Temporal Analysis of Conformers in the Course of pH Scan Directed by Urea-Urease Reaction-A "Protein Clock".

We demonstrate an analytical approach to monitor the effect of pH on protein tertiary structure. An autocatalytic enzyme reaction is used to stimulate refolding of proteins during real-time analysis. The method takes advantage of a nonlinear pH ramp generated by the urea-urease clock reaction. In this study, alterations to the structures of model proteins were monitored by mass spectrometry (charge pattern shift) and fluorometry (tryptophan fluorescence quenching). The pH measurements were conducted at different points of the sample flow line by different methods to minimize artifacts. Interestingly, different protein ions (corresponding to native and unfolded proteins) show distinct temporal mass spectral profiles, which reveal gradual refolding and concomitant deprotonation of higher charge state ions in the course of the clock reaction. Every multiply charged ion of a protein is characterized with its own "clock time". This approach does not require major modification of standard instrumentation. It enables determination of "high sensitivity" pH intervals for small and large molecules within a single experiment. Thus, it can be useful for characterizing the protein folding in response to pH change.

Triplet vs πσ* state mediated N-H dissociation of aniline.

UV-excited aromatic molecules with N-H/O-H moieties often possess an important nonradiative relaxation pathway, from an optically bright ππ* state to a dark dissociative πσ* state. We apply a new time-selected photofragment translational spectroscopy method to disclose a previously unknown triplet-mediated N-H dissociation of aniline prevented by the multiphoton dissociative ionization in conventional methods. We further determined the branching fractions of aniline dissociated in the πσ*, triplet, and ground states at 248 nm. Additionally, we selectively captured the population changes in the singlet and triplet states with ionization from different laser wavelengths, 355 or 266 nm, in time-resolved photoion yields. The combination of experimental data enables us to uniquely determine the relative ionization cross sections of the singlet and triplet states at an ionization laser wavelength of 266 nm and allows us to extensively measure the rate constants of intersystem crossing and the branching fractions at various excitation wavelengths.

Communication: Mode-dependent excited-state lifetime of phenol under the S1/S2 conical intersection.

Phenol can serve as a model for examining the deactivation of the aromatic amino acid tyrosine following UV excitation, which mainly occurs through a repulsive πσ* state along the O-H bond. The reaction barrier formed by the conical intersection between the optically bright S1 (ππ*) state and the dissociative S2 (πσ*) state does not inhibit O-H bond rupture even though the excitation energy is below the barrier height. To examine the O-H bond-rupture dynamics in association with the initial excited vibrational modes, we used a picosecond laser to investigate the vibrational-mode-dependent excited-state lifetime of phenol under the S1/S2 conical intersection. Unexpectedly short lifetimes were observed in the S1 state for a″ symmetric vibrational modes (including v4, v16a, τOH, and v5). These results clarify recent theoretical calculations showing that the relaxation from S1 to S2 either occurs via symmetry-allowed non-adiabatic transitions or is topographically linked to a lower energy minimum on the multidimensional potential energy surface.

Excited-state dissociation dynamics of phenol studied by a new time-resolved technique.

Phenol is an important model molecule for the theoretical and experimental investigation of dissociation in the multistate potential energy surfaces. Recent theoretical calculations [X. Xu et al., J. Am. Chem. Soc. 136, 16378 (2014)] suggest that the phenoxyl radical produced in both the X and A states from the O-H bond fission in phenol can contribute substantially to the slow component of photofragment translational energy distribution. However, current experimental techniques struggle to separate the contributions from different dissociation pathways. A new type of time-resolved pump-probe experiment is described that enables the selection of the products generated from a specific time window after molecules are excited by a pump laser pulse and can quantitatively characterize the translational energy distribution and branching ratio of each dissociation pathway. This method modifies conventional photofragment translational spectroscopy by reducing the acceptance angles of the detection region and changing the interaction region of the pump laser beam and the molecular beam along the molecular beam axis. The translational energy distributions and branching ratios of the phenoxyl radicals produced in the X, A, and B states from the photodissociation of phenol at 213 and 193 nm are reported. Unlike other techniques, this method has no interference from the undissociated hot molecules. It can ultimately become a standard pump-probe technique for the study of large molecule photodissociation in multistates.

