Genome-wide functional annotation and structural verification of metabolic ORFeome of Chlamydomonas reinhardtii

<p>Abstract</p> <p>Background</p> <p>Recent advances in the field of metabolic engineering have been expedited by the availability of genome sequences and metabolic modelling approaches. The complete sequencing of the <it>C. reinhardtii</it> genome has made this unicellular alga a good candidate for metabolic engineering studies; however, the annotation of the relevant genes has not been validated and the much-needed metabolic ORFeome is currently unavailable. We describe our efforts on the functional annotation of the ORF models released by the Joint Genome Institute (JGI), prediction of their subcellular localizations, and experimental verification of their structural annotation at the genome scale.</p> <p>Results</p> <p>We assigned enzymatic functions to the translated JGI ORF models of <it>C. reinhardtii</it> by reciprocal BLAST searches of the putative proteome against the UniProt and AraCyc enzyme databases. The best match for each translated ORF was identified and the EC numbers were transferred onto the ORF models. Enzymatic functional assignment was extended to the paralogs of the ORFs by clustering ORFs using BLASTCLUST.</p> <p>In total, we assigned 911 enzymatic functions, including 886 EC numbers, to 1,427 transcripts. We further annotated the enzymatic ORFs by prediction of their subcellular localization. The majority of the ORFs are predicted to be compartmentalized in the cytosol and chloroplast. We verified the structure of the metabolism-related ORF models by reverse transcription-PCR of the functionally annotated ORFs. Following amplification and cloning, we carried out 454FLX and Sanger sequencing of the ORFs. Based on alignment of the 454FLX reads to the ORF predicted sequences, we obtained more than 90% coverage for more than 80% of the ORFs. In total, 1,087 ORF models were verified by 454 and Sanger sequencing methods. We obtained expression evidence for 98% of the metabolic ORFs in the algal cells grown under constant light in the presence of acetate.</p> <p>Conclusions</p> <p>We functionally annotated approximately 1,400 JGI predicted metabolic ORFs that can facilitate the reconstruction and refinement of a genome-scale metabolic network. The unveiling of the metabolic potential of this organism, along with structural verification of the relevant ORFs, facilitates the selection of metabolic engineering targets with applications in bioenergy and biopharmaceuticals. The ORF clones are a resource for downstream studies.</p

Comparison of disease resistance of maize varieties from the 1950s to the 2000s in China

The objective of this investigation was to analyze trends in diseases resistance along with genetic gain. Experimental materials consisted of maize varieties selected from each decade beginning with the 1950s. These varieties were evaluated for resistance to maize dwarf mosaic virus (MDMV), maize rough dwarf virus (MRDV), common smut disease (CSD) and head smut disease (HSD) in several different locations. Artificial inoculation was adopted for infection with MDMV and HSD, whereas natural infection was used for infection MRDV and CSD. Results indicated that resistance of the newer varieties to MDMV, CSD, and HSD was greater than that of older varieties, but the correlation to decades was not significant. To date, no variety tested in China has shown resistance to MRDV, which is likely due to a lack of maize germplasm resources resistant to MRDV in China. So the next goal will be to import new germplasm resources and select resistant germplasm as the basis of breeding resistant varieties

SH3 interactome conserves general function over specific form

Src homology 3 (SH3) domains bind peptides to mediate protein–protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form

Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism

Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases

A reference map of the human binary protein interactome.

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships(1,2). Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome(3), transcriptome(4) and proteome(5) data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes

Achieving enhanced cryogenic toughness in a 1 GPa grade HSLA steel through reverse transformation of martensite

The effect of microstructure on crack resistance and cryogenic toughening in a 3.5 wt% Ni high-strength low-alloy (HSLA) steel was investigated. Multistage heat treatments involving quenching (Q), lamellarization (L), and tempering (T) were applied to prepare the HSLA steels with various microstructures, focusing on the reverse transformation and reconfiguration of martensite, as well as its influence on impact crack formation and propagation behavior by multi-scale characterizations. The results indicate that lamellarization treatment has little influence on tensile properties, but significantly improves impact toughness in the QL and QLT specimens, which exhibit over 30 % increment in Charpy V-notch (CVN) absorbed energy Et tested in range from RT to −196 °C, over 25 °C decrement in ductile-brittle transition temperature (DBTT), and much higher crack propagation energy Ep and higher ratio of Ep/Et, as compared with the as-quenched (AQ) and QT specimens. The lamellarization treatment also contributes to a significant refinement effect on martensitic block size, caused by fresh martensite transformation from the reversed austenite, resulting in an increment in high angle grain boundaries (HAGBs), with introduction of a small amount of retained austenite (RA) as well. Therefore, the impact crack resistance and cryogenic toughness is improved in specimens processed with lamellarization treatment, due to the enhancement in crack deflection and hindering effect of the HAGBs, as well as toughening effect by in situ austenite-to-martensite transformation of the RA. Based on the present study, a 1 GPa grade HSLA steel with high ductility and excellent cryogenic toughness can be produced

