Specific fate decisions in adult hepatic progenitor cells driven by MET and EGFR signaling.

The relative contribution of hepatocyte growth factor (HGF)/MET and epidermal growth factor (EGF)/EGF receptor (EGFR), two key signal transduction systems in the normal and diseased liver, to fate decisions of adult hepatic progenitor cells (HPCs) has not been resolved. Here, we developed a robust culture system that permitted expansion and genetic manipulation of cells capable of multilineage differentiation in vitro and in vivo to examine the individual roles of HGF/MET and EGF/EGFR in HPC self-renewal and binary cell fate decision. By employing loss-of-function and rescue experiments in vitro, we showed that both receptors collaborate to increase the self-renewal of HPCs through activation of the extracellular signal-regulated kinase (ERK) pathway. MET was a strong inducer of hepatocyte differentiation by activating AKT and signal transducer and activator of transcription (STAT3). Conversely, EGFR selectively induced NOTCH1 to promote cholangiocyte specification and branching morphogenesis while concomitantly suppressing hepatocyte commitment. Furthermore, unlike the deleterious effects of MET deletion, the liver-specific conditional loss of Egfr facilitated rather than suppressed progenitor-mediated liver regeneration by switching progenitor cell differentiation toward hepatocyte lineage. These data provide new insight into the mechanisms regulating the stemness properties of adult HPCs and reveal a previously unrecognized link between EGFR and NOTCH1 in directing cholangiocyte differentiation.

Atorvastatin Inhibits Endothelial PAI-1-Mediated Monocyte Migration and Alleviates Radiation-Induced Enteropathy.

Intestinal injury is observed in cancer patients after radiotherapy and in individuals exposed to radiation after a nuclear accident. Radiation disrupts normal vascular homeostasis in the gastrointestinal system by inducing endothelial damage and senescence. Despite advances in medical technology, the toxicity of radiation to healthy tissue remains an issue. To address this issue, we investigated the effect of atorvastatin, a commonly prescribed hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor of cholesterol synthesis, on radiation-induced enteropathy and inflammatory responses. We selected atorvastatin based on its pleiotropic anti-fibrotic and anti-inflammatory effects. We found that atorvastatin mitigated radiation-induced endothelial damage by regulating plasminogen activator inhibitor-1 (PAI-1) using human umbilical vein endothelial cells (HUVECs) and mouse model. PAI-1 secreted by HUVECs contributed to endothelial dysfunction and trans-endothelial monocyte migration after radiation exposure. We observed that PAI-1 production and secretion was inhibited by atorvastatin in irradiated HUVECs and radiation-induced enteropathy mouse model. More specifically, atorvastatin inhibited PAI-1 production following radiation through the JNK/c-Jun signaling pathway. Together, our findings suggest that atorvastatin alleviates radiation-induced enteropathy and supports the investigation of atorvastatin as a radio-mitigator in patients receiving radiotherapy.

Mapping of a major quantitative trait locus for bakanae disease resistance in rice by genome resequencing.

Bakanae disease (BD) has emerged as a serious threat in almost all rice cultivation regions worldwide. Nampyeong is a Korean japonica rice variety known to be resistant to BD. In this study, quantitative trait locus (QTL) mapping was performed with F2 and F3 plants derived from a cross between the Nampyeong variety and a susceptible Korean japonica line, DongjinAD. First, resequencing of Nampyeong and DongjinAD was performed, which identified 171,035 single nucleotide polymorphisms (SNPs) between the two parental varieties. Using these SNPs, 161 cleaved amplified polymorphic sequence (CAPS) markers and six derived CAPS markers were developed; then, a genetic map was constructed from the genotypes of 180 plants from the DongjinAD/Nampyeong F2 plants. The total length of the constructed genetic map was 1386 cM, with an average interval of 8.9 cM between markers. The BD mortality rates of each F3 family were measured by testing 40 F3 progenies using in vitro seedling screening method. QTL analysis based on the genetic map and mortality rate data revealed a major QTL, qFfR1, on rice chromosome 1. qFfR1 was located at 89.8 cM with a logarithm of the odds (LOD) score of 22.7. Further, there were three markers at this point: JNS01033, JNS01037, and JNS01041. A total of 15 genes were identified with annotations related to defense against plant diseases among the 179 genes in the qFfR1 interval at 95% probability, thereby providing potential candidate genes for qFfR1. qFfR1 and its closely linked markers will be useful in breeding rice varieties resistant to BD.

