Energy landscapes of dynamic ensembles of rolling triplet repeat bulge loops: implications for DNA expansion associated with disease states

DNA repeat domains can form ensembles of canonical and noncanonical states, including stable and metastable DNA secondary structures. Such sequence-induced structural diversity creates complex conformational landscapes for DNA processing pathways, including those triplet expansion events that accompany replication, recombination, and/or repair. Here we demonstrate further levels of conformational complexity within repeat domains. Specifically, we show that bulge loop structures within an extended repeat domain can form dynamic ensembles containing a distribution of loop positions, thereby yielding families of positional loop isomers, which we designate as "rollamers". Our fluorescence, absorbance, and calorimetric data are consistent with loop migration/translocation between sites within the repeat domain ("rollamerization"). We demonstrate that such "rollameric" migration of bulge loops within repeat sequences can invade and disrupt previously formed base-paired domains via an isoenthalpic, entropy-driven process. We further demonstrate that destabilizing abasic lesions alter the loop distributions so as to favor "rollamers" with the lesion positioned at the duplex/loop junction, sites where the flexibility of the abasic "universal hinge" relaxes unfavorable interactions and/or facilitates topological accommodation. Another strategic siting of an abasic site induces directed loop migration toward denaturing domains, a phenomenon that merges destabilizing domains. In the aggregate, our data reveal that dynamic ensembles within repeat domains profoundly impact the overall energetics of such DNA constructs as well as the distribution of states by which they denature/renature. These static and dynamic influences within triplet repeat domains expand the conformational space available for selection and targeting by the DNA processing machinery. We propose that such dynamic ensembles and their associated impact on DNA properties influence pathways that lead to DNA expansion.     

A secretory phospholipase A2-mediated neuroprotection and anti-apoptosis

Background  

Phospholipase A₂ liberates free fatty acids and lysophospholipids upon hydrolysis of phospholipids and these products are often associated with detrimental effects such as inflammation and cerebral ischemia. The neuroprotective effect of neutral phospholipase from snake venom has been investigated.     

Rotations of Periodic Strings and Short Superstrings

This paper presents two simple approximation algorithms for the shortest superstring problem with approximation ratios 2 ( ≈ 2.67) and( ≈ 2.596). The framework of our improved algorithms is similar to that of previous algorithms in the sense that they construct a superstring by computing some optimal cycle covers on the distance graph of the given strings and then break and merge the cycles to finally obtain a Hamiltonian path, but we make use of new bounds on the overlap between two strings. We prove that for each periodic semiinfinite string α = a₁a₂··· of periodq, there exists an integerk, such that forany(finite) stringsof periodpwhich isinequivalentto α, the overlap betweensand therotationα[k] = a_ka_{k + 1}··· is at mostp + q. Moreover, ifp ≤ q, then the overlap betweensand α[k] is not larger than (p + q). The bounds are tight. In the previous shortest superstring algorithmsp + qwas used as the standard (tight) bound on overlap between two strings with periodspandq.     

VACUUM ULTRAVIOLET CIRCULAR DICHROISM OF DOUBLE STRANDED NUCLEIC ACIDS

The vacuum ultraviolet (VUV) circular dichroism (CD) of double stranded DNA and RNA is greater in amplitude than the CD of these molecules for wavelengths longer than 200 nm. The amplitude of the VUV-CD depends on the base composition of DNA, with guanine-cytosine base pairs contributing more intensity than adenine-thymine base pairs. The shape and amplitude of the VUV-CO are better indicators of nucleic acid conformation (A, B or Z) than are those of the longer wavelength CD. We illustrate the unique features of VUV-CD with specific examples. In the presence of Cs⁺ and ethanol, VUV-CD reveals that poly(dA-dC)poly(dG-dT) forms a right handed double helix despite the inversion of the longer wavelength CD, which usually is used as a benchmark for the left-handed form. The greater magnitude of the VUV-CD of DNA and RNA compared to longer wavelengths means that the VUV-CD is less susceptible to distortion by the induced CD of UV-absorbing ligands like mitomycin C and N-2-acetylaminofluorene.     

Shrinky-Dink microfluidics: rapid generation of deep and rounded patterns

We present a rapid and non-photolithographic approach to microfluidic pattern generation by leveraging the inherent shrinkage properties of biaxially oriented polystyrene thermoplastic sheets. This novel approach yields channels deep enough for mammalian cell assays, with demonstrated heights up to 80 microm. Moreover, we can consistently and easily achieve rounded channels, multi-height channels, and channels as thin as 65 microm in width. Finally, we demonstrate the utility of this simple microfabrication approach by fabricating a functional gradient generator. The whole process--from device design conception to working device--can be completed within minutes.     

Integrated microfluidic cell culture and lysis on a chip

We present an integrated microfluidic cell culture and lysis platform for automated cell analysis that improves on systems which require multiple reagents and manual procedures. Through the combination of previous technologies developed in our lab (namely, on-chip cell culture and electrochemical cell lysis) we have designed, fabricated, and characterized an integrated microfluidic platform capable of culturing HeLa, MCF-7, Jurkat, and CHO-K1 cells for up to five days and subsequently lysing the cells without the need to add lysing reagents. On-demand lysis was accomplished by local hydroxide ion generation within microfluidic chambers, releasing both proteinacious (GFP) and genetic (Hoescht-stained DNA) material. Sample proteins exposed to the electrochemical lysis conditions were immunodetectable (p53) and their enzymatic activity (HRP) was investigated.     

Construction of a wheat-flour state diagram

We use pressure-variable differential scanning calorimetry to detect and characterize thermally induced transitions (glass, melting, gelatinization) in pre- and post-extruded wheat flour. The resulting data allow us to construct a two-dimensional state diagram which maps the physical states that pre- and post-extruded wheat flour can assume, at constant pressure, as a function of moisture content, temperature, and the specific mechanical energy, SME, generated in the extruder. We describe how this state diagram can be used to map the path of extrusion processing, to assess the impact of extrusion conditions, and, ultimately, to design formulations and processing conditions that result in desired end-product attributes. For the extrudates, we find that the extent of processing-induced fragmentation, as monitored by reductions in the extrudate glass transition temperature,T _g, increases with the SME generated in the extruder. We demonstrate that a wheat-flour state diagram, which includes the glass curve of the wheat-flour extrudates produced at various SME values, allows one to predict and control the impact of processing conditions on extrudate properties.     

Shrinky-Dink microfluidics: 3D polystyrene chips

We present a novel approach for the ultra-rapid direct patterning of complex three-dimensional, stacked polystyrene (PS) microfluidic chips. By leveraging the inherent shrinkage properties of biaxially pre-stressed thermoplastic sheets, microfluidic channels become thinner and deeper upon heating. Design conception to fully functional chips can thus be completed within minutes.     

Trans-membrane transport of 1-anilino-8-naphthalenesulfonate in simple cationic surfactant vesicles

The title fluorescent probe crosses dihexadecyldimethylammonium bromide vesicular membranes 1–2 orders of magnitude faster than it crosses the liposomal membranes of dipalmitoylphosphatidylcholine.