Central sleep apnea treated by a constant low-dose CO2 supplied by a novel device.

CO2 inhalation has been previously reported as a treatment for central sleep apnea both when associated with heart failure or where the cause is unknown. Here, we evaluated a novel CO2 supply system using a novel open mask capable of comfortably delivering a constantly inspired fraction of CO2 ([Formula: see text]) during sleep. We recruited 18 patients with central sleep apnea (13 patients with cardiac disease, and 5 patients idiopathic) diagnosed by diaphragm electromyogram (EMG) recordings made during overnight full polysomnography (PSG) (night 1). In each case, the optimal [Formula: see text] was determined by an overnight manual titration with PSG (night 2). Titration commenced at 1% CO2 and increased by 0.2% increments until central sleep apnea (CSA) disappeared. Patients were then treated on the third night (night 3) with the lowest therapeutically effective concentration of CO2 derived from night 2. Comparing night 1 and night 3, both apnea-hypopnea index (AHI; 31 ± 14 vs. 6 ± 3 events/h, P < 0.01) and arousal index (22 ± 8 vs. 15 ± 8 events/h, P < 0.01) were significantly improved during CO2 treatment. Sleep efficiency improved from 71 ± 18 to 80 ± 11%, P < 0.05, and sleep latency was shorter (23 ± 18 vs. 10 ± 10 min, P < 0.01). Heart rate was not different between night 1 and night 3. Our data confirm the feasibility of our CO2 delivery system and indicate that individually titrated CO2 supplementation with a novel device including a special open mask can reduce sleep disordered breathing severity and improve sleep quality. Randomized controlled studies should now be undertaken to assess therapeutic benefit for patients with CSA.NEW & NOTEWORTHY A novel device using a special mask was developed and proved that CO2 therapy using the device could eliminate central sleep apnea (CSA) events and improve sleep quality including reducing arousal index in patients with heart failure. The device would become a useful clinical treatment for heart failure patients with CSA.

Differential regulation of G protein signaling in Arabidopsis through two distinct pathways that internalize AtRGS1.

In animals, endocytosis of a seven-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic G protein-mediated signaling, depending on the bias of the receptor or ligand, which determines how much one transduction pathway is used compared to another. In Arabidopsis thaliana, GPCRs are not required for G protein-coupled signaling because the heterotrimeric G protein complex spontaneously exchanges nucleotide. Instead, the seven-transmembrane protein AtRGS1 modulates G protein signaling through ligand-dependent endocytosis, which initiates derepression of signaling without the involvement of canonical arrestins. Here, we found that endocytosis of AtRGS1 initiated from two separate pools of plasma membrane: sterol-dependent domains and a clathrin-accessible neighborhood, each with a select set of discriminators, activators, and candidate arrestin-like adaptors. Ligand identity (either the pathogen-associated molecular pattern flg22 or the sugar glucose) determined the origin of AtRGS1 endocytosis. Different trafficking origins and trajectories led to different cellular outcomes. Thus, in this system, compartmentation with its associated signalosome architecture drives biased signaling.

Stem vacuole-targetted sucrose isomerase enhances sugar content in sorghum.

BACKGROUND: Sugar content is critically important in determining sugar crop productivity. However, improvement in sugar content has been stagnant among sugar crops for decades. Sorghum, especially sweet sorghum with high biomass, shown great potential for biofuel, has lower sugar content than sugarcane. To enhance sugar content, the sucrose isomerase (SI) gene, driven by stem-specific promoters (A2 or LSG) with a vacuole-targetted signal peptide, was transformed into the sorghum inbred line (T×430). RESULTS: The study demonstrated that transgenic lines of grain sorghum, containing 50-60% isomaltulose, accumulated up to eightfold (1000 mM) more total sugar than the control T×430 did (118 mM) in stalks of T0 generation. Subsequently, the elite engineered lines (A5, and LSG9) were crossed with sweet sorghum (Rio, and R9188). Total sugar contents (over 750 mM), were notably higher in F1, and F2 progenies than the control Rio (480 mM). The sugar contents of the engineered lines (over 750 mM), including T0, T1, F1, and F2, are surprisingly higher than that of the field-grown sugarcane (normal range 600-700 mmol/L). Additionally, analysis of physiological characterization demonstrated that the superior progenies had notably higher rates of photosynthesis, sucrose transportation, and sink strength than the controls. CONCLUSIONS: The genetic engineering approach has dramatically enhanced total sugar content in grain sorghum (T0, and T1) and hybrid sorghum (F1, and F2), demonstrating that sorghum can accumulate as high or higher sugar content than sugarcane. This research illustrates that the SI gene has enormous potential on improvement of sugar content in sorghum, particularly in hybirds and sweet sorghum. The substantial increase on sugar content would lead to significant financial benefits for industrial utilization. This study could have a substantial impact on renewable bioenergy. More importantly, our results demonstrated that the phenotype of high sugar content is inheritable and shed light on improvement for other sugar crops.

Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3--high copy number group, TST-1 and PRR-1--medium copy number group, P4H-1, APRT-2 and CYC-2--low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

Establishment and application of a loop-mediated isothermal amplification (LAMP) system for detection of cry1Ac transgenic sugarcane.

To meet the demand for detection of foreign genes in genetically modified (GM) sugarcane necessary for regulation of gene technology, an efficient method with high specificity and rapidity was developed for the cry1Ac gene, based on loop-mediated isothermal amplification (LAMP). A set of four primers was designed using the sequence of cry1Ac along with optimized reaction conditions: 5.25 mM of Mg(2+), 4:1 ratio of inner primer to outer primer, 2.0 U of Bst DNA polymerase in a reaction volume of 25.0 µL. Three post-LAMP detection methods (precipitation, calcein (0.60 mM) with Mn(2+) (0.05 mM) complex and SYBR Green I visualization), were shown to be effective. The sensitivity of the LAMP method was tenfold higher than that of conventional PCR when using templates of the recombinant cry1Ac plasmid or genomic DNA from cry1Ac transgenic sugarcane plants. More importantly, this system allowed detection of the foreign gene on-site when screening GM sugarcane without complex and expensive instruments, using the naked eye. This method can not only provide technological support for detection of cry1Ac, but can also further facilitate the use of this detection technique for other transgenes in GM sugarcane.

Molecular cloning and characterization of two pathogenesis-related ß-1,3-glucanase genes ScGluA1 and ScGluD1 from sugarcane infected by Sporisorium scitamineum.

KEY MESSAGE: Two ß-1,3-glucanase genes from sugarcane were cloned and characterized. They were all located in apoplast and involves in different expression patterns in biotic and abiotic stress. Smut caused by Sporisorium scitamineum is a serious disease in the sugarcane industry. ß-1,3-Glucanase, a typical pathogenesis-related protein, has been shown to express during plant-pathogen interaction and involves in sugarcane defense response. In this study, ß-1,3-glucanase enzyme activity in the resistant variety increased faster and lasted longer than that of the susceptible one when inoculated with S. scitamineum, along with a positive correlation between the activity of the ß-1,3-glucanase and smut resistance. Furthermore, two ß-1,3-glucanase genes from S. scitamineum infected sugarcane, ScGluA1 (GenBank Accession No. KC848050) and ScGluD1 (GenBank Accession No. KC848051) were cloned and characterized. Phylogenetic analysis suggested that ScGluA1 and ScGluD1 clustered within subfamily A and subfamily D, respectively. Subcellular localization analysis demonstrated that both gene products were targeted to apoplast. Escherichia coli Rosetta (DE3) cells expressing ScGluA1 and ScGluD1 showed varying degrees of tolerance to NaCl, CdCl2, PEG, CuCl2 and ZnSO4. Q-PCR analysis showed up-regulation of ScGluA1 and slight down-regulation of ScGluD1 in response to S. scitamineum infection. It suggested that ScGluA1 may be involved in the defense reaction of the sugarcane to the smut, while it is likely that ScGluD1 was inhibited. The gene expression patterns of ScGluA1 and ScGluD1, in response to abiotic stresses, were similar to sugarcane response against smut infection. Together, ß-1,3-glucanase may function in sugarcane defense mechanism for S. scitamineum. The positive responses of ScGluA1 and the negative responses of ScGluD1 to biotic and abiotic stresses indicate they play different roles in interaction between sugarcane and biotic or abiotic stresses.

Field performance of transgenic sugarcane expressing isomaltulose synthase.