ABCA1-Mediated EMT Promotes Papillary Thyroid Cancer Malignancy through the ERK/Fra-1/ZEB1 Pathway.

Papillary thyroid cancer (PTC) is the most prevalent histological type of thyroid cancer (TC) worldwide. Although tumor metastasis occurs in regional lymph nodes, distant metastasis (DM) may also occur. Radioactive iodine (RAI) therapy is an effective treatment for TC; however, resistance to RAI occurs in patients with DM. Therefore, in this study, we investigated the efficacy of DM-related biomarkers as therapeutic targets for PTC therapy. ABCA1 expression was higher in aggressive BCPAP cells than in other PTC cells in terms of migration and invasion capacity. The knockdown of ABCA1 substantially decreased the expression of the epithelial-mesenchymal transition (EMT) marker, N-cadherin, and EMT regulator (ZEB1), resulting in suppressed migration and invasion of BCPAP cells. ABCA1 knockdown also reduced ERK activity and Fra-1 expression, which correlated with the effects of an ERK inhibitor or siRNA-mediated inhibition of ERK or Fra-1 expression. Furthermore, ABCA1-knocked-down BCPAP cells suppressed cell migration and invasion by reducing Fra-1 recruitment to Zeb1 promoter; lung metastasis was not observed in mice injected with ABCA1-knocked-down cells. Overall, our findings suggest that ABCA1 regulates lung metastasis in TC cells.

Complete plastid genome sequences of three Rosids (Castanea, Prunus, Theobroma): evidence for at least two independent transfers of rpl22 to the nucleus.

Functional gene transfer from the plastid to the nucleus is rare among land plants despite evidence that DNA transfer to the nucleus is relatively frequent. During the course of sequencing plastid genomes from representative species from three rosid genera (Castanea, Prunus, Theobroma) and ongoing projects focusing on the Fagaceae and Passifloraceae, we identified putative losses of rpl22 in these two angiosperm families. We further characterized rpl22 from three species of Passiflora and one species of Quercus and identified sequences that likely represent pseudogenes. In Castanea and Quercus, both members of the Fagaceae, we identified a nuclear copy of rpl22, which consisted of two exons separated by an intron. Exon 1 encodes a transit peptide that likely targets the protein product back to the plastid and exon 2 encodes rpl22. We performed phylogenetic analyses of 97 taxa, including 93 angiosperms and four gymnosperm outgroups using alignments of 81 plastid genes to examine the phylogenetic distribution of rpl22 loss and transfer to the nucleus. Our results indicate that within rosids there have been independent transfers of rpl22 to the nucleus in Fabaceae and Fagaceae and a putative third transfer in Passiflora. The high level of sequence divergence between the transit peptides in Fabaceae and Fagaceae strongly suggest that these represent independent transfers. Furthermore, Blast searches did not identify the "donor" genes of the transit peptides, suggesting a de novo origin. We also performed phylogenetic analyses of rpl22 for 87 angiosperms and four gymnosperms, including nuclear-encoded copies for five species of Fabaceae and Fagaceae. The resulting trees indicated that the transfer of rpl22 to the nucleus does not predate the origin of angiosperms as suggested in an earlier study. Using previously published angiosperm divergence time estimates, we suggest that these transfers occurred approximately 56-58, 34-37, and 26-27 Ma for the Fabaceae, Fagaceae, and Passifloraceae, respectively.

