Tomato expressing Arabidopsis glutaredoxin gene AtGRXS17 confers tolerance to chilling stress via modulating cold responsive components

Chilling stress is a production constraint of tomato, a tropical origin, chilling-sensitive horticultural crop. The development of chilling tolerant tomato thus has significant potential to impact tomato production. Glutaredoxins (GRXs) are ubiquitous oxidoreductases, which utilize the reducing power of glutathione to reduce disulfide bonds of substrate proteins and maintain cellular redox homeostasis. Here, we report that tomato expressing Arabidopsis GRX gene AtGRXS17 conferred tolerance to chilling stress without adverse effects on growth and development. AtGRXS17-expressing tomato plants displayed lower ion leakage, higher maximal photochemical efficiency of photosystem II (Fv/Fm) and increased accumulation of soluble sugar compared with wild-type plants after the chilling stress challenge. Furthermore, chilling tolerance was correlated with increased antioxidant enzyme activities and reduced H(2)O(2) accumulation. At the same time, temporal expression patterns of the endogenous C-repeat/DRE-binding factor 1 (SlCBF1) and CBF mediated-cold regulated genes were not altered in AtGRXS17-expressing plants when compared with wild-type plants, and proline concentrations remained unchanged relative to wild-type plants under chilling stress. Green fluorescent protein -AtGRXS17 fusion proteins, which were initially localized in the cytoplasm, migrated into the nucleus during chilling stress, reflecting a possible role of AtGRXS17 in nuclear signaling of chilling stress responses. Together, our findings demonstrate that genetically engineered tomato plants expressing AtGRXS17 can enhance chilling tolerance and suggest a genetic engineering strategy to improve chilling tolerance without yield penalty across different crop species

O-GlcNAc modulation at Akt1 Ser473 correlates with apoptosis of murine pancreatic β cells

O-GlcNAc transferase (OGT)-mediated modification of protein Ser/Thr residues withO-GlcNAc influences protein activity, similar to the effects of phosphorylation. The anti-apoptotic Akt1 is both activated by phosphorylation and modified with O-GlcNAc. However, the nature and significance of the Akt1 O-GlcNAc modification is unknown. The relationship of O-GlcNAc modification and phosphorylation at Akt1 Ser473 was examined with respect to apoptosis of murine β-pancreatic cells. Glucosamine treatment induced apoptosis, which correlated with enhanced O-GlcNAc modification of Akt1 and concomitant reduction in Ser473 phosphorylation. Pharmacological inhibition of OGT or O-GlcNAcase revealed an inverse correlation between O-GlcNAcmodification and Ser473 phosphorylation of Akt1. MALDI-TOF/TOFmass spectrometry analysis of Akt1 immunoprecipitates fromglucosamine-treated cells, but not untreated controls, showed a peptide containing S473/T479 that was presumably modified withO-GlcNAc. Furthermore, in vitroO-GlcNAc-modification analysis of wildtype and mutant Akt1 revealed that S473 was targeted by recombinant OGT. A S473A Akt1 mutant demonstrated reduced basal and glucosamine-induced Akt1 O-GlcNAc modification compared with wildtype Akt1. Furthermore, wildtype Akt1, but not the S473A mutant, appeared to be associated with OGT following glucosamine treatment. Together, these observations suggest that Akt1 Ser473 may undergo both phosphorylation and O-GlcNAc modification, and the balance between these may regulatemurine β-pancreatic cell fate

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including "safe harboring" techniques shown in other organisms142561sciescopu