Advantage of spatial map ion imaging in the study of large molecule photodissociation.

The original ion imaging technique has low velocity resolution, and currently, photodissociation is mostly investigated using velocity map ion imaging. However, separating signals from the background (resulting from undissociated excited parent molecules) is difficult when velocity map ion imaging is used for the photodissociation of large molecules (number of atoms ≥ 10). In this study, we used the photodissociation of phenol at the S1 band origin as an example to demonstrate how our multimass ion imaging technique, based on modified spatial map ion imaging, can overcome this difficulty. The photofragment translational energy distribution obtained when multimass ion imaging was used differed considerably from that obtained when velocity map ion imaging and Rydberg atom tagging were used. We used conventional translational spectroscopy as a second method to further confirm the experimental results, and we conclude that data should be interpreted carefully when velocity map ion imaging or Rydberg atom tagging is used in the photodissociation of large molecules. Finally, we propose a modified velocity map ion imaging technique without the disadvantages of the current velocity map ion imaging technique.

Is energy pooling necessary in ultraviolet matrix-assisted laser desorption/ionization?

RATIONALE: Energy pooling has been suggested as the key process for generating the primary ions during ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI). In previous studies, decreases in fluorescence quantum yields as laser fluence increased for 2-aminobenzoic acid, 2,5-dihydroxybenzoic acid (2,5-DHB), and 3-hydroxypicolinic acid were used as evidence of energy pooling. This work extends the research to other matrices and addresses whether energy pooling is a universal property in UV-MALDI. METHODS: Energy pooling was investigated in a time-resolved fluorescence experiment by using a short laser pulse (355 nm, 20 ps pulse width) for excitation and a streak camera (1 ps time resolution) for fluorescence detection. RESULTS: The excited-state lifetime of 2,5-DHB decreased with increases in laser fluence. This suggests that a reaction occurs between two excited molecules, and that energy pooling may be one of the possible reactions. However, the excited-state lifetime of 2,4,6-trihydroxyacetophenone (THAP) did not change with increases in laser fluence. The upper limit of the energy pooling rate constant for THAP is estimated to be approximately 100-500 times smaller than that of 2,5-DHB. CONCLUSIONS: The small energy pooling rate constant for THAP indicates that the potential contribution of the energy pooling mechanism to the generation of THAP matrix primary ions should be reconsidered.

Near-edge X-ray absorption fine structure spectra and site-selective dissociation of phenol.

A time-of-flight mass spectrometer with orthogonal acceleration and soft X-rays from synchrotron radiation were utilized to measure near-edge X-ray absorption fine structure (NEXAFS) spectra of carbon and oxygen in phenol and the corresponding ionic fragments following core excitation. The photon energies were in the range of 284-298 eV for the carbon K-edge and 529.5-554.5 eV for the oxygen K-edge. The total ion yield, ion intensity for each ionic fragment, and ion intensity ratio, defined as ion intensity divided by total ion yield, were measured as a function of photon energy. Possible mechanisms of dissociation are proposed and enhancements of specific products of dissociation are reported. In general, the enhancement of these specific products is small in the carbon K-edge region but is clear for some products at the oxygen K-edge. In particular, elimination of the H atom from the hydroxyl group was observed only at the oxygen K-edge. One remarkable result is that an excitation of a core-level electron of oxygen greatly enhanced the cleavage of specific C-C bonds.

Core excitation, specific dissociation, and the effect of the size of aromatic molecules connected to oxygen: phenyl ether and 1,3-diphenoxybenzene.