A case of severe gastroparesis: indigestion and weight loss after catheter ablation of atrial fibrillation.

We describe a patient with gastroparesis after radiofrequency catheter ablation (RFCA) as a result of vagus nerve injury. A 42-year-old man underwent redo-RFCA due to recurrent drug-resistant symptomatic atrial fibrillation. The patient complained of indigestion and early satiety 2 weeks after the second procedure. There was also weight loss of approximately 5 kg for 2 months. He underwent endoscopy during which food material was noticed. In the upper gastrointestinal series, most contrast material still remained in the stomach on the 2-hour delayed images, suggesting delayed gastric emptying time.

Classification of 17 species Aegilops using DNA barcoding and SNPs, reveals gene flow among Aegilops biuncialis, Aegilops juvenalis, and Aegilops columnaris.

Rapid changes in agricultural environments caused by global warming pose a major challenge to food production and safety. Common wheat (Triticum aestivum) is a hexaploid plant (AABBDD) that shares large numbers of quantitative traits and resistance genes with B and D genomes of Aegilops species, which are responsible for several metabolic functions and biosynthetic processes, particularly in plant adaptation to biotic as well as abiotic stresses. Comparatively, the abundance of the Aegilops gene pool is much higher than that of Triticum. Therefore, we used four universal DNA barcodes for plants (ITS2, matK, rbcL, and psbM-petN) to construct a phylogenetic tree to classify the genus Aegilops. Fourteen species were distinguished among a total of 17 representative species. Aegilops biuncialis, Aegilops juvenalis, and Aegilops umbellulata could not be grouped into any of the clusters in the phylogenetic tree, indicating that these three species could not be distinguished by four DNA barcodes. Therefore, from 2408 SNPs obtained using genotyping by sequencing (GBS), we manually screened 30 SNPs that could be potentially used to classify these three species. The results of gene flow and genetic differentiation index (Fst) showed that the genetic differentiation among the three species was small, and there was bidirectional horizontal gene transfer between the three species, which was consistent with our results that the three species were difficult to classify by DNA barcode.

Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections.

Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC101 and PG1 accumulated up to 32%-38% and 17%∼26% of the total soluble protein. Both RC101 and PG1 were cleaved from GFP by corresponding proteases in vitro, and Factor Xa-like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6-fold higher yield of RC101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to tobacco mosaic virus infections, confirming antiviral activity. Because RC101 and PG1 have not yet been produced in other cell culture or microbial systems, chloroplasts can be used as bioreactors for producing these proteins. Adequate yield of purified antimicrobial peptides from transplastomic plants should facilitate further preclinical studies.

Analysis of 81 genes from 64 plastid genomes resolves relationships in angiosperms and identifies genome-scale evolutionary patterns.

Angiosperms are the largest and most successful clade of land plants with >250,000 species distributed in nearly every terrestrial habitat. Many phylogenetic studies have been based on DNA sequences of one to several genes, but, despite decades of intensive efforts, relationships among early diverging lineages and several of the major clades remain either incompletely resolved or weakly supported. We performed phylogenetic analyses of 81 plastid genes in 64 sequenced genomes, including 13 new genomes, to estimate relationships among the major angiosperm clades, and the resulting trees are used to examine the evolution of gene and intron content. Phylogenetic trees from multiple methods, including model-based approaches, provide strong support for the position of Amborella as the earliest diverging lineage of flowering plants, followed by Nymphaeales and Austrobaileyales. The plastid genome trees also provide strong support for a sister relationship between eudicots and monocots, and this group is sister to a clade that includes Chloranthales and magnoliids. Resolution of relationships among the major clades of angiosperms provides the necessary framework for addressing numerous evolutionary questions regarding the rapid diversification of angiosperms. Gene and intron content are highly conserved among the early diverging angiosperms and basal eudicots, but 62 independent gene and intron losses are limited to the more derived monocot and eudicot clades. Moreover, a lineage-specific correlation was detected between rates of nucleotide substitutions, indels, and genomic rearrangements.

Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae).

Chickpea (Cicerarietinum, Leguminosae), an important grain legume, is widely used for food and fodder throughout the world. We sequenced the complete plastid genome of chickpea, which is 125,319bp in size, and contains only one copy of the inverted repeat (IR). The genome encodes 108 genes, including 4 rRNAs, 29 tRNAs, and 75 proteins. The genes rps16, infA, and ycf4 are absent in the chickpea plastid genome, and ndhB has an internal stop codon in the 5'exon, similar to other legumes. Two genes have lost their introns, one in the 3'exon of the transpliced gene rps12, and the one between exons 1 and 2 of clpP; this represents the first documented case of the loss of introns from both of these genes in the same plastid genome. An extensive phylogenetic survey of these intron losses was performed on 302 taxa across legumes and the related family Polygalaceae. The clpP intron has been lost exclusively in taxa from the temperate "IR-lacking clade" (IRLC), whereas the rps12 intron has been lost in most members of the IRLC (with the exception of Wisteria, Callerya, Afgekia, and certain species of Millettia, which represent the earliest diverging lineages of this clade), and in the tribe Desmodieae, which is closely related to the tribes Phaseoleae and Psoraleeae. Data provided here suggest that the loss of the rps12 intron occurred after the loss of the IR. The two new genomic changes identified in the present study provide additional support of the monophyly of the IR-loss clade, and resolution of the pattern of the earliest-branching lineages in this clade. The availability of the complete chickpea plastid genome sequence also provides valuable information on intergenic spacer regions among legumes and endogenous regulatory sequences for plastid genetic engineering.

Hypoxic condition- and high cell density-induced expression of Redd1 is regulated by activation of hypoxia-inducible factor-1alpha and Sp1 through the phosphatidylinositol 3-kinase/Akt signaling pathway.

Redd1, a recently discovered stress-response gene, is regulated by hypoxia via hypoxia-inducible factor 1 (HIF-1) and by DNA damage via p53/p63; however, the signaling pathway by which its expression is induced by hypoxia has not been elucidated. In the present study, we demonstrated that the expression of Redd1 in response to hypoxia (1% O(2)), hypoxia-mimetic agent, cobalt chloride (CoCl(2)) and high cell density (HCD) requires coactivation of HIF-1alpha and Sp1. CoCl(2) and HCD induced the activation of HIF-1alpha and Sp1 in HeLa cells, and siRNAs targeting HIF-1alpha and Sp1 abrogated Redd1 expression. Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002 and by a dominant-negative PI3K mutant reduced the expression of Redd1 and activation of HIF-1alpha and Sp1 by CoCl(2) and HCD. Also, suppression of Akt activation blocked the expression of Redd1 and the activation of HIF-1alpha and Sp1 by CoCl(2) and HCD. Furthermore, we found that the induction of Redd1 expression by CoCl(2) can be mediated by activation of Sp1 in HIF-1alpha-deficient cells but that a higher level of Redd1 expression is achieved when these cells are transfected with HIF-1alpha. These results demonstrate that hypoxic condition-and HCD-induced expression of Redd1 is mediated by coactivation of Sp1 and HIF-1alpha downstream of the PI3K/Akt signaling pathway.

Rice WRKY11 Plays a Role in Pathogen Defense and Drought Tolerance.

