The Uncoiling of Supercoiled Plasmid DNA Over Time Observed by Atomic Force Microscopy

Long term stability of DNA structures in a cell is critical to sustaining life. The DNA structures could be degraded biologically (e.g. enzymes), chemically (e.g. drugs), and physically (e.g. thermal agitation process) with time. The DNA structures are maintained by being regenerated and/or being recovered by proteins within a cell. However, even though it is important, it is difficult to observe the time-evolution of DNA structures for extended periods at a molecular resolution. Here, we observed the time evolution of DNA structures for two months, in order to understand the long term stability of DNA structures. For this study, we used purified plasmid DNA molecules extracted from Escherichia coli (E-coli) as a sample. We also employed atomic force microscopy (AFM) to observe the plasmid DNA structures at a molecular resolution. The purified plasmid DNA molecules were diluted with pure water, deposited on a mica surface, and observed by an AFM on a regular basis in an ambient environment for two months. The sequential AFM images show the plasmid DNA formed globular structures at the beginning and transformed into uncoiled plasmid DNA network structures after two months. The globular structures appeared to be the supercoiled state of plasmid DNA, a well-known strategy to store genetic information in a confined space for bacterial systems. The observed DNA network structures are believed to be results of long periods of unwinding and rejoining processes of the supercoiled plasmid DNA. The unwinding and rejoining processes would have been caused by small residual proteins (or enzymes) possibly present in the plasmid DNA solution. This study reveals DNA stability is dramatically influenced by prolonged (~ a few months) exposure to small amounts of residual proteins (or enzymes). The result also suggests the AFM is a powerful tool in observing the biological process at the molecular level over extended periods of time

Long-term Structural Change in Plasmid DNA

Long-term stability of plasmid DNA (pDNA) conformations is critical in many research areas, especially those concerning future gene therapy. Despite its importance, the timeevolution of pDNA structures has rarely been studied at a molecular resolution. Here, the time-evolution of pDNA solutions spanning four years was observed with atomic force microscopy (AFM). The AFM data show that the pDNA molecules evolved from isolated supercoiled structures; to aggregated supercoiled structures; to thin, branched network structures; and finally to wider, branched network structures. Additional topographical analysis of the AFM data suggests the actions of residual proteins could be the main mechanism for the structural changes in our laboratory prepared pDNA

Starch‐binding domain shuffling in Aspergillus niger glucoamylase

Aspergillus niger glucoamylase (GA) consists mainly of two forms, GAI [from the N‐terminus, catalytic domain + linker + starch‐binding domain (SBD)] and GAII (catalytic domain + linker). These domains were shuffled to make RGAI (SBD + linker + catalytic domain), RGAIΔL (SBD + catalytic domain) and RGAII (linker + catalytic domain), with domains defined by function rather than by tertiary structure. In addition, Paenibacillus macerans cyclomaltodextrin glucanotransferase SBD replaced the closely related A.niger GA SBD to give GAE. Soluble starch hydrolysis rates decreased as RGAII ≈ GAII ≈ GAI \u3e RGAIΔL ≈ RGAI ≈ GAE. Insoluble starch hydrolysis rates were GAI \u3e RGAIΔL \u3e RGAI \u3e\u3e GAE ≈ RGAII \u3e GAII, while insoluble starch‐binding capacities were GAI \u3e RGAI \u3e RGAIΔL \u3e RGAII \u3e GAII \u3e GAE. These results indicate that: (i) moving the SBD to the N‐terminus or replacing the native SBD somewhat affects soluble starch hydrolysis; (ii) SBD location significantly affects insoluble starch binding and hydrolysis; (iii) insoluble starch hydrolysis is imperfectly correlated with its binding by the SBD; and (iv) placing the P.maceranscyclomaltodextrin glucanotransferase SBD at the end of a linker, instead of closely associated with the rest of the enzyme, severely reduces its ability to bind and hydrolyze insoluble starch

On orbital allotments for geostationary satellites

The following satellite synthesis problem is addressed: communication satellites are to be allotted positions on the geostationary arc so that interference does not exceed a given acceptable level by enforcing conservative pairwise satellite separation. A desired location is specified for each satellite, and the objective is to minimize the sum of the deviations between the satellites' prescribed and desired locations. Two mixed integer programming models for the satellite synthesis problem are presented. Four solution strategies, branch-and-bound, Benders' decomposition, linear programming with restricted basis entry, and a switching heuristic, are used to find solutions to example synthesis problems. Computational results indicate the switching algorithm yields solutions of good quality in reasonable execution times when compared to the other solution methods. It is demonstrated that the switching algorithm can be applied to synthesis problems with the objective of minimizing the largest deviation between a prescribed location and the corresponding desired location. Furthermore, it is shown that the switching heuristic can use no conservative, location-dependent satellite separations in order to satisfy interference criteria

Substrate Binding by the Catalytic Domain and Carbohydrate Binding Module of Ruminococcus flavefaciens FD-1 Xyloglucanase/ Endoglucanase

