Search

Polycrystalline mercuric iodide nuclear radiation detectors have been produced in a novel technology. Unlike the normal single-crystal technology, there is no intrinsic limit to the surface on which these detectors can be produced. Detectors with areas up to about 1.5 [cm.sup.2], thicknesses from 30 to 600 [[micro]meter], and with single electrodes as well as microstrip and pixel contacts have been fabricated and successfully tested with photons in the range of 40-660 keV, [Beta] particle's emitted from a Sr-Y source, and high energy (100 GeV) muons. Results on both charge collection and counting efficiency are reported as well as some very preliminary imaging results. The experimental results on charge collection have been compared with simulation, and a combined [Mu][Tau] product [10.sup.-7] [cm.sup.2]/V for electrons has been estimated. Index Terms - Imaging, mercuric iodide, radiation detectors.

Objective: In 2016, the American Academy of Pediatrics published the Brief Resolved Unexplained Event (BRUE) Clinical Practice Guideline (CPG). A multicenter quality improvement (QI) collaborative aimed to improve CPG adherence., Methods: A QI collaborative of 15 hospitals aimed to improve testing adherence, the hospitalization of lower-risk infants, the correct use of diagnostic criteria, and risk classification. Interventions included CPG education, documentation practices, clinical pathways, and electronic medical record integration. By using medical record review, care of emergency department (ED) and inpatient patients meeting BRUE criteria was displayed via control or run charts for 3 time periods: pre-CPG publication (October 2015 to June 2016), post-CPG publication (July 2016 to September 2018), and collaborative (April 2019 to June 2020). Collaborative learning was used to identify and mitigate barriers to iterative improvement., Results: A total of 1756 infants met BRUE criteria. After CPG publication, testing adherence improved from 56% to 64% and hospitalization decreased from 49% to 27% for lower-risk infants, but additional improvements were not demonstrated during the collaborative period. During the collaborative period, correct risk classification for hospitalized infants improved from 26% to 49% (ED) and 15% to 33% (inpatient) and the documentation of BRUE risk factors for hospitalized infants improved from 84% to 91% (ED)., Conclusions: A national BRUE QI collaborative enhanced BRUE-related hospital outcomes and processes. Sites did not improve testing and hospitalization beyond the gains made after CPG publication, but they did shift the BRUE definition and risk classification. The incorporation of caregiver perspectives and the use of shared decision-making tools may further improve care., (Copyright © 2024 by the American Academy of Pediatrics.)

Background and objectives Current guidelines for the management of community-acquired pneumonia (CAP) in children recommend obtaining a blood culture for children with moderate to severe pneumonia; yet, there is no guidance to assess the severity of the disease. Thus, a blood culture is obtained for the majority of children admitted with CAP, regardless of the severity of their symptoms. The study was designed to investigate and identify the prevalence of bacteremia in pediatric patients hospitalized with CAP and to evaluate the clinical and laboratory variables associated with bacteremia. Methods We conducted a medical record review of children aged from two months to 18 years diagnosed with CAP between January 1, 2013, and December 31, 2017, at our two urban tertiary centers. We used binary logistic regression analysis and chi-square tests to look at factors associated with blood culture positivity. Results A total of 464 patients were admitted with CAP. Blood cultures were obtained in 357 (76.9%) patients; 23 patients had repeated cultures. Fifteen patients had positive cultures: 5/380 (1.3%) were considered true positive results and 10/380 (2.6%) were considered contaminants. Intensive care unit (ICU) admission (OR 5.6 with 95% CI (1- 31), p<0.03), toxic appearance (OR 12.8 with 95% CI (1.3-125), p<0.01), and significantly elevated C-reactive protein (CRP) (>300 mg/L (p<0.01) were associated with bacteremia. Conclusion The prevalence of bacteremia among children admitted for CAP is low. The use of routine blood cultures should be reserved for children with moderate to severe pneumonia. Further studies are required to better risk-stratify children with CAP., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020, Youssef et al.)

Proteorhodopsin is an integral membrane light-harvesting proton pump that is found in bacteria distributed throughout global surface waters. Here, we present a protocol for functional in vitro production of pR using a commercial cell-free synthesis system yielding 1.0mg purified protein per milliliter of cell lysate. We also present an optimized protocol for in vivo over-expression of pR in Escherichia coli, and a two-step purification yielding 5mg of essentially pure functional protein per liter of culture. Both approaches are straightforward, rapid, and easily scalable. Thus either may facilitate the exploitation of pR for commercial biotechnological applications. Finally, the implications of some observations of the in vitro synthesis behavior, as well as preliminary results towards a structural determination of pR are discussed.