Near-edge X-ray absorption fine structure (NEXAFS) spectra of phenyl ether at the carbon K-edge and 1,3-diphenoxybenzene at both the carbon and oxygen K-edges were measured in the total ion yield mode using X-rays from a synchrotron and a reflectron time-of-flight mass spectrometer. Time-dependent density functional theory was adopted to calculate the carbon and oxygen K-edge NEXAFS spectra of phenol, phenyl ether, and 1,3-diphenoxybenzene. The assignments and a comparison of the experimental and calculated spectra are presented. The mass spectra of ionic products formed after X-ray absorption at various excitation energies are also reported. Specific dissociations were observed for the 1s → π* transition of phenyl ether. In comparison with phenol and phenyl ether, the dependence of the fragmentation on the excitation site and destination state was weak in 1,3-diphenoxybenzene, likely as a result of delocalization of the valence electrons and rapid randomization of energy.

Fluorescence spectroscopy of UV-MALDI matrices and implications of ionization mechanisms.

Matrix-assisted laser desorption ionization (MALDI) has been widely used in the mass analysis of biomolecules; however, there are a lot of debates about the ionization mechanisms. Previous studies have indicated that S1-S1 annihilation might be a key process in the generation of primary ions. This study investigates S1-S1 annihilation by examining the time-resolved fluorescence spectra of 12 matrices. No S1-S1 annihilation was observed in six of these matrices (3-hydroxy-picolinic acid, 6-aza-2-thiothymine, 2,4-dihydroxy-acetophenone, 2,6-dihydroxy-acetophenone, 2,4,6-trihydroxy-acetophenone, and ferulic acid). We observed two matrix molecules reacting in an electronically excited state (S1) in five of these matrices (2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxy-acetophenone, 2,3-dihydroxybenzoic acid, and 2,6-dihydroxybenzoic acid), and S1-S1 annihilation was a possible reaction. Among these five matrices, no S1-S1 annihilation was observed for 2,3-dihydroxybenzoic acid in typical peak power region of nanosecond laser pulses in MALDI, but a very small value of reaction rate constant was observed only in the high peak power region. The excited-state lifetime of sinapinic acid was too short to determine whether the molecules reacted in an electronically excited state. No correlation was observed between the ion generation efficiency of MALDI and S1-S1 annihilation. The results indicate that the proposal of S1-S1 annihilation is unnecessary in MALDI and energy pooling model for MALDI ionization mechanism has to be modified.

Far-UV absorption spectra of SiH2 and dibridged Si2H2 isolated in solid argon.

We employ electron bombardment during the deposition of an Ar matrix containing a small proportion of SiH4 to generate various silicon hydrides. Subsequently, the irradiation of a matrix sample at 365 nm decomposes SiH2 and dibridged Si2H2 in solid Ar, which we identify through infrared spectroscopy. We further recorded the corresponding ultraviolet absorption spectra at each experimental stage. An intense band observed in the range of 170-203 nm is largely destroyed upon 365-nm photolysis, which is assigned to the C1B2 â† X1A1 transition of SiH2. Moreover, a moderate band observed in the region of 217-236 nm is reduced slightly, which is assigned to the 31B2 â† X1A1 transition of dibridged Si2H2. These assignments are made based on the observed photolytic behavior, and the prediction of the vertical excitation energies with the corresponding oscillator strengths by using time-dependent density functional theory and equation-of-motion coupled cluster theory.

IR absorption spectra of aniline cation, anilino radical, and phenylnitrene isolated in solid argon.

Electron bombardment of aniline (PhNH2) in an Ar matrix mainly generated the aniline cation (PhNH2+), anilino (PhNH) and phenyl (Ph) radicals, and phenylnitrene (PhN). Further irradiation of the electron-bombarded matrix sample at 365 nm depleted PhNH2+ and PhN, and resulted in the formation of PhNH2, PhNH, and Ph. In separate experiments, irradiation of the PhNH2/Ar matrix samples at 265 or 160 nm mainly generated PhNH and Ph radicals, but without the formation of PhNH2+ and PhN. According to the observed photochemical behaviors, quantum-chemically predicted harmonic vibrational wavenumbers of each species, and the information reported in previous photodissociation studies, we unambiguously characterized the IR features of the aromatic species. The information of the vibrational fundamentals of PhNH is new and the formation mechanism is discussed.

