Asymmetric split H-shape nanoantennas for molecular sensing.

In this paper we report on a very sensitive biosensor based on gold asymmetric nanoantennas that are capable of enhancing the molecular resonances of C-H bonds. The nanoantennas are arranged as arrays of asymmetric-split H-shape (ASH) structures, tuned to produce plasmonic resonances with reflectance double peaks within the mid-infrared vibrational resonances of C-H bonds for the assay of deposited films of the molecule 17ß-estradiol (E2), used as an analyte. Measurements and numerical simulations of the reflectance spectra have enabled an estimated enhancement factor on the order of 105 to be obtained for a thin film of E2 on the ASH array. A high sensitivity value of 2335 nm/RIU was achieved, together with a figure of merit of approximately 8. Our experimental results were corroborated using numerical simulations for the C-H stretch vibrational resonances from the analyte, superimposed on the plasmonic resonances of the ASH nanoantennas.

Split isobaric analog state in 55Ni: case of strong isospin mixing.

Study of ß+ decay of the exotic Tz=-3/2 nucleus 55Cu, via delayed γ rays, has revealed a strongly isospin mixed doublet (4599-4579 keV) in 55Ni, which represents the fragmented and previously unknown isobaric analog of the ground state of 55Cu. The observed small log ft values to both states in the doublet confirm the superallowed Fermi ß decay. The near degeneracy of a pair of 3/2- levels in 55Ni results in the strong isospin mixing. The isospin mixing matrix element between the T=3/2 and T=1/2 levels is inferred from the experiment to be 9(1) keV, which agrees well with the matrix element of the charge symmetry breaking shell model Hamiltonian of Ormand and Brown. A precise value of the half-life of 55Cu at 57(3) ms was also obtained.

The impact of feeding diets of high or low energy concentration on carcass measurements and the weight of primal and subprimal lean cuts.

Pigs from four sire lines were allocated to a series of low energy (LE, 3.15 to 3.21 Mcal ME/kg) corn-soybean meal-based diets with 16% wheat midds or high energy diets (HE, 3.41 to 3.45 Mcal ME/kg) with 4.5 to 4.95% choice white grease. All diets contained 6% DDGS. The HE and LE diets of each of the four phases were formulated to have equal lysine:Mcal ME ratios. Barrows (N = 2,178) and gilts (N = 2,274) were fed either high energy (HE) or low energy (LE) diets from 27 kg BW to target BWs of 118, 127, 131.5 and 140.6 kg. Carcass primal and subprimal cut weights were collected. The cut weights and carcass measurements were fitted to allometric functions (Y = A CW(B)) of carcass weight. The significance of diet, sex or sire line with A and B was evaluated by linearizing the equations by log to log transformation. The effect of diet on A and B did not interact with sex or sire line. Thus, the final model was (B)) where Diet = -0.5 for the LE and 0.5 for HE diets and A and B are sire line-sex specific parameters. cut weight = (1+bD(Diet)) A(CW Diet had no affect on loin, Boston butt, picnic, baby back rib, or sparerib weights (p>0.10, bD = -0.003, -0.0029, 0.0002, 0.0047, -0.0025, respectively). Diet affected ham weight (bD = -0.0046, p = 0.01), belly weight (bD = 0.0188, p = 0.001) three-muscle ham weight (bD = -0.014, p = 0.001), boneless loin weight (bD = -0.010, p = 0.001), tenderloin weight (bD = -0.023, p = 0.001), sirloin weight (bD = -0.009, p = 0.034), and fat-free lean mass (bD = -0.0145, p = 0.001). Overall, feeding the LE diets had little impact on primal cut weight except to decrease belly weight. Feeding LE diets increased the weight of lean trimmed cuts by 1 to 2 percent at the same carcass weight.

Intruder configurations in the A=33 isobars: 33Mg and 33Al.

The beta decay of 33Mg (N=21) presented in this Letter reveals intruder configurations in both the parent and the daughter nucleus. The lowest excited states in the N=20 daughter nucleus, 33Al, are found to have nearly 2p-2h intruder configuration, thus extending the "island of inversion" beyond Mg. The allowed direct beta-decay branch to the 5/2{+} ground state of the daughter nucleus 33Al implies positive parity for the ground state of the parent 33Mg, contrary to an earlier suggestion of negative parity from a g-factor measurement. An admixture of 1p-1h and 3p-3h configurations is proposed for the ground state of 33Mg to explain all of the experimental observables.

Determination of the N=16 shell closure at the oxygen drip line.