CRISPR/Cas9-induced knockout and knock-in mutations in Chlamydomonas reinhardtii

Genome editing is crucial for genetic engineering of organisms for improved traits, particularly in microalgae due to the urgent necessity for the next generation biofuel production. The most advanced CRISPR/Cas9 system is simple, efficient and accurate in some organisms; however, it has proven extremely difficult in microalgae including the model alga Chlamydomonas. We solved this problem by delivering Cas9 ribonucleoproteins (RNPs) comprising the Cas9 protein and sgRNAs to avoid cytotoxicity and off-targeting associated with vector-driven expression of Cas9. We obtained CRISPR/Cas9-induced mutations at three loci including MAA7, CpSRP43 and ChlM, and targeted mutagenic efficiency was improved up to 100 fold compared to the first report of transgenic Cas9-induced mutagenesis. Interestingly, we found that unrelated vectors used for the selection purpose were predominantly integrated at the Cas9 cut site, indicative of NHEJ-mediated knock-in events. As expected with Cas9 RNPs, no off-targeting was found in one of the mutagenic screens. In conclusion, we improved the knockout efficiency by using Cas9 RNPs, which opens great opportunities not only for biological research but also industrial applications in Chlamydomonas and other microalgae. Findings of the NHEJ-mediated knock-in events will allow applications of the CRISPR/Cas9 system in microalgae, including safe harboring techniques shown in other organisms.

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

Canagliflozin and renal outcomes in type 2 diabetes and nephropathy

BACKGROUND Type 2 diabetes mellitus is the leading cause of kidney failure worldwide, but few effective long-term treatments are available. In cardiovascular trials of inhibitors of sodium–glucose cotransporter 2 (SGLT2), exploratory results have suggested that such drugs may improve renal outcomes in patients with type 2 diabetes. METHODS In this double-blind, randomized trial, we assigned patients with type 2 diabetes and albuminuric chronic kidney disease to receive canagliflozin, an oral SGLT2 inhibitor, at a dose of 100 mg daily or placebo. All the patients had an estimated glomerular filtration rate (GFR) of 30 to <90 ml per minute per 1.73 m2 of body-surface area and albuminuria (ratio of albumin [mg] to creatinine [g], >300 to 5000) and were treated with renin–angiotensin system blockade. The primary outcome was a composite of end-stage kidney disease (dialysis, transplantation, or a sustained estimated GFR of <15 ml per minute per 1.73 m2), a doubling of the serum creatinine level, or death from renal or cardiovascular causes. Prespecified secondary outcomes were tested hierarchically. RESULTS The trial was stopped early after a planned interim analysis on the recommendation of the data and safety monitoring committee. At that time, 4401 patients had undergone randomization, with a median follow-up of 2.62 years. The relative risk of the primary outcome was 30% lower in the canagliflozin group than in the placebo group, with event rates of 43.2 and 61.2 per 1000 patient-years, respectively (hazard ratio, 0.70; 95% confidence interval [CI], 0.59 to 0.82; P=0.00001). The relative risk of the renal-specific composite of end-stage kidney disease, a doubling of the creatinine level, or death from renal causes was lower by 34% (hazard ratio, 0.66; 95% CI, 0.53 to 0.81; P<0.001), and the relative risk of end-stage kidney disease was lower by 32% (hazard ratio, 0.68; 95% CI, 0.54 to 0.86; P=0.002). The canagliflozin group also had a lower risk of cardiovascular death, myocardial infarction, or stroke (hazard ratio, 0.80; 95% CI, 0.67 to 0.95; P=0.01) and hospitalization for heart failure (hazard ratio, 0.61; 95% CI, 0.47 to 0.80; P<0.001). There were no significant differences in rates of amputation or fracture. CONCLUSIONS In patients with type 2 diabetes and kidney disease, the risk of kidney failure and cardiovascular events was lower in the canagliflozin group than in the placebo group at a median follow-up of 2.62 years