Photostability of amino acids: photodissociation dynamics of phenylalanine chromophores.

The theoretical prediction of H atom elimination on the excited state of phenol, imidazole and indole, the respective chromophores for the amino acids tyrosine, histidine and tryptophan, and the confirmation of theoretical prediction by experimental observations have a great impact on the explanation of photostability of amino acids upon irradiation with UV photons. On the other hand, no theoretical prediction of the excited state photodissociation dynamics has been made on the other aromatic amino acid, phenylalanine. In this work, photodissociation dynamics for various phenylalanine chromophores, including, phenylethylamine, N-methyl-phenylethylamine, and N-acetyl phenylalanine methyl ester was investigated in a molecular beam at 248 and 193 nm using multimass ion imaging techniques. The major dissociation channel for these compounds is the C-C bond cleavage. However, the photofragment translational energy distribution of phenylethylamine contains two components. The slow component corresponds to the dissociation on the ground state surface after internal conversion, and the fast component represents the dissociation from an excited state with a large exit barrier. The competition between the dissociation on the ground state and on the excited state changes as the size of chromophores increases. Internal conversion to the ground state prior to dissociation becomes the major nonradiative process for large chromophores. This study reveals the size-dependent photostability for these amino acid chromophores.

Photodissociation dynamics of tryptophan and the implication of asymmetric photolysis.

Photodissociation of amino acid tryptophan in a molecular beam at wavelengths of 212.8 and 193 nm, corresponding to excitation to the second and third absorption bands, was investigated using multimass ion imaging techniques. The respective wavelengths also represent excitation to the edge of a positive circular dichroism band and the center of a negative circular dichroism band of L-tryptophan. Only one dissociation channel was observed at both photolysis wavelengths: C(8)NH(6)CH(2)CHNH(2)COOH-->C(8)NH(6)CH(2)+CHNH(2)COOH. Dissociation rates were found to be 1.3x10(6) and 5x10(6) s(-1) at the respective wavelengths. Comparison to theoretical calculation indicates that dissociation occurs on the ground state after internal conversion. Implication of asymmetric photolysis is discussed.

Comparison of Clinical Characteristics Between Febrile and Afebrile Seizures Associated With Acute Gastroenteritis in Childhood.

Background: Acute gastroenteritis (AGE) accompanied by seizures is not a rare scenario in childhood. We investigated the clinical features of children with febrile or afebrile seizures during AGE and aimed to identify the impact of fever in this situation-related seizure. Methods: We retrospectively reviewed the medical charts of children admitted due to seizures associated with mild AGE between January 2008 and December 2017. These consecutive patients were divided into two groups: an "afebrile group" whose diagnosis was compatible with "benign convulsion with mild gastroenteritis (CwG)" and a "febrile group" who had a fever within 24 h of the onset of an AGE-related seizure. We compared the two groups' clinical and laboratory characteristics, electroencephalograms (EEG), neuroimaging, and outcomes. Results: Of the children suffering from AGE and seizures, 41 were afebrile and 30 were febrile, with a mean age of 32.2 ± 27.6 months. The gender, seizure semiology, frequency, duration of seizures, the time interval between AGE symptoms onset and first seizure, and levels of serum sodium, and hepatic enzymes were significantly different between the two groups. The most frequently identified enteropathogen was rotavirus (33%), especially in the male and febrile subjects. Afebrile patients had more EEG abnormalities initially, but all returned to normal later. All cases had an uneventful outcome. Of note, seizure clusters (≥2 episodes) occurred more frequently in the afebrile patients who had a duration of AGE symptoms lasting 2 days or more, or white blood cell counts ≥ 10,000/µL (p-values: 0.05 and 0.04, respectively). In comparison with seven similar studies, all showed more seizure clusters, partial seizures, and a shorter interval between AGE onset and seizures in afebrile patients than in febrile patients. Contrarily, afebrile patients had longer seizure duration and lower serum hepatic transaminases than febrile patients. Conclusion: Although fever partially influenced the clinical features of AGE-related seizures, febrile CwG might have pathophysiology distinctly different from that of febrile seizures. Comprehensive knowledge in discerning febrile and afebrile CwG can help to avoid unnecessary diagnostics tests, and anticonvulsants use.