BACKGROUND: Plants are frequently subjected to abiotic and biotic stresses, and WRKY proteins play a pivotal role in the response to such stress. OsWRKY11 is induced by pathogens, drought, and heat, suggesting a function in biotic and abiotic stress responses. RESULTS: This study identified OsWRKY11, a member of WRKY group IIc. It is a transcriptional activator that localized to the nucleus. Ectopic expression of OsWRKY11 resulted in enhanced resistance to a bacterial pathogen, Xanthomonas oryzae pv. oryzae; resistance was compromised in transgenic lines under-expressing OsWRKY11. Ectopic expression of OsWRKY11 resulted in constitutive expression of defense-associated genes, whereas knock-down (kd) of OsWRKY11 reduced expression of defense-associated genes during pathogen attack, suggesting that OsWRKY11 activates defense responses. OsWRKY11 bound directly to the promoter of CHITINASE 2, a gene associated with defense, and activated its transcription. In addition, ectopic expression of OsWRKY11 enhanced tolerance to drought stress and induced constitutive expression of drought-responsive genes. Induction of drought-responsive genes was compromised in OsWRKY11-kd plants. OsWRKY11 also bound directly to the promoter of a drought-responsive gene, RAB21, activating its transcription. In addition, OsWRKY11 protein levels were controlled by the ubiquitin-proteasome system. CONCLUSION: OsWRKY11 integrates plant responses to pathogens and abiotic stresses by positively modulating the expression of biotic and abiotic stress-related genes.

The complete nucleotide sequence of the coffee (Coffea arabica L.) chloroplast genome: organization and implications for biotechnology and phylogenetic relationships amongst angiosperms.

The chloroplast genome sequence of Coffea arabica L., the first sequenced member of the fourth largest family of angiosperms, Rubiaceae, is reported. The genome is 155 189 bp in length, including a pair of inverted repeats of 25,943 bp. Of the 130 genes present, 112 are distinct and 18 are duplicated in the inverted repeat. The coding region comprises 79 protein genes, 29 transfer RNA genes, four ribosomal RNA genes and 18 genes containing introns (three with three exons). Repeat analysis revealed five direct and three inverted repeats of 30 bp or longer with a sequence identity of 90% or more. Comparisons of the coffee chloroplast genome with sequenced genomes of the closely related family Solanaceae indicated that coffee has a portion of rps19 duplicated in the inverted repeat and an intact copy of infA. Furthermore, whole-genome comparisons identified large indels (> 500 bp) in several intergenic spacer regions and introns in the Solanaceae, including trnE (UUC)-trnT (GGU) spacer, ycf4-cemA spacer, trnI (GAU) intron and rrn5-trnR (ACG) spacer. Phylogenetic analyses based on the DNA sequences of 61 protein-coding genes for 35 taxa, performed using both maximum parsimony and maximum likelihood methods, strongly supported the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids, asterids, eurosids II, and euasterids I and II. Coffea (Rubiaceae, Gentianales) is only the second order sampled from the euasterid I clade. The availability of the complete chloroplast genome of coffee provides regulatory and intergenic spacer sequences for utilization in chloroplast genetic engineering to improve this important crop.

Effects of combinations of ROS scavengers on oxidative DNA damage caused by visible-light-activated camphorquinone/N,N-dimethyl-p-toluidine.

The objective of this investigation was to analyze whether various combinations of the ROS scavengers glutathione (GSH), N-acetyl-cysteine (NAC), and vitamins C and E decrease DNA damage due to visible-light-irradiated (VL-irradiated) camphorquinone/N,N-dimethyl-p-toluidine (CQ/DMT) compared with individual vitamin C or E. PhiX-174 RF plasmid DNA was used to determine single and double strand breaks as parameters of DNA damage. Individual ROS scavengers and combinations of the antioxidants were added to plasmid DNA treated with VL-irradiated CQ/DMT/Cu (II). After incubation, DNA was loaded into a 1% agarose gel. Following electrophoresis, gels stained with 0.5 microg/mL ethidium bromide were photographed under ultraviolet illumination and analyzed with NIH ImageJ software. Results were evaluated between groups for statistical significance using Student's paired t-test (p < 0.05). Glutathione significantly reduced oxidative DNA damage at all test concentrations when combined with vitamin C or vitamin E. The concentration of damaged DNA observed in the presence of combinations of GSH with vitamin C or vitamin E was significantly lower compared with all other combinations of antioxidants investigated in our study (p < 0.05). In contrast to GSH, NAC was not able to compensate the pro-oxidative effects of vitamin C and vitamin E. Only at a concentration of 2 mM, NAC combined with vitamin C efficiently prevented CQ/DMT/Cu (II)-associated DNA damage. Our data indicate that solely the combinations of GSH with vitamin C or vitamin E significantly reduce the severity of oxidative DNA damage caused by CQ/DMT, whereas NAC may even increase the pro-oxidant activity of vitamin C and vitamin E.

