Increasing Reduction Potentials of Type 1 Copper Center and Catalytic Efficiency of Small Laccase from Streptomyces coelicolor through Secondary Coordination Sphere Mutations.

The key to type 1 copper (T1Cu) function lies in the fine tuning of the CuII/I reduction potential (E°'T1Cu ) to match those of its redox partners, enabling efficient electron transfer in a wide range of biological systems. While the secondary coordination sphere (SCS) effects have been used to tune E°'T1Cu in azurin over a wide range, these principles are yet to be generalized to other T1Cu-containing proteins to tune catalytic properties. To this end, we have examined the effects of Y229F, V290N and S292F mutations around the T1Cu of small laccase (SLAC) from Streptomyces coelicolor to match the high E°'T1Cu of fungal laccases. Using ultraviolet-visible absorption and electron paramagnetic resonance spectroscopies, together with X-ray crystallography and redox titrations, we have probed the influence of SCS mutations on the T1Cu and corresponding E°'T1Cu . While minimal and small E°'T1Cu increases are observed in Y229F- and S292F-SLAC, the V290N mutant exhibits a major E°'T1Cu increase. Moreover, the influence of these mutations on E°'T1Cu is additive, culminating in a triple mutant Y229F/V290N/S292F-SLAC with the highest E°'T1Cu of 556 mV vs. SHE reported to date. Further activity assays indicate that all mutants retain oxygen reduction reaction activity, and display improved catalytic efficiencies (kcat /KM ) relative to WT-SLAC.

The Impact of High-Flow Nasal Cannula Device, Nebulizer Type, and Placement on Trans-Nasal Aerosol Drug Delivery.

BACKGROUND: Aerosol delivery via high-flow nasal cannula (HFNC) has been increasingly used in recent years. However, the effects of different HFNC devices, nebulizer types, and placement on aerosol deposition remain largely unknown. METHODS: An adult manikin with anatomically correct upper airway was used with a collection filter placed between the manikin's trachea and a breathing simulator, composed of a dual-chamber model lung driven by a critical care ventilator. Three HFNC device configurations were compared, with vibrating mesh nebulizer and small-volume nebulizer placed at the humidifier (inlet for Optiflow and outlet for Airvo 2) and proximal to the nasal cannula at gas flows of 10, 20, 40 and 60 L/min, in quiet and distressed breathing patterns. Albuterol (2.5 mg) was nebulized for each condition (no. = 3). The drug was eluted from the collection filter and assayed with ultraviolet spectrophotometry (276 nm). RESULTS: At all settings, except when a nebulizer was placed proximal to the nasal cannula with the Optiflow and when the HFNC flow was set at 60 L/min, the vibrating mesh nebulizer generated a higher inhaled dose than did the small-volume nebulizer (all P < .05). With the exception of distressed breathing at an HFNC flow of 10 L/min, the inhaled dose with the vibrating mesh nebulizer placed at the humidifier was greater than with the vibrating mesh nebulizer placed proximal to the nasal cannula (all P < .05), Optiflow provided a higher inhaled dose than did Airvo 2 with either AirSpiral or 900PT501 circuits with the vibrating mesh nebulizer placed at the humidifier (all P < .05). CONCLUSIONS: During transnasal aerosol delivery, the vibrating mesh nebulizer generated a higher inhaled dose than did the small-volume nebulizer when the nebulizer was placed at the humidifier. With the vibrating mesh nebulizer placed at the humidifier and an HFNC flow > 10 L/min, the inhaled dose was higher than with the vibrating mesh nebulizer placed proximal to the nasal cannula, and the inhaled dose was higher with Optiflow than with Airvo 2.

Educational intervention to improve quality of care in Barrett's esophagus: the AQUIRE randomized controlled trial.

BACKGROUND AND AIMS: Despite quality measures in upper endoscopy (EGD) for Barrett's esophagus (BE), considerable variability remains in practice among gastroenterologists. This randomized controlled trial evaluated the role of structured intensive training on the quality of EGD in BE. METHODS: In this multicenter study, 8 sites (from the GI Quality Consortium) were cluster randomized (1:1) to receive AQUIRE (A Quality Improvement program in cancer care during Endoscopy) training (intervention) or continue local standard practices (control). The primary outcome was compliance with the Seattle biopsy protocol. Secondary outcomes were change in knowledge of BE detection and sampling assessed by questionnaire and dysplasia detection rate (DDR) before and after completion of the 6-month study period. RESULTS: The intervention sites (n = 4) had 31 gastroenterologists and the control sites (n = 4) had 34. There was a significant improvement in the compliance rates with the Seattle biopsy protocol from baseline to the end of the study in the intervention sites (64.8%-73.2%, P = .002) but not in the control sites (69.5%-69.4%, P = .953). The accurate response rate on the questionnaire at the intervention sites increased from 73% at baseline to 88% after AQUIRE training (difference, 14.8%; standard deviation, 18.7; P = .008). DDR did not change significantly from baseline to 6 months in either the control or intervention groups (P = .06). CONCLUSIONS: This study confirms the capacity of a structured educational intervention to improve utilization of a standard biopsy protocol and knowledge of standards of care in BE but without significant change in DDR.