Binding and thermodynamic properties of a carbohydrate binding module (CBM) and a glycoside hydrolase family 44 xyloglucanase/endoglucanase catalytic domain (CD) fromRuminococcus flavefaciens, both when separate and when linked to each other, have been quantified when binding various β-1,4-linked glucans and xylans. The three constructs bind cellotetraose, cellopentaose, and cellohexaose with association constants that increase with chain length. The CBM does not bind xylotetraose, xylopentaose, or xylohexaose. The CD appears to bind carboxymethylcellulose (CMC) and xylan only weakly, while the CBM and the CD/CBM bind them much more strongly than they bind the cellooligosaccharides. CMC is bound to a much greater degree than is xylan. Association constants for the cellooligosaccharides are in the order CBM CD \u3c CD/CBM, while those on CMC and xylan are CD CBM CD/CBM. A synergistic effect was observed for the association constants of cellopentaose and cellohexaose with the CD/CBM when compared to the CD and CBM alone. Binding of all ligands by all three constructs is energetically favorable, enthalpy-driven, and subject to enthalpy–entropy compensation

Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1

Two forms of Ruminococcus flavefaciens FD-1 endoglucanase B, a member of glycoside hydrolase family 44, one with only a catalytic domain and the other with a catalytic domain and a carbohydrate binding domain (CBM), were produced. Both forms hydrolyzed cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, xyloglucan, lichenan, and Avicel but not cellobiose, cellotriose, mannan, or pullulan. Addition of the CBM increased catalytic efficiencies on both CMC and birchwood xylan but not on xyloglucan, and it decreased rates of cellopentaose and cellohexaose hydrolysis. Catalytic efficiencies were much higher on xyloglucan than on other polysaccharides. Hydrolysis rates increased with increasing cellooligosaccharide chain length. Cellotetraose hydrolysis yielded only cellotriose and glucose. Hydrolysis of cellopentaose gave large amounts of cellotetraose and glucose, somewhat more of the former than of the latter, and much smaller amounts of cellobiose and cellotriose. Cellohexaose hydrolysis yielded much more cellotetraose than cellobiose and small amounts of glucose and cellotriose, along with a low and transient amount of cellopentaose

Replacement and deletion mutations in the catalytic domain and belt region of Aspergillus awamori glucoamylase to enhance thermostability

Three single-residue mutations, Asp71→Asn, Gln409→Pro and Gly447→Ser, two long-to-short loop replacement mutations, Gly23-Ala24-Asp25-Gly26-Ala27-Trp28-Val29-Ser30→Asn-Pro-Pro (23–30 replacement) and Asp297-Ser298-Glu299-Ala300-Val301→Ala-Gly-Ala (297–301 replacement) and one deletion mutation removing Glu439, Thr440 and Ser441 (Δ439–441), all based on amino acid sequence alignments, were made to improve Aspergillus awamori glucoamylase thermostability. The first and second single-residue mutations were designed to introduce a potential N-glycosylation site and to restrict backbone bond rotation, respectively, and therefore to decrease entropy during protein unfolding. The third single-residue mutation was made to decrease flexibility and increase O-glycosylation in the already highly O-glycosylated belt region that extends around the globular catalytic domain. The 23–30 replacement mutation was designed to eliminate a very thermolabile extended loop on the catalytic domain surface and to bring the remainder of this region closer to the rest of the catalytic domain, therefore preventing it from unfolding. The 297–301 replacement mutant GA was made to understand the function of the random coil region between α-helices 9 and 10. Δ439–441 was constructed to decrease belt flexibility. All six mutations increased glucoamylase thermostability without significantly changing enzyme kinetic properties, with the 23–30 replacement mutation increasing the activation free energy for thermoinactivation by about 4 kJ/mol, which leads to a 4°C increase in operating temperature at constant thermostability

Rights-based approaches and beyond : challenges of linking rights and participation

As more and more development and human rights organisations critically assess their impact and strategies, there is growing convergence in the questions they raise about how to be most effective in addressing structural, systemic causes of poverty and exclusion and thus, make a positive difference in the lives of poor and marginalised people. This paper explores the growing trend of “rights-based approaches” (RBA) to development, drawing from interviews with a range of primarily US-based international human rights and development organisations as well as from insights through the authors’ years of experience working with development and rights groups in the global south. While the theory of RBA has been broadly embraced as key to getting at the root causes of poverty, many organisations are struggling to make sense of the significance of RBA in practice. We begin to unravel some key concerns with a brief discussion on critical considerations for groups as they advance rights-based work. Next, we focus on clarifying meanings, offering our own definitions of what seem to be critical components of RBA, namely participation, rights, and power. Next we summarise some of the current thinking and practice among international human rights and development organisations that are deepening their work in RBA. This includes some of the key tensions, challenges and opportunities they are encountering. Finally, in building on forgotten experiences and innovations we look at a handful of practical experiences from the past that offer valuable insights and lessons as groups seek to maximise the full practical potential of RBA. Keywords: rights, democracy, governance, participation

Observing sub-microsecond telegraph noise with the radio frequency single electron transistor

Telegraph noise, which originates from the switching of charge between meta-stable trapping sites, becomes increasingly important as device sizes approach the nano-scale. For charge-based quantum computing, this noise may lead to decoherence and loss of read out fidelity. Here we use a radio frequency single electron transistor (rf-SET) to probe the telegraph noise present in a typical semiconductor-based quantum computer architecture. We frequently observe micro-second telegraph noise, which is a strong function of the local electrostatic potential defined by surface gate biases. We present a method for studying telegraph noise using the rf-SET and show results for a charge trap in which the capture and emission of a single electron is controlled by the bias applied to a surface gate.Comment: Accepted for publication in Journal of Applied Physics. Comments always welcome, email [email protected], [email protected]