Establishment of dsDNA-dsDNA interactions by the condensin complex

Condensin is a structural maintenance of chromosomes (SMC) complex family member thought to build mitotic chromosomes by DNA loop extrusion. However, condensin variants unable to extrude loops, yet proficient in chromosome formation, were recently described. Here, we explore how condensin might alternatively build chromosomes. Using bulk biochemical and single-molecule experiments with purified fission yeast condensin, we observe that individual condensins sequentially and topologically entrap two double-stranded DNAs (dsDNAs). Condensin loading transitions through a state requiring DNA bending, as proposed for the related cohesin complex. While cohesin then favors the capture of a second single-stranded DNA (ssDNA), second dsDNA capture emerges as a defining feature of condensin. We provide complementary in vivo evidence for DNA-DNA capture in the form of condensin-dependent chromatin contacts within, as well as between, chromosomes. Our results support a “diffusion capture” model in which condensin acts in mitotic chromosome formation by sequential dsDNA-dsDNA capture

The molecular mechanism of condensin in mitotic chromosome formation

The rearrangement of interphase chromatin into mitotic chromosomes is crucial for faithful chromosome segregation. Defects in this process may cause chromosome entanglement, decompaction, chromosome bridges during anaphase, and eventually genome instability and cell death. The key player for this process was discovered to be condensin, which is a member of the structural maintenance of chromosome (SMC) complex with a conserved pentameric ring structure. However, the exact role of condensin in mitotic chromosome formation is still under debate. Three prevalent models were proposed to explain the role of condensin in mitotic chromosome condensation, namely the diffusion capture model, the torsion-mediated compaction model, and the loop extrusion model. The diffusion capture model proposes that condensin stabilizes stochastic chromatin interactions, via sequential topological entrapment or condensincondensin interaction, thereby forming and maintaining chromatin loops that compact chromatin. The torsion-mediated compaction model proposes that condensin introduces and maintains torsional strain that structures chromatin into a series of plectonemes, thereby compacting the chromatin. The loop extrusion model proposes that condensin and/or other DNA translocators enlarges chromatin loops anchored by condensin, thereby shrinking the chromatin lengthwise. Each model has its own weak points that still require further characterization. Therefore, my PhD project focuses on characterizing the condensin-DNA interaction in vitro with the hope to provide evidence for one or more of these models. Using purified fission yeast condensin, I reconstituted topological condensin loading onto DNA in vitro. I found that topologically loaded condensin can be subsequently unloaded from DNA in an ATP-dependent manner, recapitulating the regulated turnover of condensin on chromatin in vivo. Importantly, I discovered that condensin can sequentially topologically entrap two dsDNA molecules in vitro. Using single-molecule microscopy, I later confirmed that both the topological DNA loading and second dsDNA capture could be mediated by a single condensin complex. These observations provided a solid ground for the diffusion capture model of mitotic chromosome assembly by condensin

Structural characterization of theStreptococcus pneumoniaecarbohydrate substrate-binding protein SP0092

San Diego Freeway, with downtown in the distance; The development of skyscrapers over 300 feet (91 m) in San Diego is attributed to the construction of the El Cortez Apartment Hotel in 1927, the tallest building in the city from 1927 to 1963. As time went on multiple buildings claimed the title of San Diego's tallest skyscraper, including the Union Bank of California Building and Symphony Towers. Currently the tallest building in San Diego is One America Plaza, standing 500 feet (150 m) tall, which was completed in 1991. The downtown skyline contains no super-talls, as a regulation put in place by the Federal Aviation Administration in the 1970s set a 500 feet (152 m) limit on the height of buildings due to the proximity of San Diego International Airport. Source: Wikipedia; http://en.wikipedia.org/wiki/Main_Page (accessed 7/15/2012

Synthesis and Protective Effect of Scutellarein on Focal Cerebral Ischemia/Reperfusion in Rats

Scutellarein, the main metabolite of scutellarin in vivo, has relatively better solubility, bioavailability and bio-activity than scutellarin. However, compared with scutellarin, it is very difficult to obtain scutellarein from Nature. Therefore, the present study focused on establishing an efficient route for the synthesis of scutellarein by hydrolyzing scutellarin. Neurological deficit score and cerebral infarction volume with the administration of scutellarein were then used to compare its neuroprotective effects on focal cerebral ischemia/reperfusion in rats induced by middle cerebral artery occlusion (MCAO) with those of scutellarin. The results showed that scutellarein had better protective effect on focal cerebral ischemia/reperfusion than scutellarin, which laid the foundation for further research and development of scutellarein as a promising candidate for ischemic cerebro-vascular disease

Synthesis and Protective Effect of Scutellarein on Focal Cerebral Ischemia/Reperfusion in Rats

Scutellarein, the main metabolite of scutellarin <em>in vivo</em>, has relatively better solubility, bioavailability and bio-activity than scutellarin. However, compared with scutellarin, it is very difficult to obtain scutellarein from Nature. Therefore, the present study focused on establishing an efficient route for the synthesis of scutellarein by hydrolyzing scutellarin. Neurological deficit score and cerebral infarction volume with the administration of scutellarein were then used to compare its neuroprotective effects on focal cerebral ischemia/reperfusion in rats induced by middle cerebral artery occlusion (MCAO) with those of scutellarin. The results showed that scutellarein had better protective effect on focal cerebral ischemia/reperfusion than scutellarin, which laid the foundation for further research and development of scutellarein as a promising candidate for ischemic cerebro-vascular disease

A Comparative Evaluation of eDNA Metabarcoding Primers in Fish Community Monitoring in the East Lake

East Lake in Wuhan, China, harbors a high number of freshwater fish species of great conservation value, concurrently serving as vital resources for local livelihoods. However, the ecosystem is threatened by an array of anthropogenic activities, thus requiring consistent monitoring of the local fish community to enable more efficacious conservation management. In place of conventional surveying methods, we undertook the first analysis of the fish distribution within East Lake via metabarcoding of environmental DNA (eDNA). The accuracy and efficacy of eDNA metabarcoding rely heavily upon selecting an appropriate primer set for PCR amplification. Given the varying environmental conditions and taxonomic diversity across distinct study systems, it remains a challenge to propose an optimal genetic marker for universal use. Thus, it becomes necessary to select PCR primers suitable for the composition of fish in the East Lake. Here, we evaluated the performance of two primer sets, Mifish-U and Metafish, designed to amplify 12S rRNA barcoding genes in fishes. Our results detected a total of 116 taxonomic units and 51 fish species, with beta diversity analysis indicating significant differences in community structure diversity between the six sampling locations encompassing East Lake. While it was difficult to accurately compare the species-level discriminatory power and amplification bias of the two primers, Mifish outperformed Metafish in terms of taxonomic specificity for fish taxa and reproducibility. These findings will assist with primer selection for eDNA-based fish monitoring and biodiversity conservation in the East Lake and other freshwater ecosystems