Utility of lab-on-a-chip technology for high-throughput nucleic acid and protein analysis

On-chip electrophoresis can provide size separations of nucleic acids and proteins similar to more traditional slab gel electrophoresis. Lab-on-a-chip (LoaC) systems utilize on-chip electrophoresis in conjunction with sizing calibration, sensitive detection schemes, and sophisticated data analysis to achieve rapid analysis times (<120 s). This work describes the utility of LoaC systems to enable and augment systems biology investigations. RNA quality, as assessed by an RNA integrity number score, is compared to existing quality control (QC) measurements. High-throughput DNA analysis of multiplex PCR samples is used to stratify gene sets for disease discovery. Finally, the applicability of a high-throughput LoaC system for assessing protein purification is demonstrated. The improvements in workflow processes, speed of analysis, data accuracy and reproducibility, and automated data analysis are illustrated.     

Mössbauer studies of the iron-sulfur cluster-free hydrogenase: the electronic state of the mononuclear Fe active site

The iron-sulfur cluster-free hydrogenase (Hmd) from methanogenic archaea harbors an iron-containing, light-sensitive cofactor of still unknown structure as prosthetic group. The enzyme is reversibly inhibited by CO and cyanide and is EPR silent. We report here on M?ssbauer spectra of the (57)Fe-labeled enzyme and of the isolated cofactor. The spectrum of the holoenzyme measured at 80 K revealed a doublet peak with an isomer shift delta = 0.06 mm.s(-)(1) and a quadrupole splitting of DeltaE(Q) = 0.65 mm.s(-)(1) (at pH 8.0). The signal intensity corresponded to the enzyme concentration assuming 1 Fe per mol active site. Upon addition of CO or cyanide to the enzyme, the isomer shift decreased to -0.03 mm.s(-)(1) and -0.00(1) mm.s(-)(1), and the quadrupole splitting increased to 1.38 mm.s(-)(1) and 1.75 mm.s(-)(1), respectively. The three spectra could be perfectly simulated assuming the presence of only one type of iron in Hmd. The low isomer shift is characteristic for Fe in a low oxidation state (0, +1, +2). When the spectra of the holoenzyme and of the CO- or cyanide-inhibited enzyme were measured at 4 K in a magnetic field of 4 and 7 T, the spectra obtained could be simulated assuming the presence of only the external magnetic field, which excludes that the iron in the active site of Hmd is Fe(I), high-spin Fe(0), or high-spin Fe(II). M?ssbauer spectra of the isolated Hmd cofactor are also reported.     

Lattice dynamics of KCN and NaCN in the disordered cubic phase

The rotational degrees of freedom of the CN^- dumbbell have been included in a shell model treatment of the lattice dynamics of KCN and NaCN. Recent experiments are discussed and compared with the model.     

Quantitative analysis of organic mixtures, part one: General principles : By

Two pro-ligands ((R)LH) comprised of an o,p-di-tert-butyl-substituted phenol covalently bonded to a benzimidazole ((Bz)LH) or a 4,5-di-p-methoxyphenyl substituted imidazole ((PhOMe)LH), have been structurally characterised. Each possesses an intramolecular O-H[dot dot dot]N hydrogen bond between the phenolic O-H group and an imidazole nitrogen atom and (1)H NMR studies show that this bond is retained in solution. Each (R)LH undergoes an electrochemically reversible, one-electron, oxidation to form the [(R)LH] (+) radical cation that is considered to be stabilised by an intramolecular O...H-N hydrogen bond. The (R)LH pro-ligands react with M(BF(4))(2).H(2)O (M = Cu or Zn) in the presence of Et(3)N to form the corresponding [M((R)L)(2)] compound. [Cu((Bz)L)(2)] (), [Cu((PhOMe)L)(2)] (), [Zn((Bz)L)(2)] and [Zn((PhOMe)L)(2)] have been isolated and the structures of .4MeCN, .2MeOH, .2MeCN and .2MeCN determined by X-ray crystallography. In each compound the metal possesses an N(2)O(2)-coordination sphere: in .4MeCN and .2MeOH the {CuN(2)O(2)} centre has a distorted square planar geometry; in .2MeCN and .2MeCN the {ZnN(2)O(2)} centre has a distorted tetrahedral geometry. The X-band EPR spectra of both and , in CH(2)Cl(2)-DMF (9 : 1) solution at 77 K, are consistent with the presence of a Cu(ii) complex having the structure identified by X-ray crystallography. Electrochemical studies have shown that each undergo two, one-electron, oxidations; the potentials of these processes and the UV/vis and EPR properties of the products indicate that each oxidation is ligand-based. The first oxidation produces [M(II)((R)L)((R)L )](+), comprising a M(ii) centre bound to a phenoxide ((R)L) and a phenoxyl radical ((R)L ) ligand; these cations have been generated electrochemically and, for R = PhOMe, chemically by oxidation with Ag[BF(4)]. The second oxidation produces [M(II)((R)L )(2)](2+). The information obtained from these investigations shows that a suitable pro-ligand design allows a relatively inert phenoxyl radical to be generated, stabilised by either a hydrogen bond, as in [(R)LH] (+) (R = Bz or PhOMe), or by coordination to a metal, as in [M(II)((R)L)((R)L )](+) (M = Cu or Zn; R = Bz or PhOMe). Coordination to a metal is more effective than hydrogen bonding in stabilising a phenoxyl radical and Cu(ii) is slightly more effective than Zn(II) in this respect.     