Transgenic sugarcane plants expressing a vacuole-targeted isomaltulose (IM) synthase in seven recipient genotypes (elite cultivars) were evaluated over 3 years at a field site typical of commercial cane growing conditions in the Burdekin district of Australia. IM concentration typically increased with internode maturity and comprised up to 217 mm (33% of total sugars) in whole-cane juice. There was generally a comparable decrease in sucrose concentration, with no overall decrease in total sugars. Sugarcane is vegetatively propagated from stem cuttings known as setts. Culture-derived plants were slower to establish and generally gave shorter and thinner stalks at harvest than those grown from field-sourced setts in the initial field generations. However, after several cycles of field propagation, selections were obtained with cane yields similar to the recipient genotypes. There was no apparent adverse effect of IM accumulation on vigour assessed by stalk height and diameter or other visual indicators including germination of setts and establishment of stools. There was some inconsistency in IM levels in juice, between samplings of the vegetatively propagated transgenic lines. Until the causes are resolved, it is prudent to selectively propagate from stalks with higher IM levels in the initial vegetative field generations. Pol/Brix ratio allowed rapid identification of lines with high IM levels, using common sugar industry instruments. Sucrose isomerase activity was low in these transgenic lines, and the results indicate strong potential to develop sugarcane for commercial-scale production of IM if higher activity can be engineered in appropriate developmental patterns.

Isomaltulose is actively metabolized in plant cells.

Isomaltulose is a structural isomer of sucrose (Suc). It has been widely used as a nonmetabolized sugar in physiological studies aimed at better understanding the regulatory roles and transport of sugars in plants. It is increasingly used as a nutritional human food, with some beneficial properties including low glycemic index and acariogenicity. Cloning of genes for Suc isomerases opened the way for direct commercial production in plants. The understanding that plants lack catabolic capabilities for isomaltulose indicated a possibility of enhanced yields relative to Suc. However, this understanding was based primarily on the treatment of intact cells with exogenous isomaltulose. Here, we show that sugarcane (Saccharum interspecific hybrids), like other tested plants, does not readily import or catabolize extracellular isomaltulose. However, among intracellular enzymes, cytosolic Suc synthase (in the breakage direction) and vacuolar soluble acid invertase (SAI) substantially catabolize isomaltulose. From kinetic studies, the specificity constant of SAI for isomaltulose is about 10% of that for Suc. Activity varied against other Suc isomers, with V(max) for leucrose about 6-fold that for Suc. SAI activities from other plant species varied substantially in substrate specificity against Suc and its isomers. Therefore, in physiological studies, the blanket notion of Suc isomers including isomaltulose as nonmetabolized sugars must be discarded. For example, lysis of a few cells may result in the substantial hydrolysis of exogenous isomaltulose, with profound downstream signal effects. In plant biotechnology, different V(max) and V(max)/K(m) ratios for Suc isomers may yet be exploited, in combination with appropriate developmental expression and compartmentation, for enhanced sugar yields.

Doubled sugar content in sugarcane plants modified to produce a sucrose isomer.

Sucrose is the feedstock for more than half of the world's fuel ethanol production and a major human food. It is harvested primarily from sugarcane and beet. Despite attempts through conventional and molecular breeding, the stored sugar concentration in elite sugarcane cultivars has not been increased for several decades. Recently, genes have been cloned for bacterial isomerase enzymes that convert sucrose into sugars which are not metabolized by plants, but which are digested by humans, with health benefits over sucrose. We hypothesized that an appropriate sucrose isomerase (SI) expression pattern might simultaneously provide a valuable source of beneficial sugars and overcome the sugar yield ceiling in plants. The introduction of an SI gene tailored for vacuolar compartmentation resulted in sugarcane lines with remarkable increases in total stored sugar levels. The high-value sugar isomaltulose was accumulated in storage tissues without any decrease in stored sucrose concentration, resulting in up to doubled total sugar concentrations in harvested juice. The lines with enhanced sugar accumulation also showed increased photosynthesis, sucrose transport and sink strength. This remarkable step above the former ceiling in stored sugar concentration provides a new perspective into plant source-sink relationships, and has substantial potential for enhanced food and biofuel production.

Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene.

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35 degrees C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50 degrees C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35 degrees C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the Km was 39.9 mM, the Vmax was 638 U mg(-1), and the Kcat/Km was 1.79 x 10(4) M(-1) s(-1), compared to a Km of 76.0 mM, a Vmax of 423 U mg(-1), and a Kcat/Km of 0.62 x 10(4) M(-1) s(-1) for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.