Geographic distribution, source analysis, and ecological risk assessment of PTEs in the topsoil of different land uses around the antimony tailings tank: A case study of Longwangchi tailings pond, Hunan, China

Few studies have addressed the contamination of surface soils around antimony tailings ponds, and studying the contamination levels and sources of potentially toxic elements (PTEs) in soils around antimony tailings ponds and assessing the associated environmental risks are key steps in conducting environmental protection. Therefore, this study is the first to investigate the current status, spatial distribution, potential sources and ecological risks of Pb, Sb, Cr, Zn, Cd, As and Cu contamination in surface soils in woodlands, grasslands, farmlands and construction areas around the Longwangchi antimony tailings pond in Hunan, China. According to the analytic results of soil samples, the order of PTEs in terms of average concentration is: Sb > Zn > Cu > Pb > As > Cr > Cd. The cumulative index of land was applied to define Sb and Cd as extremely heavy pollution, and PLI divides the overall pollution level of soil into serious pollution levels. The ecological risk index was used to evaluate the risk characteristics of PTEs. The results showed that the ecological risk of Sb was particularly prominent, and 95% of the study area had reached a very high risk level. A quantitative comparison of ecological risk levels between different land uses revealed that there was a clear dividing line between regions, with built-up areas contributing the most to higher ecological risk at 8.89%, followed by 1.72% for grassland, 1.15% for agricultural land, and 0.57% for woodland. The visualization results of spatial distribution of PTEs exhibited that severe pollution occurred in the middle, north and southeast of the study area, and these distribution characteristics are mainly dictated by the polluted runoffs from tailings ponds and human activities. Principal component analysis (PCA) and positive matrix decomposition (PMF) were employed to identify the exact pollution sources, and the closer the determination coefficient (R2) of PMF was to 1, the finer the pollution sources analyzed. A total of six potential sources of PTEs were analyzed by PMF: 36% of tailings pond contamination and mining operations, 8% atmospheric deposition, 19% traffic emissions, 9% combustion emissions, 11% natural sources, and 17% inputs related to agricultural production. This study is a complement to the study of environmental pollution around antimony tailing ponds, providing evaluation criteria for the pollution identification indicators of antimony tailing ponds

Enhanced free surface mobility facilitates the release of free-volume holes in thin-film polymer glasses

The concept of free volume underpins the variations in polymer dynamics. Annihilation of free-volume holes through diffusion to surfaces has been proposed to determine the vitrification of the confined polymers. In this work, we assessed the hole diffusion dynamics in glassy thin poly (ethylene terephthalate) films with various thicknesses using a strategy based on covering the film surface with crystals of different sizes, which allows for changing the hole diffusion efficiency, thus tuning the glass transition temperature of the thin film (Tgfilm). The apparent activation energy for hole diffusion (Ea,hole) in the thin films was elucidated by modeling the correlation between Tgfilm and surface coverage of crystals using the free-volume hole diffusion model. Our results demonstrated a trend of linear decrease in Ea,hole with the increasing inverse film thickness (h–1), suggesting that the presence of a free surface with faster dynamics promotes hole diffusion in thin films. This finding demonstrates that the free volume release in thin films and thus the confined polymer dynamics can be modulated by changing the free surface properties of the films.B.Z. acknowledges financial support from Natural Science Foundation of China (Grant nos. 21973083) and Projects of International Cooperation and Exchanges NSFC (Grant nos. 22011530456). D.C. acknowledges financial support from the project PGC2018-094548-B-I00 (MICINN-Spain and FEDER-UE) and the project IT-1175-19 (Basque Government).Peer reviewe

Effects of Land Use/Cover Changes and Urban Forest Configuration on Urban Heat Islands in a Loess Hilly Region: Case Study Based on Yan’an City, China

In this study Yan’an City, a typical hilly valley city, was considered as the study area in order to explain the relationships between the surface urban heat island (SUHI) and land use/land cover (LULC) types, the landscape pattern metrics of LULC types and land surface temperature (LST) and remote sensing indexes were retrieved from Landsat data during 1990–2015, and to find factors contributed to the green space cool island intensity (GSCI) through field measurements of 34 green spaces. The results showed that during 1990–2015, because of local anthropogenic activities, SUHI was mainly located in lower vegetation cover areas. There was a significant suburban-urban gradient in the average LST, as well as its heterogeneity and fluctuations. Six landscape metrics comprising the fractal dimension index, percentage of landscape, aggregation index, division index, Shannon’s diversity index, and expansion intensity of the classified LST spatiotemporal changes were paralleled to LULC changes, especially for construction land, during the past 25 years. In the urban area, an index-based built-up index was the key positive factor for explaining LST increases, whereas the normalized difference vegetation index and modified normalized difference water index were crucial factors for explaining LST decreases during the study periods. In terms of the heat mitigation performance of green spaces, mixed forest was better than pure forest, and the urban forest configuration had positive effects on GSCI. The results of this study provide insights into the importance of species choice and the spatial design of green spaces for cooling the environment