Whole genome mapping in a wheat doubled haploid population using SSRs and TRAPs and the identification of QTL for agronomic traits

Genetic maps are useful for detecting quantitative trait loci (QTL) associated with quantitative traits and for marker-assisted selection (MAS) in breeding. In this research, we used the wheat × maize method to develop a doubled haploid (DH) population derived from the synthetic hexaploid wheat (SHW) line TA4152-60 and the North Dakota hard red spring wheat line ND495. The population consisted of 213 lines, of which a subset of 120 lines was randomly selected and used to construct linkage maps of all 21 chromosomes and for QTL detection. The whole genome maps consisted of 632 markers including 410 SSRs, 218 TRAPs, 1 RFLP, and 3 phenotypic markers, and spanned 3,811.5 cM with an average density of one marker per 6.03 cM. Telomere sequence-based TRAPs allowed us to define the ends of seven linkage groups. Analysis revealed major QTLs associated with the traits of days to heading on chromosomes 5A and 5B, plant height on chromosomes 4D and 5A, and spike characteristics on chromosomes 3D, 4A, 4D, 5A and 5B. The DH population and genetic map will be a useful tool for the identification of disease resistance QTL and agronomically important loci, and will aid in the identification and development of markers for MAS. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.     

A combined computational-experimental analyses of selected metabolic enzymes in Pseudomonas species

Comparative genomic analysis has revolutionized our ability to predict the metabolic subsystems that occur in newly sequenced genomes, and to explore the functional roles of the set of genes within each subsystem. These computational predictions can considerably reduce the volume of experimental studies required to assess basic metabolic properties of multiple bacterial species. However, experimental validations are still required to resolve the apparent inconsistencies in the predictions by multiple resources. Here, we present combined computational-experimental analyses on eight completely sequenced Pseudomonas species. Comparative pathway analyses reveal that several pathways within the Pseudomonas species show high plasticity and versatility. Potential bypasses in 11 metabolic pathways were identified. We further confirmed the presence of the enzyme O-acetyl homoserine (thiol) lyase (EC: 2.5.1.49) in P. syringae pv. tomato that revealed inconsistent annotations in KEGG and in the recently published SYSTOMONAS database. These analyses connect and integrate systematic data generation, computational data interpretation, and experimental validation and represent a synergistic and powerful means for conducting biological research.     

Challenges and opportunities for photochemists on the verge of solar energy conversion.

Has photochemistry missed the boat on solar energy conversion? Certainly not, but it is time to reach out and make a difference if we do not want to have to choose between feeding our families or our thirst for fuel. Compared to other initiatives, such as biofuels or nuclear fusion, direct conversion of solar energy into electricity or fuels is lagging behind in terms of funding, and this is slowing progress on overcoming critical bottlenecks. This perspective outlines some of the key fundamental issues in solar energy conversion based on organic photovoltaic devices or artificial photosynthesis where being a photochemist can make a difference.     

Cellular conditioning with trichostatin A enhances the anti-stress response through up-regulation of HDAC4 and down-regulation of the IGF/Akt pathway

Evidence is accumulating that chromatin plays a major role in the control of cellular response to stress. This is best illustrated by the recent findings that chromatin-modifying factors of class III histone deacetylases (sirtuins) are capable of protecting cells from oxidative and genotoxic stress. In particular, Sirt1 has been shown to mimic the action of caloric restriction for the prevention of aging-associated diseases. In the present study, we have investigated the potential role of class I and II histone deacetylases (HDACs) in cellular protection against various stresses, including those caused by nutrient deprivation. For this, we utilized a cellular model in which expression of class I and II HDACs was altered as a result of cellular adaptation to trichostatin A (TSA), a selective inhibitor of these deacetylases. Our results indicated that TSA-resistant cells also developed resistance to H₂O₂, DNA-damaging agents, and to nutrient deprivation. Interestingly, the insulin signaling pathway mediated by Akt was inhibited in the TSA-resistant cells, mirroring the effect of glucose deprivation on this pathway. Since expression of HDAC4 was consistently enhanced in the TSA-resistant cell lines, we suggest that this enzyme may contribute to their anti-stress response. In agreement with this, siRNA-mediated knockdown of HDAC4 in stress-resistant cells enhanced their sensitivity to the DNA-damaging drug doxorubicin and also to glucose deprivation. Akt phosphorylation was also up-regulated in response to HDC4 knockdown. Together, these findings suggest that cellular conditioning with TSA may represent a useful approach to mimic the effects of caloric restriction.     

Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes

In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte-keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.     

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.     

Discovery of a series of aminopiperidines as novel iNOS inhibitors

Nitric oxide (NO), a mediator of various physiological and pathophysiological processes, is synthesized by three isozymes of nitric oxide synthase (NOS). Potential candidate clinical drugs should be devoid of inhibitory activity against endothelial NOS (eNOS), since eNOS plays an important role in maintaining normal blood pressure and flow. A new series of aminopiperidines as potent inhibitors of iNOS were identified from a HTS lead. From this study, we identified compound 33 as a potent iNOS inhibitor, with >25-fold selectivity over eNOS and 16-fold selectivity over nNOS.     

Discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors

The design of amide and heteroaryl amide isosteres as replacements for the carbamate substructure in previously disclosed 2,6-disubstituted piperidine N-arylsulfonamides is described. In several cases, amides lessened CYP liabilities in this class of gamma-secretase inhibitors. Selected compounds showed significant reduction of Abeta levels upon oral dosing in a transgenic murine model of Alzheimer's disease.     

Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties

Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.