Photodissociation dynamics of N-methylindole, N-methylpyrrole, and anisole.

Photodissociation experiments employing molecular beams of N-methylindole, N-methylpyrrole, and anisole at 193 and 248 nm, respectively, have been conducted using multimass ion imaging techniques. We find that CH3 elimination is the sole dissociation channel for the studied molecules at both 193 and 248 nm. The photofragment translational energy distribution of anisole is found to contain both fast and slow components at the two wavelengths. On the other hand, a fast component (large recoil velocity) is dominant for N-methylindole at 248 nm, and a slow component (small recoil velocity) is dominant at 193 nm. The absorption coefficient of N-methylpyrrole is too weak for study at 248 nm. The photofragment translational energy distribution at 193 nm includes a large portion of the slow component and a small portion of the fast component. The findings indicate that the fast component corresponds to dissociation from the repulsive excited state and the slow component corresponding to dissociation from the ground electronic state. A comparison with the photodissociation dynamics of phenol, pyrrole, and indole suggests that replacement of the H atom by CH3 does not change the dissociation channels on the excited state. However, the respective dissociation channels for anisole and N-methylpyrrole on the ground state differ significantly from that for phenol and pyrrole.

Photodissociation dynamics of 2,5-dihydroxyacetophenone.

Photodissociation of 2,5-dihydroxyacetophenone (DHAP), an important matrix compound in matrix-assisted laser desorption/ionization (MALDI), was studied in a molecular beam at 193 nm using multimass ion imaging techniques. Two major dissociation channels were observed, including (1) C(6)H(3)(OH)(2)COCH(3) --> OC(6)H(3)(OH)COCH(3) + H and (2) C(6)H(3)(OH)(2)COCH(3) --> C(6)H(3)(OH)(2) + COCH(3). The minor channels include C(6)H(3)(OH)(2)COCH(3) --> C(6)H(3)(OH)(2)CO + CH(3) and/or C(6)H(3)(OH)(2)COCH(3) --> C(6)H(3)(OH)(2) + CO + CH(3). The photofragment translational energy distribution suggests that reaction 1 occurs at an excited state in which the potential along the O-H bond distance is repulsive. Comparison to the branching ratios from RRKM calculations suggests that reaction 2 does not occur at either the ground state or the triplet state or that if it does occur at one of these states it must not follow the RRKM model. A comparison to the photodissociation dynamics of acetophenone and phenol and its derivatives was made.

355 nm multiphoton dissociation and ionization of 2, 5-dihydroxyacetophenone.

Multiphoton dissociation and ionization of 2,5-dihydroxyacetophenone (DHAP), an important matrix compound in UV matrix-assisted laser desorption/ionization (MALDI), is studied in a molecular beam at 355 nm using multimass ion imaging mass spectrometer and time-of-flight mass spectrometry. For laser fluence larger than 130 mJ/cm(2), nearly all of the irradiated molecules absorb at least one photon. The absorption cross section was found to be sigma = 1.3(+/-0.2) x 10(-17)cm(2). Molecules excited by two photons quickly dissociate into fragments. The major channels are (1) C(6)H(3)(OH)(2)COCH(3) --> C(6)H(3)(OH)(2)CO + CH(3) and (2) C(6)H(3)(OH)(2)COCH(3) --> C(6)H(3)(OH)(2) + COCH(3). Molecules absorbing three or more photons become parent ions or crack into smaller ionic fragments. The concentration ratio of ions (parent ions and ionic fragments) to neutral fragments is about 10(-6):1. Changing the molecular beam carrier gas from He at 250 Torr to Ar at 300 Torr results in molecular beam clustering (dimers and trimers). Multiphoton ionization of clusters by a 355 nm laser beam produces only dimer cations, (C(6)H(3)(OH)(2)COCH(3))(2)(+). Protonated clusters or negatively charged ions, observed from a solid sample of DHAP using 355 nm multiphoton ionization, were not found in the molecular beam. The experimental results indicate that the photoionization occurs in the gas phase after DHAP vaporizes from the solid phase may not play an important role in the MALDI process.