Structural Shielding Evaluation: A Case Study of the Radiography Room of a Rural Hospital in Jos, Nigeria.

INTRODUCTION: Most Nigerian rural hospitals constructed before the publication of the 2005 National Council on Radiation Protection and Measurements Report No. 147 did not undergo a performance assessment at the time of construction. To avoid overexposure to ionizing radiation and to ensure adequate protection of patients, workers, and the public, the shielding barriers need to be evaluated to ascertain that they conform to this standard. METHODS: This study evaluates the shielding barriers for the general radiography room in a rural hospital in Jos, Nigeria. The workload information, generator voltage waveform, anode material, filtration, and anode angle with XRAYBARR calculation model were used to estimate the thickness of lead, concrete, gypsum, steel, plate glass, and wood required to shield the X-ray facility installed in the hospital. The design dose limit was compared to the estimated shielded dose, and the calculated shielded barrier thickness to the design shielded barrier thickness was also compared. RESULTS: The unshielded radiation doses inside the X-ray room were high, indicating that the radiological department of the study area is not minimizing radiation doses to patients. The calculated doses beyond the barriers were greater than the design dose limit, indicating that the shielded barriers in place were not adequate and did not comply with the international standard. DISCUSSION: Hospitals must understand the type of shielding materials that can provide adequate protection and to what extent they can protect their radiography rooms. Management and radiation protection agencies need to ascertain whether these barriers are still adequate or require reinforcement through regular quality assurance testing. Due to an increase in workload associated with an increase in population and urbanization, proper policies are needed more than ever in this and other rural hospitals in Nigeria. CONCLUSION: The tested X-ray rooms did not comply with international recommendations for shielding thickness. Except for the door, console, and changing room, a 0.5 mm-thick lead reinforcement is required. It is also recommended that quality assurance testing occur on an annual basis.

IL-12 dependent/IFN gamma independent expression of CCR5 by myelin-reactive T cells correlates with encephalitogenicity.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease with similarities to multiple sclerosis (MS). It is induced in mice by the transfer of myelin-reactive T cells. Here we demonstrate that IL-12 stimulates myelin-reactive T cells to up-regulate the beta-chemokine receptor, CCR5, in correlation with the acquisition of central nervous system-infiltrating and encephalitogenic properties. These effects of IL-12 are IFN gamma-independent. The CCR5 ligands, RANTES and MIP-1 alpha, are expressed in the spinal cords of mice at EAE onset. Our results suggest that reagents that block CCR5/beta-chemokine interactions and/or antagonize IL-12 might be useful for treatment of autoimmune demyelination.

Activation of APCs through CD40 or Toll-like receptor 9 overcomes tolerance and precipitates autoimmune disease.

Some autoreactive T cells normally escape thymic selection and persist in the periphery. This is true of myelin-reactive CD4(+) T cells, the effectors of experimental autoimmune encephalomyelitis (EAE) in laboratory animals and the presumed mediators of multiple sclerosis in humans. Nonetheless, most individuals do not succumb to autoimmune disease. There is growing evidence that while peripheral APCs stimulate immune responses against foreign Ags in the setting of tissue destruction and "danger," they actually maintain tolerance against self Ags under steady state conditions. We hypothesized that tolerance against candidate autoantigens could be reversed by activation of APCs via CD40 or Toll-like receptor 9 signaling. Adult SJL mice injected i.p. with a peptide fragment of proteolipid protein (a candidate autoantigen in multiple sclerosis) emulsified in IFA fail to mount lymphoproliferative or cytokine responses and are protected from EAE upon subsequent challenge with the Ag combined with adjuvants. Here we report that tolerized proteolipid protein-specific lymph node cells regain the ability to divide, differentiate along a Th1 lineage, and transfer EAE when reactivated in the presence of agonistic Abs against CD40 or CpG oligonucleotides. The effects of both anti-CD40 and CpG oligonucleotides are dependent upon induction of IL-12. Our findings suggest two mechanisms to explain the well-documented association between infectious illnesses and flare-ups of multiple sclerosis. Microbial pathogens could 1) release molecules that bind Toll-like receptors, and/or 2) stimulate microbe-specific T cells to express CD40 ligand, thereby licensing APCs that bear both microbial and autoantigens to break tolerance.