Natural-abundance 15N NMR spectroscopy on dodecylguanidine reveals solvent and protonation effects that model those that could occur for the arginine side chain in proteins. Our results demonstrate that the 15N chemical shifts of the terminal guanine nitrogens strongly depend on the solvent chosen for measurements. A polar H-bond-donating solvent like water has strongly deshielding effects on the neutral guanidine group (with the latter acting predominantly as an H-bond acceptor). As a result, a substantial upfield shift occurs when neutral guanidine is dissolved instead in a non-H-bonding solvent (chloroform). These solvent effects can be as large as those induced by protonation changes. This limits the ability of 15N chemical shifts to distinguish the protonation state of the arginine side chain, at least without specific knowledge of its environment. These results help to reconcile previous interpretations about the protonation state arg-82 in the M state of bacteriorhodopsin based on FTIR and 15N NMR spectroscopy. That is, contrary to earlier conclusions from solid-state NMR, the side chain of arg-82 could undergo a deprotonation between the bR and M states, but only if it also experienced a significant decrease in the H-bonding character and polarity of its environment. In fact, the average 15N chemical shift of the two Neta of arg-82 in bacteriorhodopsin's M intermediate (from the previous NMR measurements) is 17 ppm upfield from the corresponding value for the deprotonated arginine side chain in aqueous solution at pH >14, but only 3 ppm upfield from the value for deprotonated dodecylguanidine in chloroform.

Proteorhodopsin (pR) is a homologue of bacteriorhodopsin (bR) that has been recently discovered in oceanic bacterioplankton. Like bR, pR functions as a light-driven proton pump. As previously characterized by laser flash induced absorption spectroscopy (Krebs, R. A.; Alexiev, U.; Partha, R.; DeVita, A. M.; Braiman, M. S. BMC Physiol. 2002, 2, 5), the pR photocycle shows evidence of light-induced H(+) release on the 10-50 micros time scale, and of substantial accumulation of the M intermediate, only at pH values above 9 and after reconstitution into phospholipid followed by extensive washing to remove detergent. We have therefore measured the time-resolved FTIR difference spectra of pR intermediates reconstituted into DMPC vesicles at pH 9.5. A mixture of K- and L-like intermediates, characterized by a 1516 cm(-1) positive band and a 1742 cm(-1) negative band respectively, appears within 20 micros after photolysis. This mixture decays to an M-like state, with a clear band at 1756 cm(-1) due to protonation of Asp-97. The 50-70 micros rise of M at pH 9.5 is similar to (but a little slower than) the rise times for M formation and H(+) release that were reported earlier based on flash photolysis measurements of pR reconstituted into phospholipids with shorter acyl chains. We conclude that, at pH 9.5, H(+) release occurs while Asp-97 is still protonated; i.e., this aspartic acid cannot be the H(+) release group observed by flash photolysis under similar conditions.

Time-resolved Fourier transform infrared (FTIR) difference spectra of the halorhodopsin (hR) photocycle have been collected from 3 micros to 100 ms in saturating concentrations of KCl or KBr. Kinetic analysis of these data revealed two decay processes, with time constants of tau(1) approximately 150 micros and tau(2) approximately 16 ms in the presence of either halide, with tau(2) describing the return to the starting (hR) state. Comparison to previous low-temperature FTIR spectra of hR intermediates confirms that characteristic hK and hL spectral features are both present before the tau(1) decay, in a state previously defined as hK(L) (Dioumaev, A., and M. Braiman. 1997. Photochem. Photobiol. 66:755-763). However, the relative sizes of these features depend on which halide is present. In Br-, the hL features are clearly more dominant than in Cl-. Therefore, the state present before tau(1) is probably best described as an hK(L)/hL(1) equilibrium, instead of a single hK(L) state. Different halides affect the relative amounts of hK(L) and hL(1) present, i.e., Cl- produces a much more significant back-reaction from hL(1) to hK(L) than does Br-. The halide dependence of this back-reaction could therefore explain the halide selectivity of the halorhodopsin anion pump.