Health Education and Symptom Flare Management Using a Video-based m-Health System for Caring Women With IC/BPS.

OBJECTIVE: To assess effectiveness of the video-based m-health system providing videos dictated by physicians for health education and symptom self-management for patients with Interstitial cystitis/bladder pain syndrome (IC/BPS). METHODS: An m-health system was designed to provide videos for weekly health education and symptom flare self-management. O'Leary-Sant index and visual analogue scale as well as SF-36 health survey were administrated to evaluate the disease severity and quality of life (QoL), respectively. A total of 60 IC/BPS patients were recruited and randomly assigned to either control group (30 patients) or study group (30 patients) in sequence depending on their orders to visit our urological clinic. Patients in both control and study groups received regular treatments, while those in the study group received additional video-based intervention. Statistical analyses were conducted to compare the outcomes between baseline and postintervention for both groups. The outcomes of video-based intervention were also compared with the text-based intervention conducted in our previous study. RESULTS: After video-based intervention, patients in the study group exhibited significant effect manifested in all disease severity and QoL assessments except the pain visual analogue scale, while no significance was found in the control group. Moreover, the study group exhibited more significant net improvements than the control group in 7 SF-36 constructs, except the mental health. The limitations include short intervention duration (8 weeks) and different study periods between text-based and video-based interventions. CONCLUSION: Video-based intervention is effective in improving the QoL of IC/BPS patients and outperforms the text-based intervention even in a short period of intervention.

Posterior Reversible Encephalopathy Syndrome With Spinal Cord Involvement in Children.

We characterized a cohort of patients with posterior reversible encephalopathy syndrome with spinal cord involvement. We encountered 2 children and identified an additional 19 patients from the internet databases. Of the 21 patients analyzed, 8 were children. The mean peak systolic blood pressure in adults was significantly higher than in children (221.8 ± 14.3 vs 191.4 ± 31.3 mm Hg; P < .01). Regardless of age, the most common clinical symptom was headache (90%) and the least common clinical symptom was seizures (28%). Atypical neuroimaging was more common in children (63%) than in adults (8%). Abnormal cerebrospinal fluid results were frequently found in children (83%). All children recovered uneventfully, but 3 adults had sequelae. A broader clinicoradiologic spectrum makes the diagnosis of children more complex than in adults. Awareness of the atypical features with a meticulous management of hypertension is imperative to avoid unnecessary invasive workups and to achieve an uneventful recovery.

Is rigorous punishment effective? A case study of lifetime license revocation in Taiwan.

This study investigated the effectiveness of administrative lifetime driver's license revocation (ALLR) and its impact on offenders, based on a two-stage survey of 768 offenders. It was found that after ALLR had been imposed, 23.4% of these offenders were still driving almost the same as before, 59.8% drove significantly less frequently, and only 16.8% of the offenders gave up driving completely. The results of logistic regression models showed that offenders' compliance with ALLR was significantly correlated with their personal characteristics (age, income), penalty status (incarceration, duration of ALLR), and the need to drive for working, commuting and shopping. Elderly and low-income offenders were more likely to abide by the ALLR restriction. The application of the generalized estimating equations (GEE) model was used to identify the determinant factors affecting offenders' driving mileage, and to effectively estimate the driving mileage reduction as a result of the ALLR. It was found that ALLR is fairly effective in keeping offenders off the road, but that it may reduce their ability to make a living, resulting in the less fortunate becoming more helpless.