The neutron unbound ground state of (25)O (Z=8, N=17) was observed for the first time in a proton knockout reaction from a (26)F beam. A single resonance was found in the invariant mass spectrum corresponding to a neutron decay energy of 770_+20(-10) keV with a total width of 172(30) keV. The N=16 shell gap was established to be 4.86(13) MeV by the energy difference between the nu1s(1/2) and nu0d(3/2) orbitals. The neutron separation energies for (25)O agree with the calculations of the universal sd shell model interaction. This interaction incorrectly predicts an (26)O ground state that is bound to two-neutron decay by 1 MeV, leading to a discrepancy between the theoretical calculations and experiment as to the particle stability of (26)O. The observed decay width was found to be on the order of a factor of 2 larger than the calculated single-particle width using a Woods-Saxon potential.

Observation of proton radioactivity of the (21+) high-spin isomer in 94Ag.

We have observed direct one-proton decay of the (21+) isomer in the N=Z nuclide 94Ag into high-spin states in 93Pd by detecting protons in coincidence with gamma-gamma correlations and applying gamma gates based on known 93Pd levels. Two decay branches have been identified, with proton energies of 0.79(3) and 1.01(3) MeV and branching ratios of 1.9(5)% and 2.2(4)%, respectively. The corresponding partial half-life values are 21(6) and 18(4) s. The Q value of the direct proton decay of the (21+) isomer was found to be 5.78(3) MeV. The very small reduced widths of the observed proton decays might reflect dominating collective configurations in the (21+) isomer, and the fine structure of the proton spectrum might indicate a strong deformation of this state.

p-sd Shell gap reduction in neutron-rich systems and cross-shell excitations in 20 O.

Excited states in 20O were populated in the reaction 10Be(14C,alpha) at Florida State University (FSU). Charged particles were detected with a particle telescope consisting of 4 annularly segmented Si surface barrier detectors and gamma radiation was detected with the FSU gamma detector array. Five new states were observed below 6 MeV from the alpha-gamma and alpha-gamma-gamma coincidence data. Shell model calculations suggest that most of the newly observed states are core-excited 1p-1h excitations across the N=Z=8 shell gap. Comparisons between experimental data and calculations for the neutron-rich O and F isotopes imply a steady reduction of the p-sd shell gap as neutrons are added.

29Na: defining the edge of the island of inversion for Z=11.

The low-energy level structure of the exotic Na isotopes (28,29)Na has been investigated through beta-delayed gamma spectroscopy. The N=20 isotones for Z=10-12 are considered to belong to the "island of inversion" where intruder configurations dominate the ground state wave function. However, it is an open question as to where and how the transition from normal to intruder dominated configurations happens in an isotopic chain. The present work, which presents the first detailed spectroscopy of (28,29)Na, clearly demonstrates that such a transition in the Na isotopes occurs between 28Na (N=17) and 29Na (N=18), supporting the smaller N=20 shell gap in neutron-rich sd shell nuclei. The evidence for inverted shell structure is found in beta-decay branching ratios, intruder dominated spectroscopy of low-lying states, and shell model analysis.

Lowest excitations in 56Ti and the predicted N=34 shell closure.

Recent experimental characterization of the subshell closure at N=32 in the Ca, Ti, and Cr isotones has stimulated shell-model calculations that indicated the possibility that the N=34 isotones of these same elements could exhibit characteristics of a shell closure, namely, a high energy for the first excited 2(+) level. To that end, we have studied the decay of 56Sc produced in fragmentation reactions and identified new gamma rays in the daughter N=34 isotone 56Ti. The first 2(+) level is found at an energy of 1127 keV, well below the expected position that would indicate the presence of an N=34 shell closure in 56Ti.

A Mutation in the gene-encoding bacteriophage T7 DNA polymerase that renders the phage temperature-sensitive.

Gene 5 of bacteriophage T7 encodes a DNA polymerase essential for phage replication. A single point mutation in gene 5 confers temperature sensitivity for phage growth. The mutation results in an alanine to valine substitution at residue 73 in the exonuclease domain. Upon infection of Escherichia coli by the temperature-sensitive phage at 42 degrees C, there is no detectable T7 DNA synthesis in vivo. DNA polymerase activity in these phage-infected cell extracts is undetectable at assay temperatures of 30 degrees C or 42 degrees C. Upon infection at 30 degrees C, both DNA synthesis in vivo and DNA polymerase activity in cell extracts assayed at 30 degrees C or 42 degrees C approach levels observed using wild-type T7 phage. The amount of soluble gene 5 protein produced at 42 degrees C is comparable to that produced at 30 degrees C, indicating that the temperature-sensitive phenotype is not due to reduced expression, stability, or solubility. Thus the polymerase induced at elevated temperatures by the temperature-sensitive phage is functionally inactive. Consistent with this observation, biochemical properties and heat inactivation profiles of the genetically altered enzyme over-produced at 30 degrees C closely resemble that of wild-type T7 DNA polymerase. It is likely that the polymerase produced at elevated temperatures is a misfolded intermediate in its folding pathway.

Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.

The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.

A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization.

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.

Enzymatic labeling of DNA.

This appendix describes the preparation of uniformly labeled radioactive probes by nick translation and random oligonucleotide-primed synthesis, as well as the end-labeling of oligonucleotides by T4 polynucleotide kinase. Accompanying these protocols are methods for removing unincorporated dNTP precursors with spin columns and for measuring the specific activity of a probe by acid precipitation.

Expression using the T7 RNA polymerase/promoter system.

This unit describes the expression of genes by placing them under the control of the bacteriophage T7 RNA polymerase. T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences and is resistant to antibiotics such as rifampicin that inhibit E. coli RNA polymerase. Consequently, the addition of rifampicin to cells that are producing T7 RNA polymerase results in the exclusive expression of genes under the control of a T7 RNA polymerase promoter (p(T7)). In the Basic Protocol, two plasmids are maintained within the same E. coli cell. One (the expression vector) contains p(T7) upstream of the gene to be expressed. The second contains the T7 RNA polymerase gene under the control of a heat-inducible E. coli promoter. Upon heat induction, the T7 RNA polymerase is produced and initiates transcription on the expression vector, resulting in turn in the expression of the gene(s) under the control of p(T7). If desired, the gene products can be uniquely labeled by carrying out the procedure in minimal medium, adding rifampicin to inhibit the E. coli RNA polymerase, and then labeling the proteins with [35S]methionine.

Phosphatases and kinases.

The reaction conditions and applications of two phosphatases and one kinase are described in this unit. Bacterial alkaline phosphatase (BAP) from E. coli and calf intestine phosphatase (CIP) from veal are commonly used in nucleic acid research. Both enzymes catalyze the hydrolysis of 5'-phosphate residues from DNA, RNA, and ribo- and deoxyribonucleoside triphosphates. The dephosphorylated products possess 5'-hydroxyl termini which can subsequently be radioactively labeled using [gamma-32P]ATP and T4 polynucleotide kinase. T4 polynucleotide kinase has 3 activities: the forward reaction efficiently catalyzes the transfer of the terminal (gamma) phosphate of ATP to the 5'-hydroxyl termini of DNA and RNA. The exchange reaction catalyzes the exchange of 5'-terminal phosphates. Lastly, T4 polynucleotide kinase is a 3' phosphatase.

Exonucleases.

Reaction conditions for numerous exonucleases are detailed in this unit along with discussions of potential applications. Single-stranded and double-stranded 5' --> 3' and 3' --> 5' exonucleases are included.

Endonucleases.

Reaction conditions for numerous endonucleases are detailed in this unit along with discussions of potential applications. Specific enzymes include BAL 31 nuclease, S1 nuclease, mung bean nuclease, micrococcal nuclease and DNase I.

DNA ligases.

DNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5' phosphate and a 3'-hydroxyl terminus in duplex DNA. This activity can repair single-stranded nicks in duplex DNA and join duplex DNA restriction fragments having either blunt ends or homologous cohesive ends. Two ligases are used for nucleic acid research and their reaction conditions and applications are described in this unit: E. coli ligase and T4 ligase. These enzymes differ in two important properties. One is the source of energy: T4 ligase uses ATP, while E. coli ligase uses NAD. Another important difference is their ability to ligate blunt ends; under normal reaction conditions, only T4 DNA ligase will ligate blunt ends.

RNA ligases.

T4 RNA ligase, the product of the phage gene 63, is purified from phage-infected cells. It catalyzes the ATP-dependent covalent joining of single-stranded 5'-phosphoryl termini of DNA or RNA to single-stranded 3'-hydroxyl termini of DNA or RNA. This unit describes specific reaction conditions as well as applications such as radioactive labeling of 3' termini of RNA, circularizing deoxy- and ribo-oligonucleotides, ligating oligomers for oligonucleotide synthesis, and stimulating the blunt-end ligation activity of T4 DNA ligase.

DNA-dependent DNA polymerases.

This unit presents characteristics and reaction conditions of the DNA-dependent DNA polymerases, including E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, native and modified T7 DNA polymerase, and Taq DNA polymerase.