Arginine-82 (R82) of bacteriorhodopsin (bR) has long been recognized as an important residue due to its absolute conservation in the archaeal rhodopsins and the effects of R82 mutations on the photocycle and proton release. However, the nature of interactions between R82 and other residues of the protein has remained difficult to decipher. Recent NMR studies showed that the two terminal nitrogens of R82 experience a highly perturbed asymmetric environment during the M state trapped at cryogenic temperatures [Petkova et al. (1999) Biochemistry 38, 1562-1572]. Although previous low-temperature FT-IR spectra of wild-type and mutant bR samples have demonstrated effects of R82 on vibrations of other amino acid side chains, no bands in these spectra were assignable to vibrations of R82 itself. We have now measured time-resolved FT-IR difference spectra of bR intermediates in the wild-type and R82A proteins, as well as in samples of the R82C mutant with and without thioethylguanidinium attached via a disulfide linkage at the unique cysteine site. Several bands in the bR --> M difference spectrum are attributable to guanidino group vibrations of R82, based on their shift upon isotope substitution of the thioethylguanidinium attached to R82C and on their disappearance in the R82A spectrum. The frequencies and intensities of these IR bands support the NMR-based conclusion that there is a significant perturbation of R82 during the bR photocycle. However, the unusually low frequencies attributable to R82 guandino group vibrations in M, approximately 1640 and approximately 1545 cm(-)(1), would require a reexamination of a previously discarded hypothesis, namely, that the perturbation of R82 involves a change in its ionization state.

Purpose: Interphotoreceptor retinoid-binding protein (IRBP) is unusual for a lipid-binding protein in that its gene is expressed uniquely by cells of photoreceptor origin and consists of four homologous repeats, each coding for a module of approximately 300 amino acid residues. All-trans retinol binding domains, which appear to be present in each module, are composed of conserved hydrophobic regions [Baer et al, Exp Eye Res 1998; 66:249-262]. Here we investigate the role of highly conserved arginines contained in these regions., Methods: To study the arginines in an individual module without the interference of ligand-binding activity elsewhere in the protein, we expressed in E. coli the fourth module of Xenopus IRBP by itself as a soluble thioredoxin fusion protein (X4IRBP). Arginines 1005, 1041, 1073 and 1122 were separately replaced by glutamine using PCR overlap extension mutagenesis. The glutamine substitutions were confirmed by liquid chromatography-tandem mass spectrometry. The binding of all-trans retinol and 9-(9-anthroyloxy)stearic acid (9-AS) to X4IRBP and each of the mutants was evaluated by fluorescence spectroscopy. Binding was followed by monitoring the enhancement of ligand fluorescence and the quenching of protein endogenous fluorescence. The ability of the recombinant proteins to protect all-trans retinol from oxidative degradation was evaluated by monitoring absorbance at 325 nm over time., Results: The substitution of Gln for Arg1005 about doubled the amount of ligand necessary to attain saturation and about doubled the level of fluorescence enhancement obtained at saturation for both all-trans retinol and 9-AS. Although there was not a significant change in the Kd, the substitution increased the calculated number of binding sites (N) from approximately 2 to approximately 4 per polypeptide. The other Arg->Gln mutants did not significantly change the Kd or N. None of the mutations compromised the ability of the module to protect all-trans retinol from degradation., Conclusions: Our data suggest that the function of the conserved arginines in IRBP is fundamentally different from that of other retinoid-binding proteins. These residues do not appear to play a role in defining the specificity of the ligand-binding domain. Rather, Arg1005 appears to play a role in defining the capacity of the domain. Our data suggest that the binding site consists of a single hydrophobic cavity promiscuous for fatty acids and all-trans retinol.

