A Blue Native-PAGE analysis of membrane protein complexes in Clostridium thermocellum

Background Clostridium thermocellum is a Gram-positive thermophilic anaerobic bacterium with the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. Identification and characterization of protein complexes in C. thermocellum are important toward understanding its metabolism and physiology. Results A two dimensional blue native/SDS-PAGE procedure was developed to separate membrane protein complexes of C. thermocellum. Proteins spots were identified by MALDI-TOF/TOF Mass spectrometry. 24 proteins were identified representing 13 distinct protein complexes, including several putative intact complexes. Interestingly, subunits of both the F1-F0-ATP synthase and the V1-V0-ATP synthase were detected in the membrane sample, indicating C. thermocellum may use alternative mechanisms for ATP generation. Conclusion Two dimensional blue native/SDS-PAGE was used to detect membrane protein complexes in C. thermocellum. More than a dozen putative protein complexes were identified, revealing the simultaneous expression of two sets of ATP synthase. The protocol developed in this work paves the way for further functional characterization of these protein complexes

(Main Section: Ecological Planning)

To rapidly and clearly define the knowledge structure, research focus, and research trends in the field of ecological planning and design, the bibliometric method was combined with visualization tools to conduct a classified statistical study of 712 articles on ecological planning and design in the core database of Web of Science (WoS) from 1992 to 2017, making a contrastive analysis of information like the author, journal, research institution, country, keywords of those articles from three perspectives, namely co-authorship analysis, co-citation analysis, and co-words analysis. Three conclusions were drawn. First, academic exchanges and cooperation in the field of ecological planning and design should be strengthened between countries and scientific research institutions, especially China and its research institutions should actively participate in such exchanges and cooperation, and scholars are expected to seize the opportunities to cooperate with each other in research and practice in this field. Second, as developed European and American countries outweigh Asian countries in terms of research results and overall influence in the field of ecological planning and design, the popularity and influence of journals from Asian countries need to be improved, particularly as they study and report on ecosystem services. Third, as research on ecological planning and design become increasingly systematic, comprehensive, and humanistic, resilience has become a research hotspot in this field in recent years, thus enough attention should be paid to this aspect. Finally, the author hopes that the combination of visualization tools and the bibliometric method can foster enlightenment and research ideas among researchers and practitioners in this field

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

Transcriptomics of Gabra4 knockout mice reveals common NMDAR pathways underlying autism, memory, and epilepsy

Autism spectrum disorder (ASD) is a neuronal developmental disorder with impaired social interaction and communication, often with abnormal intelligence and comorbidity with epilepsy. Disturbances in synaptic transmission, including the GABAergic, glutamatergic, and serotonergic systems, are known to be involved in the pathogenesis of this disorder, yet we do not know if there is a common molecular mechanism. As mutations in the GABAergic receptor subunit gene GABRA4 are reported in patients with ASD, we eliminated the Gabra4 gene in mice and found that the Gabra4 knockout mice showed autistic-like behavior, enhanced spatial memory, and attenuated susceptibility to pentylenetetrazol-induced seizures, a constellation of symptoms resembling human high-functioning autism. To search for potential molecular pathways involved in these phenotypes, we performed a hippocampal transcriptome profiling, constructed a hippocampal interactome network, and revealed an upregulation of the NMDAR system at the center of the converged pathways underlying high-functioning autism-like and anti-epilepsy phenotypes

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

The extreme Arctic warm anomaly in November 2020

In November 2020, the eastern Arctic experienced an extensive extreme warm anomaly (i.e., the second strongest case since 1979), which was followed by extreme cold conditions over East Asia in early winter. The observed Arctic warm anomaly in November 2020 was able to extend upwards to the upper troposphere, characterized as a deep Arctic warm anomaly. In autumn 2020, substantial Arctic sea-ice loss that exceeded the record held since 1979, accompanied by increased upward turbulent heat flux, was able to strongly warm the Arctic. Furthermore, there was abundant northward moisture transport into the Arctic from the North Atlantic, which was the strongest in the past four decades. This extreme moisture intrusion was able to enhance the downward longwave radiation and strongly contribute to the warm conditions in the Arctic. Further analysis indicated that the remote moisture intrusion into the Arctic was promoted by the large-scale atmospheric circulation patterns, such as the wave train propagating from the midlatitude North Atlantic to the Arctic. This process may have been linked to the warmer sea surface temperature in the midlatitude North Atlantic.publishedVersio