Phylogenetic analyses of Vitis (Vitaceae) based on complete chloroplast genome sequences: effects of taxon sampling and phylogenetic methods on resolving relationships among rosids.

BACKGROUND: The Vitaceae (grape) is an economically important family of angiosperms whose phylogenetic placement is currently unresolved. Recent phylogenetic analyses based on one to several genes have suggested several alternative placements of this family, including sister to Caryophyllales, asterids, Saxifragales, Dilleniaceae or to rest of rosids, though support for these different results has been weak. There has been a recent interest in using complete chloroplast genome sequences for resolving phylogenetic relationships among angiosperms. These studies have clarified relationships among several major lineages but they have also emphasized the importance of taxon sampling and the effects of different phylogenetic methods for obtaining accurate phylogenies. We sequenced the complete chloroplast genome of Vitis vinifera and used these data to assess relationships among 27 angiosperms, including nine taxa of rosids. RESULTS: The Vitis vinifera chloroplast genome is 160,928 bp in length, including a pair of inverted repeats of 26,358 bp that are separated by small and large single copy regions of 19,065 bp and 89,147 bp, respectively. The gene content and order of Vitis is identical to many other unrearranged angiosperm chloroplast genomes, including tobacco. Phylogenetic analyses using maximum parsimony and maximum likelihood were performed on DNA sequences of 61 protein-coding genes for two datasets with 28 or 29 taxa, including eight or nine taxa from four of the seven currently recognized major clades of rosids. Parsimony and likelihood phylogenies of both data sets provide strong support for the placement of Vitaceae as sister to the remaining rosids. However, the position of the Myrtales and support for the monophyly of the eurosid I clade differs between the two data sets and the two methods of analysis. In parsimony analyses, the inclusion of Gossypium is necessary to obtain trees that support the monophyly of the eurosid I clade. However, maximum likelihood analyses place Cucumis as sister to the Myrtales and therefore do not support the monophyly of the eurosid I clade. CONCLUSION: Phylogenies based on DNA sequences from complete chloroplast genome sequences provide strong support for the position of the Vitaceae as the earliest diverging lineage of rosids. Our phylogenetic analyses support recent assertions that inadequate taxon sampling and incorrect model specification for concatenated multi-gene data sets can mislead phylogenetic inferences when using whole chloroplast genomes for phylogeny reconstruction.

Complete plastid genome sequence of Daucus carota: implications for biotechnology and phylogeny of angiosperms.

