A glutathione-dependent formaldehyde-activating enzyme (Gfa) from Paracoccus denitrificans detected and purified via two- dimensional proton exchange NMR spectroscopy

The formation of S-hydroxymethylglutathione from formaldehyde and glutathione is a central reaction in the consumption of the cytotoxin formaldehyde in some methylotrophic bacteria as well as in many other organisms. We describe here the discovery of an enzyme from Paracoccus denitrificans that accelerates this spontaneous condensation reaction. The rates of S- hydroxymethylglutathione formation and cleavage were determined under equilibrium conditions via two-dimensional proton exchange NMR spectroscopy. The pseudo first order rate constants k(1)* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k(1)* was found to be 0.02 s(-1) for the spontaneous reaction. A 10-fold increase of the rate constant was observed upon addition of cell extract from P. denitrificans grown in the presence of methanol corresponding to a specific activity of 35 units mg(-1). Extracts of cells grown in the presence of succinate revealed a lower specific activity of 11 units mg(-1). The enzyme catalyzing the conversion of formaldehyde and glutathione was purified and named glutathione-dependent formaldehyde- activating enzyme (Gfa). The gene gfa is located directly upstream of the gene for glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of S- hydroxymethylglutathione. Putative proteins with sequence identity to Gfa from P. denitrificans are present also in Rhodobacter sphaeroides, Sinorhizobium meliloti, and Mesorhizobium loti

H1 photonic crystal cavitites for hybrid quantum information protocols

Hybrid quantum information protocols are based on local qubits, such as trapped atoms, NV centers, and quantum dots, coupled to photons. The coupling is achieved through optical cavities. Here we demonstrate far-field optimized H1 photonic crystal membrane cavities combined with an additional back reflection mirror below the membrane that meet the optical requirements for implementing hybrid quantum information protocols. Using numerical optimization we find that 80% of the light can be radiated within an objective numerical aperture of 0.8, and the coupling to a single-mode fiber can be as high as 92%. We experimentally prove the unique external mode matching properties by resonant reflection spectroscopy with a cavity mode visibility above 50%.Comment: 14 pages, 11 figure

Deterministic nano-assembly of a coupled quantum emitter - photonic crystal cavity system

The interaction of a single quantum emitter with its environment is a central theme in quantum optics. When placed in highly confined optical fields, such as those created in optical cavities or plasmonic structures, the optical properties of the emitter can change drastically. In particular, photonic crystal (PC) cavities show high quality factors combined with an extremely small mode volume. Efficiently coupling a single quantum emitter to a PC cavity is challenging because of the required positioning accuracy. Here, we demonstrate deterministic coupling of single Nitrogen-Vacancy (NV) centers to high-quality gallium phosphide PC cavities, by deterministically positioning their 50 nm-sized host nanocrystals into the cavity mode maximum with few-nanometer accuracy. The coupling results in a 25-fold enhancement of NV center emission at the cavity wavelength. With this technique, the NV center photoluminescence spectrum can be reshaped allowing for efficient generation of coherent photons, providing new opportunities for quantum science.Comment: 13 pages, 4 figure

Prescription Drug Abuse Communication: A Qualitative Analysis of Prescriber and Pharmacist Perceptions and Behaviors

Background: Interpersonal communication is inherent in a majority of strategies seeking to engage prescriber and pharmacist health care professionals (HCPs) in the reduction and prevention of prescription drug abuse (PDA). However, research on HCP PDA communication behavioral engagement and factors that influence it is limited. Objectives This study quantitatively examined communication behaviors and trait-level communication metrics, and qualitatively described prescription drug abuse-related communication perceptions and behaviors among primary care prescribers and community pharmacists. Methods: Five focus groups (N = 35) were conducted within the Appalachian Research Network (AppNET), a rural primary care practice-based research network (PBRN) in South Central Appalachia between February and October, 2014. Focus groups were structured around the administration of three previously validated trait-level communication survey instruments, and one instrument developed by the investigators to gauge HCP prescription drug abuse communication engagement and perceived communication importance. Using a grounded theory approach, focus group themes were inductively derived and coded independently by study investigators. Member-checking interviews were conducted to validate derived themes. Results: Respondents\u27 trait-level communication self-perceptions indicated low communication apprehension, high self-perceived communication competence, and average willingness to communicate as compared to instrument specific criteria and norms. Significant variation in HCP communication behavior engagement was noted specific to PDA. Two overarching themes were noted for HCP-patient communication: 1) influencers of HCP communication and prescribing/dispensing behaviors, and 2) communication behaviors. Multiple sub-themes were identified within each theme. Similarities were noted in perceptions and behaviors across both prescribers and pharmacists. Conclusions: Despite the perceived importance of engaging in PDA communication, HCPs reported that prescription drug abuse communication is uncomfortable, variable, multifactorial, and often avoided. The themes that emerged from this analysis support the utility of communication science and health behavior theories to better understand and improve PDA communication behaviors of both prescribers and pharmacists, and thereby improve engagement in PDA prevention and treatment