Alternatives to Fuel Tax: a State Level Perspective

Under the current fuel-tax-based highway financing system, the funding gap between highway needs and revenue available is expected to grow as vehicle fuel economy improves and use of alternative fuels increases. Consequently, the highway financing mechanism needs to be restructured or a different financing strategy undertaken. Previous research provided examples of successful implementation of pricing schemes in terms of design of pricing scheme, technology issues, legal and institutional issues, and public acceptability. Synthesizing this information, a methodological framework was established for evaluation of alternative user charging schemes. Based on the assessment of the current and projected highway revenue and the needs and demand of Indiana, the study developed three alternative financing schemes: (1) enhancement of the current tax system; (2) addition of new funding sources to supplement the current system; and (3) replacement of the current system with new financing schemes. The first alternative suggests an increase and/or indexing of fuel taxes and vehicle registration fees, supplemented by adjustments in other general taxes where possible. The second alternative includes an option to impose tolls, which was estimated to yield annual revenue in the range of $40-$90 million, depending on the toll rate schedules. The third alternative is to establish a distance-based charging scheme (VMT fees), the rates of which can be calculated from highway expenditure and demand information. Based on the past three-year data, it was found that on average 2.9 cents need to be charged per vehicle-mile traveled, with separate rates varying by road functional class. More rigorous analysis was conducted on the basis of a highway cost allocation study to calculate separate unit rates by vehicle class as well as road functional class. Under the suggested pricing structure, named as Indiana Statewide Comprehensive Usage-based Road Pricing (ISCURP), automobiles are charged 1.21 cents per mile, single unit trucks 9.18 cents per mile, and combination trucks 23.54 cents per mile. Each alternative was evaluated on the basis of the established criteria and compared with the other two alternatives. The third alternative, to replace the current fuel tax system with VMT fees, was found to have the best potential in achieving revenue adequacy, system efficiency, and equity. The implementation of this alternative, however, could be cost-prohibitive and could face opposition from the public. Based on evaluation results, a stepwise modification of the current system was proposed, aiming at a long-term goal of implementing ISCURP. In the short-term, it is suggested to implement the first alternative, which requires minimal cost of execution, and in the meantime, to inform and educate the public to prepare them for major changes in pricing schemes. In the medium-term, a pilot study for ISCURP could be completed. Finally, in the long-term, a structure would need to be designed whereby highway agencies could know the exact costs of preservation and maintenance by facility type and user classification so that ISCURP can be successfully implemented

Diagnosis and Prioritization of Vulnerable Areas of Urban Ecosystem Regulation Services

Rapid urbanization and population growth have led to drastic degradation of urban ecosystem regulation services (ERS). Urgently needed is the identification of vulnerable areas where ERS are being intensively deteriorated, and preparation of measures to respond to them. This study developed a framework to diagnose and prioritize vulnerable areas of urban ERS. The vulnerability of urban ERS that include carbon storage capacity, flood-risk mitigation capacity, and heat stress reduction capacity was diagnosed with a resolution of 100 m × 100 m grid. Priority areas to improve urban ERS were delineated using hot spot analysis, and the diagnosed results of the urban ERS were categorized by eight combination types including exposure, sensitivity, and adaptability. The spatial and societal problems included in the priority areas were further investigated by overlaying hot spot areas with eight combination maps. Finally, spatial management measures for the priority areas were suggested based on the analysis results. From the detailed diagnosis results of the vulnerable ERS areas, this study provides a framework to link the concept of ERS vulnerability with urban planning. Furthermore, effective spatial planning guidelines can be prepared to improve urban ERS by spatially delineating priority areas to improve urban ERS vulnerability

Diagnosis and Prioritization of Vulnerable Areas of Urban Ecosystem Regulation Services

Rapid urbanization and population growth have led to drastic degradation of urban ecosystem regulation services (ERS). Urgently needed is the identification of vulnerable areas where ERS are being intensively deteriorated, and preparation of measures to respond to them. This study developed a framework to diagnose and prioritize vulnerable areas of urban ERS. The vulnerability of urban ERS that include carbon storage capacity, flood-risk mitigation capacity, and heat stress reduction capacity was diagnosed with a resolution of 100 m × 100 m grid. Priority areas to improve urban ERS were delineated using hot spot analysis, and the diagnosed results of the urban ERS were categorized by eight combination types including exposure, sensitivity, and adaptability. The spatial and societal problems included in the priority areas were further investigated by overlaying hot spot areas with eight combination maps. Finally, spatial management measures for the priority areas were suggested based on the analysis results. From the detailed diagnosis results of the vulnerable ERS areas, this study provides a framework to link the concept of ERS vulnerability with urban planning. Furthermore, effective spatial planning guidelines can be prepared to improve urban ERS by spatially delineating priority areas to improve urban ERS vulnerability