Purpose: Interphotoreceptor retinoid-binding protein (IRBP), an extracellular protein believed to support the exchange of retinoids between the neural retina and retinal pigment epithelium (RPE) in the vertebrate eye, exhibits a modular, i.e., repeat, structure. The present study was undertaken to determine whether an individual module of IRBP has activity in retinoid transfer between the RPE and rod photoreceptors., Methods: The retinoid transfer activity of a recombinant protein corresponding to the fourth module of Xenopus laevis IRBP (X4IRBP) was examined in two ways. First, X4IRBP was tested for its ability to support the regeneration of porphyropsin in detached/reattached Xenopus retina/RPE-eyecups. Following illumination and removal of native IRBP, Xenopus eyecups supplemented with 42 microM X4IRBP or (as a control) Ringer's solution were incubated in darkness and then analyzed for regenerated porphyropsin. Second, toad (Bufo marinus) RPE-eyecup preparations were used to evaluate X4IRBP's ability to promote the release of 11-cis retinal from the RPE., Results: The regeneration of porphyropsin in X4IRBP-supplemented Xenopus retina/RPE-eyecups (0.45 +/- 0.04 nmol; mean +/- SEM, n = 11) exceeded that in controls (0.13 +/- 0.02 nmol, n = 11). For promoting the release of 11-cis retinal from the toad RPE, 42 microM X4IRBP was more effective than equimolar bovine serum albumin although considerably less than that of 26 microM native bovine IRBP., Conclusions: The results indicate a low but significant activity of IRBP's fourth module in reactions relevant to retinoid exchange.

The exchange of all-trans retinol and 11-cis retinal between the photoreceptors and retinal pigmented epithelium is mediated by interphotoreceptor retinoid-binding protein (IRBP). IRBP contains binding sites for retinoids, docosahexaenoic acid and probably cell surface and matrix receptors. IRBP arose through the quadruplication of an ancient protein, represented by its carboxy-terminal module (module 4 in amphibians and mammals). Module 4 has retinol binding activity and is composed of regions coded for by each of IRBP's four exons. Determining the function of the exons has been hampered by insoluble expression of module 4 in Escherichia coli. Here, we found that module 4 of Xenopus IRBP (X4IRBP), as well as its exon segments, can be expressed in a soluble form as thioredoxin fusion proteins. The recombinant proteins were purified by ion exchange and arsenical-based affinity chromatography. Liquid chromatography/mass spectrometry confirmed that the sequence of X4IRBP is correct. All-trans retinol binding was characterized by monitoring enhancement of retinol fluorescence, quenching of intrinsic protein fluorescence, and transfer of energy to the bound retinol. Retinol bound to X4IRBP at 2.20+/-0.29 sites with a KD=1.25+/-0.39. One of the two sites was localized to Exons(2+3) and had a KD=0.26+/-0.13 micron. This site, which supported protein quenching and energy transfer, probably contains at least one of the two conserved tryptophans present in this segment. The second site was localized to Exon 4. This site supported the enhancement of retinol fluorescence but not protein quenching or energy transfer and had a KD=1.94+/-0.20 micron. Exon 1 had no retinol binding activity. The location of the retinol binding regions correlated with the distribution of domains conserved between IRBPs and the newly recognized family of C-terminal processing proteases (CtpAs), proteins which bind and cleave non-polar carboxy termini., (Copyright 1998 Academic Press Limited.)

Step-scan Fourier transform infrared spectroscopy with 50 ns time resolution was applied to the early stages of the photocycle of halorhodopsin (hR) for the temperature range 3-42 degrees C. Kinetic data analysis with global fitting revealed two distinct kinetic processes associated with relaxations of the early red-shifted photoproduct hK; these processes have time constants tau 1 approximately equal to 280 ns and tau 2 approximately equal to 360 microns at 20 degrees C. Spectral features demonstrate that the tau 1 process corresponds to a transition between two distinct bathointermediates, hKE and hKL. The vibrational difference bands associated with both tau 1 and tau 2 transitions are spread throughout the whole 1800-900 cm-1 range. However, the largest bands correspond to ethylenic C=C stretches, fingerprint C-C stretches and hydrogen out-of-plane (HOOP) wags of the retinal chromophore. The time evolution of these difference bands indicate that both the tau 1 and tau 2 decay processes involve principally a relaxation of the chromophore and its immediate environment. The decay of the intense HOOP vibrations is nearly equally divided between the tau 1 and tau 2 processes, indicating a complex chromophore relaxation from a twisted nonrelaxed conformation in the primary (hKE) bathointermediate, to a less-twisted structure in hKL, and finally to a roughly planar structure in the hypsochromically shifted hL intermediate. This conclusion is also supported by the unexpectedly large positive entropy of activation observed for the tau 1 process. The two relaxations from hKE to hL are largely analogous to corresponding relaxations (KE-->KL-->L) in the bacteriorhodopsin photocycle, except that the second step is slowed down by over 200-fold in hR.