BACKGROUND: Carrot (Daucus carota) is a major food crop in the US and worldwide. Its capacity for storage and its lifecycle as a biennial make it an attractive species for the introduction of foreign genes, especially for oral delivery of vaccines and other therapeutic proteins. Until recently efforts to express recombinant proteins in carrot have had limited success in terms of protein accumulation in the edible tap roots. Plastid genetic engineering offers the potential to overcome this limitation, as demonstrated by the accumulation of BADH in chromoplasts of carrot taproots to confer exceedingly high levels of salt resistance. The complete plastid genome of carrot provides essential information required for genetic engineering. Additionally, the sequence data add to the rapidly growing database of plastid genomes for assessing phylogenetic relationships among angiosperms. RESULTS: The complete carrot plastid genome is 155,911 bp in length, with 115 unique genes and 21 duplicated genes within the IR. There are four ribosomal RNAs, 30 distinct tRNA genes and 18 intron-containing genes. Repeat analysis reveals 12 direct and 2 inverted repeats > or = 30 bp with a sequence identity > or = 90%. Phylogenetic analysis of nucleotide sequences for 61 protein-coding genes using both maximum parsimony (MP) and maximum likelihood (ML) were performed for 29 angiosperms. Phylogenies from both methods provide strong support for the monophyly of several major angiosperm clades, including monocots, eudicots, rosids, asterids, eurosids II, euasterids I, and euasterids II. CONCLUSION: The carrot plastid genome contains a number of dispersed direct and inverted repeats scattered throughout coding and non-coding regions. This is the first sequenced plastid genome of the family Apiaceae and only the second published genome sequence of the species-rich euasterid II clade. Both MP and ML trees provide very strong support (100% bootstrap) for the sister relationship of Daucus with Panax in the euasterid II clade. These results provide the best taxon sampling of complete chloroplast genomes and the strongest support yet for the sister relationship of Caryophyllales to the asterids. The availability of the complete plastid genome sequence should facilitate improved transformation efficiency and foreign gene expression in carrot through utilization of endogenous flanking sequences and regulatory elements.

The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms.

BACKGROUND: Cotton (Gossypium hirsutum) is the most important fiber crop grown in 90 countries. In 2004-2005, US farmers planted 79% of the 5.7-million hectares of nuclear transgenic cotton. Unfortunately, genetically modified cotton has the potential to hybridize with other cultivated and wild relatives, resulting in geographical restrictions to cultivation. However, chloroplast genetic engineering offers the possibility of containment because of maternal inheritance of transgenes. The complete chloroplast genome of cotton provides essential information required for genetic engineering. In addition, the sequence data were used to assess phylogenetic relationships among the major clades of rosids using cotton and 25 other completely sequenced angiosperm chloroplast genomes. RESULTS: The complete cotton chloroplast genome is 160,301 bp in length, with 112 unique genes and 19 duplicated genes within the IR, containing a total of 131 genes. There are four ribosomal RNAs, 30 distinct tRNA genes and 17 intron-containing genes. The gene order in cotton is identical to that of tobacco but lacks rpl22 and infA. There are 30 direct and 24 inverted repeats 30 bp or longer with a sequence identity > or = 90%. Most of the direct repeats are within intergenic spacer regions, introns and a 72 bp-long direct repeat is within the psaA and psaB genes. Comparison of protein coding sequences with expressed sequence tags (ESTs) revealed nucleotide substitutions resulting in amino acid changes in ndhC, rpl23, rpl20, rps3 and clpP. Phylogenetic analysis of a data set including 61 protein-coding genes using both maximum likelihood and maximum parsimony were performed for 28 taxa, including cotton and five other angiosperm chloroplast genomes that were not included in any previous phylogenies. CONCLUSION: Cotton chloroplast genome lacks rpl22 and infA and contains a number of dispersed direct and inverted repeats. RNA editing resulted in amino acid changes with significant impact on their hydropathy. Phylogenetic analysis provides strong support for the position of cotton in the Malvales in the eurosids II clade sister to Arabidopsis in the Brassicales. Furthermore, there is strong support for the placement of the Myrtales sister to the eurosid I clade, although expanded taxon sampling is needed to further test this relationship.

The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms.

BACKGROUND: The production of Citrus, the largest fruit crop of international economic value, has recently been imperiled due to the introduction of the bacterial disease Citrus canker. No significant improvements have been made to combat this disease by plant breeding and nuclear transgenic approaches. Chloroplast genetic engineering has a number of advantages over nuclear transformation; it not only increases transgene expression but also facilitates transgene containment, which is one of the major impediments for development of transgenic trees. We have sequenced the Citrus chloroplast genome to facilitate genetic improvement of this crop and to assess phylogenetic relationships among major lineages of angiosperms. RESULTS: The complete chloroplast genome sequence of Citrus sinensis is 160,129 bp in length, and contains 133 genes (89 protein-coding, 4 rRNAs and 30 distinct tRNAs). Genome organization is very similar to the inferred ancestral angiosperm chloroplast genome. However, in Citrus the infA gene is absent. The inverted repeat region has expanded to duplicate rps19 and the first 84 amino acids of rpl22. The rpl22 gene in the IRb region has a nonsense mutation resulting in 9 stop codons. This was confirmed by PCR amplification and sequencing using primers that flank the IR/LSC boundaries. Repeat analysis identified 29 direct and inverted repeats 30 bp or longer with a sequence identity > or = 90%. Comparison of protein-coding sequences with expressed sequence tags revealed six putative RNA edits, five of which resulted in non-synonymous modifications in petL, psbH, ycf2 and ndhA. Phylogenetic analyses using maximum parsimony (MP) and maximum likelihood (ML) methods of a dataset composed of 61 protein-coding genes for 30 taxa provide strong support for the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids and asterids. The MP and ML trees are incongruent in three areas: the position of Amborella and Nymphaeales, relationship of the magnoliid genus Calycanthus, and the monophyly of the eurosid I clade. Both MP and ML trees provide strong support for the monophyly of eurosids II and for the placement of Citrus (Sapindales) sister to a clade including the Malvales/Brassicales. CONCLUSION: This is the first complete chloroplast genome sequence for a member of the Rutaceae and Sapindales. Expansion of the inverted repeat region to include rps19 and part of rpl22 and presence of two truncated copies of rpl22 is unusual among sequenced chloroplast genomes. Availability of a complete Citrus chloroplast genome sequence provides valuable information on intergenic spacer regions and endogenous regulatory sequences for chloroplast genetic engineering. Phylogenetic analyses resolve relationships among several major clades of angiosperms and provide strong support for the monophyly of the eurosid II clade and the position of the Sapindales sister to the Brassicales/Malvales.

Plastid transformation in the monocotyledonous cereal crop, rice (Oryza sativa) and transmission of transgenes to their progeny.

The plastid transformation approach offers a number of unique advantages, including high-level transgene expression, multi-gene engineering, transgene containment, and a lack of gene silencing and position effects. The extension of plastid transformation technology to monocotyledonous cereal crops, including rice, bears great promise for the improvement of agronomic traits, and the efficient production of pharmaceutical or nutritional enhancement. Here, we report a promising step towards stable plastid transformation in rice. We produced fertile transplastomic rice plants and demonstrated transmission of the plastid-expressed green fluorescent protein (GFP) and aminoglycoside 3'-adenylyltransferase genes to the progeny of these plants. Transgenic chloroplasts were determined to have stably expressed the GFP, which was confirmed by both confocal microscopy and Western blot analyses. Although the produced rice plastid transformants were found to be heteroplastomic, and the transformation efficiency requires further improvement, this study has established a variety of parameters for the use of plastid transformation technology in cereal crops.

The effect of sling exercise on sagittal lumbosacral angle and intervertebral disc area of chronic low back pain patients.

The purpose of this study was to observe the change of lumbosacral angle and intervertebral disc (IVD) area. The study was conducted on chronic low back pain (CLBP) female patients for 12 weeks by operating sling exercise and general physical therapy. The 57 CLBP were divided into 2 groups which, sling exercise group (SEG, n=34) and general physical therapy group (PTG, n=23). The experiment was conducted three times a week for 12 weeks. The lumbosacral angle, which means the angle between the L1-L2 lumbar was measured by plain radiography. The IVD area, which means the IVD height and volume was measured by magnetic resonance imaging. The pain was measured by visual analogue scale (VAS). As a result, after 12-week exercise, VAS had decreased in all groups. The angle of L3-4 and L4-5 and the height of IVD had increased in SEG. Also, IVD height and volume has more improved in SEG compare the PTG. Therefore, the sling exercise is proper treatment for CLBP patients' recovery because It improve the lumbosacral angle and IVD area.