Phenoxyl radicals: H-bonded and coordinated to Cu(II) and Zn(II)

Two pro-ligands ((R)LH) comprised of an o,p-di-tert-butyl-substituted phenol covalently bonded to a benzimidazole ((Bz)LH) or a 4,5-di-p-methoxyphenyl substituted imidazole ((PhOMe)LH), have been structurally characterised. Each possesses an intramolecular O-H[dot dot dot]N hydrogen bond between the phenolic O-H group and an imidazole nitrogen atom and (1)H NMR studies show that this bond is retained in solution. Each (R)LH undergoes an electrochemically reversible, one-electron, oxidation to form the [(R)LH] (+) radical cation that is considered to be stabilised by an intramolecular O...H-N hydrogen bond. The (R)LH pro-ligands react with M(BF(4))(2).H(2)O (M = Cu or Zn) in the presence of Et(3)N to form the corresponding [M((R)L)(2)] compound. [Cu((Bz)L)(2)] (), [Cu((PhOMe)L)(2)] (), [Zn((Bz)L)(2)] and [Zn((PhOMe)L)(2)] have been isolated and the structures of .4MeCN, .2MeOH, .2MeCN and .2MeCN determined by X-ray crystallography. In each compound the metal possesses an N(2)O(2)-coordination sphere: in .4MeCN and .2MeOH the {CuN(2)O(2)} centre has a distorted square planar geometry; in .2MeCN and .2MeCN the {ZnN(2)O(2)} centre has a distorted tetrahedral geometry. The X-band EPR spectra of both and , in CH(2)Cl(2)-DMF (9 : 1) solution at 77 K, are consistent with the presence of a Cu(ii) complex having the structure identified by X-ray crystallography. Electrochemical studies have shown that each undergo two, one-electron, oxidations; the potentials of these processes and the UV/vis and EPR properties of the products indicate that each oxidation is ligand-based. The first oxidation produces [M(II)((R)L)((R)L )](+), comprising a M(ii) centre bound to a phenoxide ((R)L) and a phenoxyl radical ((R)L ) ligand; these cations have been generated electrochemically and, for R = PhOMe, chemically by oxidation with Ag[BF(4)]. The second oxidation produces [M(II)((R)L )(2)](2+). The information obtained from these investigations shows that a suitable pro-ligand design allows a relatively inert phenoxyl radical to be generated, stabilised by either a hydrogen bond, as in [(R)LH] (+) (R = Bz or PhOMe), or by coordination to a metal, as in [M(II)((R)L)((R)L )](+) (M = Cu or Zn; R = Bz or PhOMe). Coordination to a metal is more effective than hydrogen bonding in stabilising a phenoxyl radical and Cu(ii) is slightly more effective than Zn(II) in this respect.     

The de Rham Witt complex,cohomological kernels and pm-extensions in characteristic p

In earlier work the authors determined the Brauer kernel of extensions of degree p in characteristic $p>2$ where the Galois group is a semidirect product of order ps for $s|(p?1)$. This result is extended here and tools are developed to compute the cohomological kernels $H_{p^m}^{n+1}(E_m/F)$ for all $n\ge 0$ where $[E_m:F]=p^m$ and the Galois closure is a semidirect product of cyclic groups order $p^m$ and s where $s|(p?1)$. A six-term exact sequence describing the K-theory and cohomology of the extension is obtained. As an application it is shown that any F-division p-algebra of index $p^m$ split in $E_m$ is cyclic; a characteristic p analogue of a result of Vishne. The proofs use the de Rham Witt complex and Izhboldin groups, extending techniques developed earlier for the study of degree 4 extensions in characteristic two. The paper also provides background on the de Rham Witt Complex and Izhboldin groups difficult to track down in the literature.     

Changes in the Inter- and Intra- Fibrillar Structure of Lyocell (TENCEL®) Fibers after KOH Treatment

Lyocell fibers were treated with KOH up to 8 M which was demonstrated to distribute homogeneously at the outer zones of fiber cross section compared to NaOH which accesses more deeply but less homogenously. Both NaOH and KOH solutions can be used to lower significantly the fibrillation of lyocell fibers. However, due to intrafibrillar swelling together with deep penetration ability of alkali seen for NaOH treatments results in great fiber tensile strength loss which is not observed for KOH treatments due to its inability to penetrate the fiber completely. The porous structure of fibers was studied by inverse size exclusion chromatography (ISEC) to identify mean pore diameter, total pore area and accessible pore volume (APV). Mean pore diameter of fibers decreased after KOH treatments which did not change after NaOH treatments. Wide angle X-ray diffraction analyses (WAXD) were applied to identify the crystallinity index and crystallite size. In general, fiber properties such as water retention value, carboxyl content using methylene blue sorption method, depth of color measured after dyeing with C.I. Direct Red 81 and weight loss were distinctly different in the ranges up to 2 M, 2-5 M and 5 to 8 M KOH. KOH treatment suggests new possibilities for the pretreatment of lyocell fibers to lower fibrillation while slightly lowering elongation at break without a distinct loss in tensile strength and with less decrease in carboxyl content and weight loss without changing dyeing properties of fibers compared to NaOH treatment.     

Bio-friendly synthesis of ZnO nanoparticles in aqueous solution at near-neutral pH and low temperature

ZnO nanoparticles are synthesized using a new bio-friendly method. The experimental conditions are very mild: aqueous solution at near-neutral pH and 37 degrees C. The as-obtained nanoparticles show the stable wurtzite structure without the need of annealing. The two reagents used are aqueous solutions of zinc nitrate and buffer tris(hydroxymethyl)aminomethane. This is a standard nontoxic buffer and inert to a wide variety of chemicals and biomolecules, therefore extremely satisfactory for biochemical reactions. Furthermore, this is a polydentade ligand which adsorbs strongly on one or more surfaces of ZnO inhibiting its crystal growth and yielding nearly spherical ZnO nanoparticles. Our objective is to use the crystallization method described here for further incorporation of biomolecules as additives in the reaction solution, aiming at the formation of ZnO with new physical properties.