpha function in human kidney cell line using siRNA.

The peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPARalpha is mainly expressed in the liver, kidney, heart and muscle. PPARalpha activates fatty acid catabolism, stimulates gluconeogenesis and ketone body synthesis and is involved in the control of lipoprotein assembly. Although PPARalpha is well characterized in the liver, its physiological function is unknown in the kidney. To investigate the intimate function of PPARalpha in the kidney, we analyzed the target gene expression in human metastatic renal cell carcinoma cell line, Caki-1, using small interfering RNA (siRNA) against PPARalpha and real-time RT-PCR methods. We found that some selected genes (long-chain fatty-acid-CoA ligase (FACL1), carnitine palmitoyltransferase 1A (CPT1A), adipose differentiation-related protein (ADRP) and aquaporin 3 (AQP3)) were down-regulated by PPARalpha siRNA. These results suggest that PPARalpha regulates fatty acid metabolism and body water homeostasis in this cell line.