Engineered arrays of NV color centers in diamond based on implantation of CN- molecules through nanoapertures

We report a versatile method to engineer arrays of nitrogen-vacancy (NV) color centers in dia- mond at the nanoscale. The defects were produced in parallel by ion implantation through 80 nm diameter apertures patterned using electron beam lithography in a PMMA layer deposited on a diamond surface. The implantation was performed with CN- molecules which increased the NV defect formation yield. This method could enable the realization of a solid-state coupled-spin array and could be used for positioning an optically active NV center on a photonic microstructure.Comment: 12 pages, 3 figure

Spin dynamics in the optical cycle of single nitrogen-vacancy centres in diamond

We investigate spin-dependent decay and intersystem crossing in the optical cycle of single negatively-charged nitrogen-vacancy (NV) centres in diamond. We use spin control and pulsed optical excitation to extract both the spin-resolved lifetimes of the excited states and the degree of optically-induced spin polarization. By optically exciting the centre with a series of picosecond pulses, we determine the spin-flip probabilities per optical cycle, as well as the spin-dependent probability for intersystem crossing. This information, together with the indepedently measured decay rate of singlet population provides a full description of spin dynamics in the optical cycle of NV centres. The temperature dependence of the singlet population decay rate provides information on the number of singlet states involved in the optical cycle.Comment: 11 pages, 5 figure

Vascular perfusion and hypoxic areas in RIF-1 tumours after photodynamic therapy.

The influence of photodynamic therapy (PDT) on vascular perfusion and the development of hypoxia was investigated in the murine RIF-1 tumour. Image analysis was used to quantify changes in perfusion and hypoxia at 5 min after interstitial Photofrin-mediated PDT. The fluorescent stain Hoechst 33342 was used as an in vivo marker of functional vascular perfusion and the antibody anti-collagen type IV as a marker of the tumour vasculature. The percentage of total tumour vasculature that was perfused decreased to less than 30% of control values after PDT. For the lower light doses this decrease was more pronounced in the centre of the tumour. The observed reduction in vascular perfusion showed a good linear correlation (r = 0.98) with previously published tumour perfusion data obtained with the 86Rb extraction technique. The image analysis technique provides extra information concerning the localisation of (non)-perfused vessels. To detect hypoxic tumour areas in vivo, an immunohistochemical method was used employing NITP [7-(4'-(2-nitroimidazol-1-yl)-butyl)-theophylline]. A large increase in hypoxic areas was found for PDT-treated tumours. More than half the total tumour area was hypoxic after PDT, compared with < 4% for control tumours. Our studies illustrate the potential of image analysis systems for monitoring the functional consequences of PDT-mediated vascular damage early after treatment. This provides direct confirmation that the perfusion changes lead to tissue hypoxia, which has implications for the combined treatment of PDT with bioreductive drugs

Single and coupled L3 photonic crystal cavities for cavity-QED experiments

Here we discuss the experimental characterization of the spatial far-field profiles for the confined modes in a photonic crystal cavity of the L3 type, finding a good agreement with FDTD simulations. We then link the far-field profiles to relevant features of the cavity mode near-fields, using a simple Fabry-Perot resonator model. Finally, we describe a technique for independent all-electrical control of the wavelength of quantum dots in separated L3 cavities, coupled by a waveguide, by electrical isolation via proton implantation

The Epstein-Barr Virus (EBV)-Encoded Protein Kinase, EBV-PK, but Not the Thymidine Kinase (EBV-TK), Is Required for Ganciclovir and Acyclovir Inhibition of Lytic Viral Production

Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC50] = 1.5 μM) and ACV (IC50 = 4.1 μM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC50 = 19.6 μM) and ACV (IC50 = 36.4 μM). Expression of the EBV-PK protein in trans restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC50 = 1.2 μM) and ACV (IC50 = 2.8 μM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities