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Herein, we explore the generation and characterization of the radical cations of glycylphenylalanylglycine, or [GFG]•+, formed via dissociative electron-transfer reaction from the tripeptide to copper(II) within a ternary complex. A comprehensive investigation employing isotopic labeling, infrared multiple-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations elucidated the details and energetics in formation of the peptide radical cations as well as their dissociation products. Unlike conventional aromatic-containing peptide radical cations that primarily form canonical π-radicals, our findings reveal that 75% of the population of the experimentally produced [GFG]•+ precursors are [GFα•G]+, where the radical resides on the middle α-carbon of the phenylalanyl residue. This unexpected isomeric ion has an enthalpy of 6.8 kcal/mol above the global minimum, which has an N-terminal captodative structure, [Gα•FG]+, comprising 25% of the population. The [b₂-H]•+ product ions are also present in a ratio of 75/25 from [GFα•G]+/ [Gα•FG]+, the results of which are obtained from matches between the IRMPD action spectrum and predicted IR absorption spectra of the [b₂-H]•+ candidate structures, as well as from IRMPD isomer population analyses., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Residue-specific phosphorylation is a protein post-translational modification that regulates cellular functions. Experimental determination of the exact sites of protein phosphorylation provides an understanding of the signaling and processes at work for a given cellular state. Any experimental artifact that involves migration of the phosphate group during measurement is a concern, as the outcome can lead to erroneous conclusions that may confound studies on cellular signal transduction. Herein, we examine computationally the mechanism by which a phosphate group migrates from one serine residue to another serine in monoprotonated pentapeptides [B A -pSer-Gly-Ser-B B + H] + → [B A -Ser-Gly-pSer-B B + H] + (where B A and B B are different combinations of the three basic amino acids, histidine, lysine, and arginine). In addition to moving the phosphate group, the overall mechanism involves transferring a proton from the N-terminal amino acid, B A , to the C-terminal amino acid, B B . This is not a synchronous process, and there is a key high-energy intermediate, structure C , that is zwitterionic with both the basic amino acids protonated and the phosphate group attached to both serine residues and carrying a negative charge. The barriers to moving the phosphate group are calculated to be in the range of 219-274 kJ mol -1 at the B3LYP/6-31G(d) level. These barriers are systematically slightly lower and in good agreement with single-point energy calculations at both M06-2 X /6-311++G(d,p) and MP2/6-31++G(d,p) levels. The competitive reaction, loss of phosphoric acid from the protonated pentapeptides, has a barrier in the range of 176-202 kJ mol -1 at the B3LYP/6-31G(d) level. Extension of the theory to M06-2 X /6-311++G(d,p)//B3LYP/6-31G(d) and MP2/6-31++G(d,p)// B3LYP/6-31G(d) gives higher values for the loss of phosphoric acid, falling in the range of 196-226 kJ mol -1 ; these are comparable to the barriers against phosphate migration at the same levels of theory. For larger peptides His-pSer-(Gly) n -Ser-His, where n has values from 2 to 5, the barriers against the loss of phosphoric acid are higher than those against the phosphate group migration. This difference is most pronounced and significant when n = 4 and 5 (the differences are approximately 80 kJ mol -1 under the single-point energy calculations at the M06-2X and MP2 levels). Energy differences using two more recent functionals, M08-HX and MN15, on His-pSer-(Gly) n -Ser-His, where n = 1 and 5, are in good agreement with the M06-2X and MP2 calculations. These results provide the mechanistic rationale for phosphate migration versus other competing reactions in the gas phase under tandem mass spectrometry conditions.

Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S. meliloti with Medicago sativa and S. medicae with M. truncatula. Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bustamante et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background The pathophysiological mechanism behind adverse outcomes associated with ischemia-inducing epicardial coronary stenoses and microcirculatory dysfunction remains unclear. Wall shear stress (WSS) plays an important role in atherosclerotic plaque progression and vulnerability. We aimed to evaluate the relationship between WSS, functionally significant epicardial coronary stenoses, and microcirculatory dysfunction. Methods and Results Patients undergoing invasive coronary physiology testing were included. Fractional flow reserve, instantaneous wave-free ratio, and the index of microcirculatory resistance were measured. Quantitative coronary angiography was used to obtain the lesion percentage diameter stenosis. Computational fluid dynamics analysis was performed to calculate WSS parameters. Multiple regression analysis was performed to calculate the standardized regression coefficient (β) for the coronary physiology indices. A total of 107 vessels from 88 patients were included. Fractional flow reserve independently predicted the total area of low WSS (β=-0.44; 95% CI, -0.62 to -0.25; P <0.001) and maximum lesion WSS (β=-0.53; 95% CI, -0.70 to -0.36; P <0.001) after adjusting for percentage diameter stenosis and index of microcirculatory resistance. Similarly, instantaneous wave-free ratio also independently predicted the total area of low WSS (β=-0.45; 95% CI, -0.62 to -0.28; P <0.001) and maximum lesion WSS (β=-0.58; 95% CI, -0.73 to -0.43; P <0.001). The index of microcirculatory resistance did not predict either low or high WSS. Conclusions Fractional flow reserve and instantaneous wave-free ratio independently predicted the total burden of low WSS and maximum lesion WSS in coronary arteries. No relationship was found between microcirculatory dysfunction and WSS.

Background: Brief nonharmful ischemia, remote ischemic preconditioning (RIPC) has been proposed to confer benefit to patients with coronary artery disease via unknown mechanisms., Objectives: We aimed to investigate the effect of RIPC on circulating levels of extracellular vesicles (EVs) and global coagulation and fibrinolytic factors in patients with coronary disease., Patients/methods: Blood samples were taken from 60 patients presenting for coronary angiography enrolled in a randomized, controlled trial before and after RIPC (3 × 5 min administration of 200 mmHg sphygmomanometer on the arm, n = 31) or sham (n = 29) treatment. Most patients (n = 48) had significant coronary artery disease and all were taking at least one antiplatelet agent., Results: Remote ischemic preconditioning significantly decreased circulating levels of EVs expressing platelet markers CD41 and CD61 detected by flow cytometry in plasma, whereas no such effect was found on EVs expressing phosphatidylserine, CD62P, CD45, CD11b, CD144, CD31 + /CD41 - , or CD235a. RIPC had no effect on the overall hemostatic potential assay or circulating antigen levels of tissue plasminogen activator, urokinase, plasminogen activator inhibitor-1, or plasminogen. Sham treatment had no effect on any studied parameter. Statin use inhibited the effect of RIPC on CD61 + EVs, diabetes modified the effect of RIPC on CD45 + and CD11b + EVs, and hypertension modified the effect of RIPC on CD235a + EVs., Conclusions: Remote ischemic preconditioning decreased circulating levels of platelet-derived EVs in patients with coronary disease taking conventional antiplatelet therapy. This may reflect increased EV clearance/uptake or change in production. Clinical variables may alter the effectiveness of RIPC., (© 2021 International Society on Thrombosis and Haemostasis.)

Background: Transcatheter aortic valve implantation (TAVI) has become the standard-of-care for treatment of severe symptomatic aortic stenosis and is also being increasingly recommended for low-risk patients. While TAVI boasts positive post-procedural outcomes, it is also associated with cognitive complications, namely delirium and cognitive decline. There is a pressing need for accurate risk tools which can identify TAVI patients at risk of delirium and cognitive decline, as risk scores designed for general cardiovascular surgery fall short. The present effect-finding exploratory study will assess the utility of various measures in the context of aging and frailty in predicting who will and who will not develop delirium or cognitive impairment following TAVI. The measures we propose include gait, visual symptoms, voice, swallowing, mood and sleep. Methods: This is an observational prospective cohort study focused on identifying pre-procedural risk factors for the development of delirium and cognitive decline following TAVI. Potential risk factors will be measured prior to TAVI. Primary outcomes will be post-procedure cognitive decline and delirium. Secondary outcomes include activities of daily living, quality of life, and mortality. Delirium presence will be measured on each of the first 2 days following TAVI. All other outcomes will be assessed at 3-, 6-, and 12-months post-operatively. A series of logistic regressions will be run to investigate the relationship between potential predictors and outcomes (presence vs. absence of either delirium or cognitive decline). Discussion: This study will assess the strengths of associations between a range of measures drawn from frailty and aging literature in terms of association with cognitive decline and delirium following TAVI. Identified measures can be used in future development of TAVI risk prediction models, which are essential for the accurate identification of cognitive at-risk patients and successful application of pre-procedural interventions. Clinical Trial Registration: This trial is registered with the Australian New Zealand Clinical Trials Registry. [https://bit.ly/2PAotP5], [ACTRN12618001114235]., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ghezzi, Psaltis, Loetscher, Davis, Montarello, Lau, Delacroix, Bourke, McLoughlin, Keage and Keage.)

The number of immune-related endocrine dysfunctions (irEDs) has concurrently increased with the widespread use of immunotherapy in clinical practice and further expansion of the approved indications for immune checkpoint inhibitor (ICI) in cancer management. A retrospective analysis was conducted on consecutive patients ≥18 years of age with advanced solid malignancies who had received at least one dose of anti-programmed cell death protein 1 (anti-PD-1) and/or anti-CTLA4 antibodies between January 2014 and December 2019 at a university hospital in Hong Kong. Patients were reviewed up to two months after the last administration of an ICI. The types, onset times and grades of irEDs, including hypothyroidism, hyperthyroidism, adrenal insufficiency and immune-related diabetes mellitus, were recorded. Factors associated with irEDs were identified using multivariate analysis.&nbsp;A total of 953 patients (male: 603, 64.0%; median age: 62.0 years) were included. Of these, 580 patients (60.9%) used ICI-alone, 132 (13.9%) used dual-ICI, 187 (19.6%) used an ICI combined with chemotherapy (chemo + ICI), and 54 (5.70%) used immunotherapy with a targeted agent (targeted + ICI).&nbsp;A significantly higher proportion of patients using targeted + ICI had irEDs and hypothyroidism; in contrast, a higher proportion of patients using dual-ICI had adrenal insufficiency. There was no significant difference in the incidence of irED between the younger (<65 years) and older (≥65 years) patients. Using logistic regression, only treatment type was significantly associated with irEDs. Notably, older patients had a higher risk of having immune-related diabetes mellitus. This large, real-world cohort demonstrates that targeted + ICI has a higher risk of overall irED and hypothyroidism. Immunotherapy is safe and well-tolerated regardless of age, but close monitoring of fasting glucose is essential in older populations.

Radical cations of an aliphatic tripeptide prolyl-glycyl-glycine (PGG •+ ) and its sequence ions [a 3 + H] •+ and [b 2 - H] •+ have been generated by collision-induced dissociation of the [Cu(Phen)(PGG)] •2+ complex, where Phen = 1,10-phenanthroline. Infrared multiple photon dissociation spectroscopy, ion-molecule reaction experiments, and theoretical calculations have been used to investigate the structures of these ions. The unpaired electron in these three radical cations is located at different α-carbons. The PGG •+ radical cation has a captodative structure with the radical at the α-carbon of the proline residue and the proton on the oxygen of the first amide group. This structure is at the global minimum on the potential energy surface (PES). By contrast, the [a 3 + H] •+ and [b 2 - H] •+ ions are not the lowest-energy structures on their respective PESs, and their radicals are formally located at the C-terminal and second α-carbons, respectively. Density functional theory calculations on the structures of the ternary copper(II) complex ion suggest that the charge-solvated isomer of the metal complex is the precursor ion that dissociates to give the PGG •+ radical cation. The isomer of the complex in which PGG is bound as a zwitterion dissociates to give the [a 3 + H] •+ and [b 2 - H] •+ ions.

Background: The last visual survey of older adults in Hong Kong was a district-level study in 2002, with no assessment of behavioral and medical risk factors for visual impairment (VI). Our objectives were to determine the latest VI prevalence among older adults, significance of any spatial and temporal differences on the prevalence, and any associations of sociodemographic, behavioral and medical risk factors with VI from a multi-perspective analysis., Methods: Community-based pilot survey of residents from a suburb of Hong Kong, aged ≥50, using a standardized questionnaire, was conducted in 2016., Results: Of the 222 subjects, crude rates of bilateral and unilateral VI were 9.46 and 32.88%, respectively, or corresponding age-and-gender-adjusted rates of 6.89 and 30.5%. Older age and lower educational were associated with higher risk for unilateral VI, while older age, temporary housing, obesity and hyperlipidemia were associated with higher risk for bilateral VI. Smoking and alcohol-drinking status were not associated with unilateral or bilateral VI. Relative changes in ORs of hypertension or educational level on unilateral or bilateral VI were >  10% after adjusting for age. Interaction term between hyperlipidemia and gender or obesity was significant for unilateral VI. Gender, hypertension and cataract were not associated with unilateral or bilateral VI in general population of pooled analysis but were identified as risk factors in specific subgroups of stratified analysis. Refractive error (myopia or hyperopia) was significantly associated with VI in the eye-level analysis after adjusting the inter-eye correlation., Conclusions: Sociodemographic and medical risk factors contributed to VI, but behavioral risk factors did not. Sociodemographic disparities of visual health existed. Age was the confounders of the VI-hypertension or VI-educational level relationships. Gender and obesity were more likely to have multiplicative effect on unilateral VI when combined with hyperlipidemia. Stratified analysis should be conducted to provide further insight into the risk factors for VI in specific populations. Uncorrected refractive error remains a significant cause of impaired vision. The spatial and temporal differences in bilateral VI prevalence from the previous local study indicates a territory-wide survey is needed to assess regional differences and overall prevalence of VI in Hong Kong.

Objectives: To assess the proportions of respondents in the general community having heard or awareness, and their knowledge level, of three common eye diseases: age-related macular degeneration, cataract, and glaucoma. We also attempted to assess for risk factors that may be associated with any variations, which will help identify the areas of inadequate knowledge and demographics of potential audiences for focused health education., Methods: We conducted a community-based pilot survey for the residents from a southern suburb of Hong Kong in early 2016, by inviting all aged 50 or above to complete a standardized questionnaire in the local community hall., Results: Most of the 222 respondents have heard, or awareness, of cataract (92.79% or 81.98%, respectively), followed by glaucoma (86.94% or 52.70%, respectively), and age-related macular degeneration (51.35% or 29.28%, respectively). The results of Cronbach's alpha (α > 0.7) and Spearman's correlation coefficient (p < 0.01) suggested that the internal consistency, convergent and discriminant validities of the questionnaire were acceptable for the study population. Compared to a previous Hong Kong survey in 2002, the proportions of having heard of the three eye diseases were greater, but the overall knowledge remained limited. From a maximum knowledge score of 29, the median scores for age-related macular degeneration, cataract, and glaucoma were 9, 13, and 14, respectively. Except for the treatment of cataract, the knowledge level in most areas was low. Sociodemographic factors and medical history, rather than behavioral factors, were more likely to be associated with having a higher knowledge level. Subjects with family or friends with a history of glaucoma or age-related macular degeneration were more aware and knowledgeable, but not for subjects who were current and past smokers or alcohol drinkers. For age-related macular degeneration, gender modified the effect between age and knowledge level, while age was a confounder of having medical history, and having heard or awareness, of the disease., Conclusion: Despite a larger proportion of the community having heard or awareness since 15 years ago, much effort remains for improving health knowledge of these three eye diseases in Hong Kong. We recommend targeting respondents with higher lifestyle risks, such as current and past smokers or alcohol drinkers, as a focused audience, and utilizing family members, relatives, or friends as another way of distributing health information., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

Single-cell RNA sequencing (scRNA-seq) is the leading technique for characterizing the transcriptomes of individual cells in a sample. The latest protocols are scalable to thousands of cells and are being used to compile cell atlases of tissues, organs and organisms. However, the protocols differ substantially with respect to their RNA capture efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. In the present study, we generated benchmark datasets to systematically evaluate protocols in terms of their power to comprehensively describe cell types and states. We performed a multicenter study comparing 13 commonly used scRNA-seq and single-nucleus RNA-seq protocols applied to a heterogeneous reference sample resource. Comparative analysis revealed marked differences in protocol performance. The protocols differed in library complexity and their ability to detect cell-type markers, impacting their predictive value and suitability for integration into reference cell atlases. These results provide guidance both for individual researchers and for consortium projects such as the Human Cell Atlas.

Objective: To present the study design and the baseline data of a prospective cohort study investigating the safety, refractive correction and effectiveness of myopia control in subjects fitted with orthokeratology (ortho-k) lenses of different compression factors., Methods and Analysis: This study is a 2-year longitudinal, double-masked, partially randomised study. Myopic children aged between 6 and 10 years are recruited and they may choose to participate in either the ortho-k or spectacle-wearing group. Subjects in the ortho-k group are randomly assigned to wear ortho-k lenses of either conventional compression factor (CCF, 0.75 D) or increased compression factor (ICF, 1.75 D). For the ortho-k subjects, the time and between-group effects within the first month of lens wear were analysed., Results: Sixty-nine ortho-k subjects (CCF: 34; ICF: 35) and 30 control subjects were recruited. There were no significant differences in baseline demographic data among the three groups of subjects (p>0.19). At the 1-month visit, the first fit success rates were 97% and 100% in the CCF and ICF ortho-k group, respectively. A higher percentage of ICF subjects could achieve full correction (CCF: 88.2%; ICF: 94.3%). The change in axial length was significantly higher in the ICF group (CCF, 0.003 mm; ICF, -0.031 mm) (p<0.05). No significant between-group differences in daytime vision or in the coverage and depth of corneal staining between the two ortho-k groups (p>0.05) were observed at any visit., Conclusion: ICF did not compromise the corneal integrity and the lens centration within the first month of lens wear. The preliminary performance of ortho-k lenses with ICF of 1.00D shows that it was safe to be used in the longer term for the investigation of myopia control., Trial Registration Number: NCT02643342., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objectives: We investigated whether remote ischemic preconditioning (RIPC) inhibits agonist-induced conformational activation of platelet α IIb β 3 in patients with coronary artery disease already receiving conventional antiplatelet therapy., Patients/methods: Consecutive patients with angiographically confirmed coronary artery disease were randomized to RIPC or sham treatment. Venous blood was collected before and immediately after RIPC/sham. Platelet aggregometry (ADP, arachidonic acid) and whole blood platelet flow cytometry was performed for CD62P, CD63, active α IIb β 3 (PAC-1 binding) before and after stimulation with ADP, thrombin ± collagen, or PAR-1 thrombin receptor agonist., Results: Patients (25 RIPC, 23 sham) were well matched, 83% male, age (mean ± standard deviation) 63.3 ± 13.2 years, 95% aspirin, 81% P2Y 12 inhibitor. RIPC did not affect platelet aggregation, nor agonist-induced expression of CD62P, but selectively and significantly decreased α IIb β 3 activation after stimulation with either PAR-1 agonist peptide or the combination of thrombin + collagen, but not after ADP nor thrombin alone. The effect of RIPC on platelet α IIb β 3 activation was evident in patients receiving both aspirin and P2Y 12 inhibitor, and was not associated with an increase in vasodilator-stimulated phosphoprotein phosphorylation., Conclusions: Remote ischemic preconditioning inhibits conformational activation of platelet α IIb β 3 in response to exposure to thrombin and collagen in patients with coronary artery disease receiving dual antiplatelet therapy. These findings indicate agonist-specific inhibition of platelet activation by RIPC in coronary artery disease that is not obviated by the prior use of P2Y 12 inhibitors., (© 2020 International Society on Thrombosis and Haemostasis.)

Purpose: This retrospective longitudinal study aimed to examine the relationship between ocular higher-order aberrations (HOA) and axial eye growth in young myopic children undergoing orthokeratology (ortho-k) treatment., Methods: Axial length and ocular HOA, measured under cycloplegia annually over a 2-year period from the right eyes of myopic children, who previously completed ortho-k clinical trials, were retrieved. Linear mixed model analyses were applied to determine the association between ocular HOA, other known confounding variables (age, sex, and refractive error), and axial eye growth., Results: Data from 103 subjects were analyzed. The root-mean square (RMS) values of total ocular HOA (third to sixth orders combined), spherical (\({\rm{Z}}&#95;4^0\) and \({\rm{Z}}&#95;6^0\) combined), and comatic (\({\rm{Z}}&#95;3^{ - 1}\), \({\rm{Z}}&#95;3^1\), \({\rm{Z}}&#95;5^{ - 1}\), and \({\rm{Z}}&#95;5^1\) combined) aberrations increased by approximately 3, 9, and 2 times, respectively, after 2 years of ortho-k treatment. After adjusting for age, sex, and refractive error, higher RMS values of total HOA and spherical aberrations were associated with both longer axial length and slower axial elongation (all P < 0.01). For individual Zernike term coefficients, a higher level of positive spherical aberration (\({\rm{Z}}&#95;4^0\)) was also associated with longer axial length and slower axial elongation (both P < 0.01), after adjusting for baseline HOA., Conclusions: Ortho-k for myopia control significantly increases the Zernike coefficients and therefore the RMS values for a range of total ocular HOA terms or metrics in children. These findings suggest the potential role of HOA, particularly spherical aberration, as the possible mechanism of slowing axial elongation in ortho-k treatment.

The current study investigated the cognitive and neural substrates that underpin writing ability. We explored similarities and differences in writing numbers and words and compared these to language and manual actions in a large group of sub-acute, stroke patients (n = 740). The behavioral data showed association and dissociation in the ability to write words and numbers. Comorbidities of writing deficits with both language and motor impairments were prevalent, with less than a handful showing deficits restricted to the writing tasks. A second analysis with a subset of patients (n = 267) explored the neural networks that mediate writing abilities. Lesion to right temporal contributed to writing words, while lesions to left postcentral contributed to writing numbers. Overlapping neural mechanisms included the bilateral prefrontal cortex, right inferior parietal, left middle occipital and the right cerebellum. With the former regions associated with error pattern typical to writing based on prior knowledge (the lexical route), while lesion to left MOG was associated with errors to the phonological (non-lexical) route. Using principle components extracted from the behavioral data, we showed that right prefrontal and right parietal contributed to the ability to use pen, while lesion to bilateral prefrontal, inferior temporal and cerebellum supported unique use of pen for writing. The behavioral and imaging data suggested that writing numbers and words primarily relied on overlapping cognitive and neural functions. Incidents of pure writing deficits, in the absence of motor or language deficits were rare. Nevertheless, the PCA and neural data suggested that writing abilities were associated with some unique neuro-cognitive functions, specifically dedicated to the use of pen and the ability to transform meaning to motor command.

[a 3 + H] 2+ ions generated from Ln 3+ /tripeptide complexes, where Ln = La or Ce, have similar structures to the linear [a n ] + ions but with protonation at both the terminal NH 2 and N═CH 2 groups. Ion stability is favored by having the basic secondary amine of the proline residue at the N-terminus and by an amino acid residue accommodating one of the protons on the side chain. Dissociation of [a 3 + H] 2+ ions derived from peptides containing only aliphatic residues is by cleavage of the second amide bond to give [b 2 ] + or [a 2 ] + ions along with internal [a 1 ] + ions. For [a 3 + H] 2+ ions containing a tryptophan residue in the central location, in addition to cleavage of the amide bond, losses of neutrals NH 3 , HN═CHR, (NH 3 + CO), and HNCO were observed. Dissociations of some unsolvated Ln 3+ /tripeptide complexes gave [b 3 + H] 2+ ions in low abundance; formation of these [b 3 + H] 2+ ions was favored by the presence of a proline residue at the N-terminus and by either a histidine or tryptophan residue in the central position. Dissociation of these [b 3 + H] 2+ ions was by the loss of (H 2 O + CO) and not only CO, indicating that these ions did not have the same type of oxazolone structure as found for [b n ] + ions. Density functional theory calculations suggest that the observed [b 3 + H] 2+ ions of ProGlyGly were formed from [Ce(ProGlyGly)] 3+ complexes in which the peptide was bound to the metal ion as an enolate. Dissociation of the slightly lower-energy complex, where the peptide is bound in the keto form, would produce an oxazolone but the high barrier required to create this isomer of the [b 3 + H] 2+ ion would be sufficient to result in further dissociation. Two isomers of the [b 3 + H] 2+ ion of ProHisGly have been created, one from the [Ce(ProHisGly)] 3+ complex that characteristically dissociates by the combined loss of (H 2 O + CO) and the other by the loss of glycine from [ProHisGlyGly + 2H] 2+ . The [b 3 + H] 2+ ion derived from [ProHisGlyGly + 2H] 2+ dissociated by the loss of only CO.

Purpose: To determine the influence of compression factor upon changes in axial length and choroidal thickness during and following orthokeratology treatment., Methods: Orthokeratology lenses of different compression factors (one eye with 0.75 D and the fellow eye with 1.75 D) were randomly assigned to 28 subjects (median [range] age: 9.3 [7.8-11.0] years). Ocular biometrics were measured weekly for 1 month of lens wear and after lens cessation until the refraction stabilized (mean duration: 2.8 ± 0.4 weeks). Changes between eyes, and the associations between axial shortening and choroidal thickening with other ocular biometrics were analyzed., Results: There were no significant between-eye differences in the changes of ocular biometrics (all P > 0.05). After adjusting for paired-eye data, axial length initially decreased by 26 ± 41 μm ( P = 0.03) at week 1, then gradually returned to its original length. An approximate antiphase relationship of choroidal thickness (mean change: 9 ± 12 μm, P < 0.001) with axial length was observed. A significant rebound in axial length, but not choroidal thickness, occurred during the cessation period. Central corneal thinning and choroidal thickening accounted for 70% of initial axial shortening., Conclusions: Increasing the compression factor by 1.00 D did not affect changes in ocular biometrics in short-term orthokeratology. Significant axial shortening and choroidal thickening were observed during early treatment period. Axial shortening could not be entirely explained by central corneal thinning and choroidal thickening, which warrants further investigation., Translational Relevance: Initial axial shortening in orthokeratology is transient and therefore axial length remains useful for long-term monitoring of axial elongation in children.

Structures of [Ce(GGG)] 3+ and [Ce(GGG ? H)] 2+ have been investigated by DFT calculations. The two lowest-energy structures of the triply charged metal complex have the peptide in either the iminol or conventional zwitterionic form, and these ions have almost identical energies. In the doubly charged complex, the iminol and charge-solvated structures are the best structures on the potential energy surface, but the latter is favored. In both iminol structures, the metal ion coordinates to the iminol oxygen rather than to the nitrogen, unlike in previously reported iminol-containing complexes. Triply charged [Ce(peptide)] 3+ complexes are fragile and not easily isolated in a mass spectrometer, whereas the doubly charged [Ce(peptide ? H)] 2+ complexes are more robust. Here, we studied the fragmentations of 37 [Ce(peptide ? H)] 2+ and 30 [Ce(peptide)(peptide ? H)] 2+ complexes and the results are systematically summarized. Losses of CO and/or H 2 O are the most commonly observed fragmentation channels for [Ce(peptide ? H)] 2+ complexes and these dissociation pathways are modeled by DFT calculations. For [Ce(peptide)(peptide ? H)] 2+ complexes the neutral peptide plays the role of a solvent molecule but, unlike in the dissociations of [Ce(CH 3 CN)(peptide ? H)] 2+ complexes, the loss of the solvent molecule is not observed. Instead, fragmentation occurs by cleavage of the second amide bond of the solvating peptide molecule.

Objectives: Anaemia is associated with increased mortality in acute pulmonary embolism (PE) patients. However, prior studies have not examined the prognostic impact of trends in plasma haemoglobin during admission. This study investigates the impact of changes in haemoglobin level on mortality during hospital stay in acute PE., Study Design: A retrospective observational study., Setting: Tertiary-referral centre in Australia., Participants: Consecutive patients from 2000 to 2012 admitted with confirmed acute PE were identified from a dedicated PE database. Haemoglobin levels on days 1, 3-4, 5-6 and 7 of admission were retrieved. Patients without both baseline haemoglobin and subsequent haemoglobin levels were excluded (n=327), leaving 1099 patients as the study cohort. Anaemia was defined as haemoglobin <130 g/L for men and <120 g/L for women. There were 576 patients without anaemia throughout admission, 65 with transient anaemia (anaemic on day 1, but subsequently normalised during admission), 122 with acquired anaemia (normal on day 1 but developed anaemia during admission) and 336 with persistent anaemia. A total of 71 patients received blood transfusion during admission., Main Outcome Measure: 6-month mortality was tracked from a state-wide death database and analysed using multivariable modelling., Results: After adjusting for transfusion, patietns with persistent anaemia had a significantly increased 6-month mortality risk (adjusted HR 1.97, 95% CI 1.26 to 3.09, p=0.003) compared with patients without anaemia. There was no difference in mortality between patients with transient or acquired anaemia and patients without anaemia., Conclusion: Among patients who had anaemia during their admission for acute PE, only the subgroup with persistent anaemia demonstrated worse outcomes., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: To investigate the repeatability of choroidal thickness measurements determined from enhanced depth imaging optical coherence tomography (EDI-OCT) images of eyes after wearing single-vision spectacles (SV) and orthokeratology (ortho-k) lenses., Methods and Analysis: Two EDI-OCT images of 40 children (SV: 20, ortho-k: 20) taken at a single visit were analysed twice. Subjects in the ortho-k group had been wearing ortho-k for 1-4 weeks. The choroidal thickness was determined from each image using a graph theory-based software and, where appropriate, manual correction of choroidal boundaries was undertaken by an experienced examiner., Results: The mean (±SD) choroidal thickness was 227.3±42.2 µm for the SV subjects and 251.1±54.4 µm for the ortho-k subjects. The interimage differences in choroidal thickness were -0.99±3.54 and -1.14±5.03 µm for the SV and ortho-k subjects, respectively, and the limits of agreement were +5.96 to -7.93 and +8.72 to -11.00 µm, respectively., Conclusion: The coefficients of repeatability of choroidal thickness measurements from two EDI-OCT images taken at a single visit were 7.08 µm (SV) and 10.06 µm (ortho-k), suggesting that a change in choroidal thickness of less than 10 µm may not indicate a real change resulting from ortho-k lens wear., Competing Interests: Competing interests: None declared.

Background Remote ischemic preconditioning (RIPC) attenuates myocardial damage during elective and primary percutaneous coronary intervention. Recent studies suggest that coronary microcirculatory function is an important determinant of clinical outcome. The aim of this study was to assess the effect of RIPC on markers of microcirculatory function. Methods and Results Patients referred for cardiac catheterization and fractional flow reserve measurement were randomized to RIPC or sham. Operators and patients were blinded to treatment allocation. Comprehensive physiological assessments were performed before and after RIPC/sham including the index of microcirculatory resistance and coronary flow reserve after intracoronary glyceryl trinitrate and during the infusion of intravenous adenosine. Thirty patients were included (87% male; mean age: 63.1±10.0 years). RIPC and sham groups were similar with respect to baseline characteristics. RIPC decreased the calculated index of microcirculatory resistance (median, before RIPC: 22.6 [interquartile range [IQR]: 17.9-25.6]; after RIPC: 17.5 [IQR: 14.5-21.3]; P=0.007) and increased coronary flow reserve (2.6±0.9 versus 3.8±1.7, P=0.001). These RIPC-mediated changes were associated with a reduction in hyperemic transit time (median: 0.33 [IQR: 0.26-0.40] versus 0.25 [IQR: 0.20-0.30]; P=0.010). RIPC resulted in a significant decrease in the calculated index of microcirculatory resistance compared with sham (relative change with treatment [mean±SD] was -18.1±24.8% versus +6.1±37.5; P=0.047) and a significant increase in coronary flow reserve (+41.2% [IQR: 20.0-61.7] versus -7.8% [IQR: -19.1 to 10.3]; P<0.001). Conclusions The index of microcirculatory resistance and coronary flow reserve are acutely improved by remote ischemic preconditioning. This raises the possibility that RIPC confers cardioprotection during percutaneous coronary intervention as a result of an improvement in coronary microcirculatory function. Clinical Trial Registration URL: www.anzctr.org.au/ . Unique identifier: CTRN12616000486426.

Essentials Procoagulant platelets can be detected using GSAO in human whole blood. Stable coronary artery disease is associated with a heightened procoagulant platelet response. Agonist-induced procoagulant platelet response is not inhibited by aspirin alone. Collagen plus thrombin induced procoagulant platelet response is partially resistant to clopidogrel., Summary: Background Procoagulant platelets are a subset of highly activated platelets with a critical role in thrombin generation. Evaluation of their clinical utility in thrombotic disorders, such as coronary artery disease (CAD), has been thwarted by the lack of a sensitive and specific whole blood assay. Objectives We developed a novel assay, utilizing the cell death marker, GSAO [(4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid], and the platelet activation marker, P-selectin, to identify procoagulant platelets in whole blood by flow cytometry. Patients/Methods Using this assay, we characterized the procoagulant platelet population in healthy controls and a cohort of patients undergoing elective coronary angiography. Results In patients with CAD, compared with patients without CAD, there was a heightened procoagulant platelet response to thrombin (25.2% vs. 12.2%), adenosine diphosphate (ADP) (7.8% vs. 2.7%) and thrombin plus collagen (27.2% vs. 18.3%). The heightened procoagulant platelet potential in CAD patients was not associated with other markers of platelet function, including aggregation, dense granule release and activation of α 2b β 3 integrin. Although dual antiplatelet therapy (DAPT) was associated with partial suppression of procoagulant platelets, this inhibitory effect on a patient level could not be predicted by aggregation response to ADP and was not fully suppressed by clopidogrel. Conclusions We report for the first time that procoagulant platelets can be efficiently detected in a few microliters of whole blood using the cell death marker, GSAO, and the platelet activation marker, P-selectin. A heightened procoagulant platelet response may provide insight into the thrombotic risk of CAD and help identify a novel target for antiplatelet therapies in CAD., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

This retrospective longitudinal analysis aimed to investigate the association between ocular higher-order aberrations (HOAs) and axial eye growth in Hong Kong children. Measures of axial length and ocular HOAs under cycloplegia were obtained annually over a two-year period from 137 subjects aged 8.8 ± 1.4 years with mean spherical equivalent refraction of -2.04 ± 2.38 D. A significant negative association was observed between the RMS of total HOAs and axial eye growth (P = 0.03), after adjusting for other significant predictors of axial length including age, sex and refractive error. Similar negative associations with axial elongation were found for the RMS of spherical aberrations ([Formula: see text] and [Formula: see text] combined) (P = 0.037). Another linear mixed model also showed that greater levels of vertical trefoil [Formula: see text], primary spherical aberration [Formula: see text] and negative oblique trefoil [Formula: see text] were associated with slower axial elongation and longer axial length (all P < 0.05). These findings support the potential role of HOAs, image quality and a vision-dependent mechanism in childhood eye growth.

Collision-induced dissociations of isotopically labeled protonated tetraglycines establish that the [b 4 ] + ion formed by loss of water from the second amide bond (structure II) rearranges to form N 1 -protonated 3,5-dihydro-4H-imidazol-4-one (structure I), the product of water loss from the first amide bond. Structure II is slightly higher in energy than I (ΔH at 0 K is 5.1 kJ mol -1 , as calculated at M06-2X/6-311++G-(d,p)), and the barrier to interconversion is 139.8 kJ mol -1 above I. The dominant dissociation pathway is the loss of methanimine (HN=CH 2 ) from ion I with a barrier of 167.1 kJ mol -1 , giving [GlyGlyGlyGly + H - H 2 O - HN=CH 2 ] + , ion III; a minor channel, loss of NH 3 , has a slightly higher barrier (181.5 kJ mol -1 ). Using labeled glycine ( 13 C α ) it was determined that loss of the imine is from the same residue as that from which water was initially lost. The collision-induced dissociation spectra of ion III derived from both I and II were identical, and their energy-resolved curves were also very similar. Ion III fragments by losses of a glycine molecule (the dominant channel), a water molecule, and a glycine residue (57 Da), giving ions IV, V, and VII, respectively. Isotopic labeling established the origins of each of the neutral molecules that are lost. Using glycine (2,2 D 2 ), rapid deuterium exchange was observed for both ions I and II for the α-hydrogens that are from the same residue as that from which the water had been eliminated.

In this case, prompt stenting of the dissection entry flap allowed for stabilization and eventual healing of a severe catheter-induced aortic dissection, without resort to surgical intervention.

Discordance between angiography-based anatomical assessment of coronary stenosis severity and fractional flow reserve (FFR) has been attributed to several factors including lesion length and irregularity, and the myocardial territory supplied by the target vessel. We sought to examine if coronary arterial distensibility is an independent contributor to this discordance. There were two parts to this study. The first consisted of "in silico" models of 26 human coronary arteries. Computational fluid dynamics-derived FFR was calculated for fully rigid, partially distensible and fully distensible models of the 26 arteries. The second part of the study consisted of 104 patients who underwent coronary angiography and FFR measurement. Distensibility at the lesion site (DistensibilityMLA) and for the reference vessel (DistensibilityRef) was determined by analysing three-dimensional angiography images during end-systole and end-diastole. Computational fluid dynamics-derived FFR was 0.67±0.19, 0.70±0.18 and 0.75±0.17 (P<0.001) in the fully rigid, partially distensible and fully distensible models respectively. FFR correlated with both DistensibilityMLA (r = 0.36, P<0.001) and DistensibilityRef (r = 0.44, P<0.001). Two-way ANCOVA analysis revealed that DistensibilityMLA (F (1, 100) = 4.17, p = 0.031) and percentage diameter stenosis (F (1, 100) = 60.30, p < 0.01) were both independent predictors of FFR. Coronary arterial distensibility is a novel, independent determinant of FFR, and an important factor contributing to the discordance between anatomical and functional assessment of stenosis severity.

Background: Pulmonary embolism continues to be a significant cause of death. The aim was to derive and validate a risk prediction model for in-hospital death after acute pulmonary embolism to identify low risk patients suitable for outpatient management., Methods: A confirmed acute pulmonary embolism database of 1,426 consecutive patients admitted to a tertiary-center (2000-2012) was analyzed, with odd and even years as derivation and validation cohorts respectively. Risk stratification for in-hospital death was performed using multivariable logistic-regression modelling. Models were compared using receiver-operating characteristic-curve and decision curve analyses., Results: In-hospital mortality was 3.6% in the derivation cohort (n = 693). Adding day-1 sodium and bicarbonate to simplified Pulmonary Embolism Severity Index (sPESI) significantly increased the C-statistic for predicting in-hospital death (0.71 to 0.86, P = 0.001). The validation cohort yielded similar results (n = 733, C-statistic 0.85). The new model was associated with a net reclassification improvement of 0.613, and an integrated discrimination improvement of 0.067. The new model also increased the C-statistic for predicting 30-day mortality compared to sPESI alone (0.74 to 0.83, P = 0.002). Decision curve analysis demonstrated superior clinical benefit with the use of the new model to guide admission for pulmonary embolism, resulting in 43 fewer admissions per 100 presentations based on a risk threshold for admission of 2%., Conclusions: A risk model incorporating sodium, bicarbonate, and the sPESI provides accurate risk prediction of acute in-hospital mortality after pulmonary embolism. Our novel model identifies patients with pulmonary embolism who are at low risk and who may be suitable for outpatient management.

Background: Acute pulmonary embolism (PE) carries an increased risk of death. Using transthoracic echocardiography (TTE) to assist diagnosis and risk stratification is recommended in current guidelines. However, its utilization in real-world clinical practice is unknown. We conducted a retrospective observational study to delineate the prevalence of inpatient TTE use following confirmed acute PE, identify predictors for its use and its impact on patient's outcome., Methods: Clinical details of consecutive patients (2000 to 2012) from two tertiary-referral hospitals were retrieved from dedicated PE databases. All-cause and cause-specific mortality was tracked from a state-wide death registry., Results: In total, 2306 patients were admitted with confirmed PE, of whom 687 (29.8%) had inpatient TTE (39.3% vs 14.4% between sites, P<0.001). Site to which patient presented, older age, cardiac failure, atrial fibrillation and diabetes were independent predictors for inpatient TTE use, while malignancy was a negative predictor. Overall mortality was 41.4% (mean follow-up 66.5±49.5months). Though inpatient TTE use was not an independent predictor for all-cause or cardiovascular mortality in multivariable analysis, in the inpatient TTE subgroup, right ventricle-right atrial pressure gradient (hazard ratio [HR] 1.02 per-1mmHg increase, 95% confidence interval [CI] 1.01-1.03) and moderate/severe aortic stenosis (HR 2.26, 95% CI 1.20-4.27) independently predicted all-cause mortality., Conclusions: Inpatient TTE is used infrequently in real-world clinical settings following acute PE despite its usefulness in risk stratification, prognostication and assessing comorbid cardiac pathologies. Identifying patients that will benefit most from a TTE assessment following an acute PE episode and reducing barriers in accessing TTE should be explored., Competing Interests: The authors have declared that no competing interests exist.

Background: Symptomatic pulmonary embolism (PE) is a major cause of cardiovascular death and morbidity. Estimated prevalence and incidence of atrial fibrillation (AF) in developed countries are between 388-661 per 100,000, and 90-123 per 100,000 person-years respectively. However, the prevalence and incidence of AF in patients presenting with an acute PE and its predictors are not clear., Methods: Individual patient clinical details were retrieved from a database containing all confirmed acute PE presentations to a tertiary institution from 2001-2012. Prevalence and incidence of AF was tracked from a population registry by systematically searching for AF during any hospital admission (2000-2013) based on International Classification of Disease (ICD-10) code., Results: Of the 1,142 patients included in this study, 935 (81.9%) had no AF during index PE admission whilst 207 patients had documented baseline AF (prevalence rate 18,126 per 100,000; age-adjusted 4,672 per 100,000). Of the 935 patients without AF, 126 developed AF post-PE (incidence rate 2,778 per 100,000 person-years; age-adjusted 984 per 100,000 person-years). Mean time from PE to subsequent AF was 3.4 ± 2.9 years. Total mortality (mean follow-up 5.0 ± 3.7 years) was 42% (n = 478): 35% (n = 283), 59% (n = 119) and 60% (n = 76) in the no AF, baseline AF and subsequent AF cohorts respectively. Independent predictors for subsequent AF after acute PE include age (hazard ratio [HR] 1.06, 95% confidence interval [CI] 1.04-1.08, p<0.001), history of congestive cardiac failure (HR 1.88, 95% CI 1.12-3.16, p = 0.02), diabetes (HR 1.72, 95% CI 1.07-2.77, p = 0.02), obstructive sleep apnea (HR 4.83, 1.48-15.8, p = 0.009) and day-1 serum sodium level during index PE admission (HR 0.94, 95% CI 0.90-0.98, p = 0.002)., Conclusions: Patients presenting with acute PE have a markedly increased age-adjusted prevalence and subsequent incidence of AF. Screening for AF may be of importance post-PE.

Background: There are no currently effective cognitive assessment tools for patients who have suffered stroke in the People's Republic of China. The Birmingham Cognitive Screen (BCoS) has been shown to be a promising tool for revealing patients' poststroke cognitive deficits in specific domains, which facilitates more individually designed rehabilitation in the long run. Hence we examined the reliability and validity of a Cantonese version BCoS in patients with acute ischemic stroke, in Guangzhou., Method: A total of 98 patients with acute ischemic stroke were assessed with the Cantonese version of the BCoS, and an additional 133 healthy individuals were recruited as controls. Apart from the BCoS, the patients also completed a number of external cognitive tests, including the Montreal Cognitive Assessment Test (MoCA), Mini Mental State Examination (MMSE), Albert's cancellation test, the Rey-Osterrieth Complex Figure Test, and six gesture matching tasks. Cutoff scores for failing each subtest, ie, deficits, were computed based on the performance of the controls. The validity and reliability of the Cantonese BCoS were examined, as well as interrater and test-retest reliability. We also compared the proportions of cases being classified as deficits in controlled attention, memory, character writing, and praxis, between patients with and without spoken language impairment., Results: Analyses showed high test-retest reliability and agreement across independent raters on the qualitative aspects of measurement. Significant correlations were observed between the subtests of the Cantonese BCoS and the other external cognitive tests, providing evidence for convergent validity of the Cantonese BCoS. The screen was also able to generate measures of cognitive functions that were relatively uncontaminated by the presence of aphasia., Conclusion: This study suggests good reliability and validity of the Cantonese version of the BCoS. The Cantonese BCoS is a very promising tool for the detection of cognitive problems in Cantonese speakers.

The objective of this study was to identify the current status of stream water-quality assessment and reporting methods for four states in the Ohio River basin (Indiana, Ohio, Tennessee, and Virginia), as required by the 305(b) section of the United States (US) Clean Water Act. Specifically, we clarified the discrepancies that exist among stream-impairment status classified by benthic macroinvertebrate multimetric indices (MMIs) and depicted using Geographic Information Systems shapefiles. In addition, we provided guidance in solving some of the comparability problems that arise when developing state-specific MMIs and depicting stream-impairment status using Geographic Information Systems technology. The MMI variation among states and differences in shapefile formats resulted in a nationwide dataset, which cannot be directly compared. Incorporating the changes suggested in this study allow for a uniform assessment and reporting method nationwide. Successful implementation of these changes would strengthen the US Environmental Protection Agency efforts to identify impaired streams and sources of those impairments without the limitations of state-by-state .developed assessment methods.

We report a lesion-symptom mapping analysis of visual speech production deficits in a large group (280) of stroke patients at the sub-acute stage (<120 days post-stroke). Performance on object naming was evaluated alongside three other tests of visual speech production, namely sentence production to a picture, sentence reading and nonword reading. A principal component analysis was performed on all these tests' scores and revealed a 'shared' component that loaded across all the visual speech production tasks and a 'unique' component that isolated object naming from the other three tasks. Regions for the shared component were observed in the left fronto-temporal cortices, fusiform gyrus and bilateral visual cortices. Lesions in these regions linked to both poor object naming and impairment in general visual-speech production. On the other hand, the unique naming component was potentially associated with the bilateral anterior temporal poles, hippocampus and cerebellar areas. This is in line with the models proposing that object naming relies on a left-lateralised language dominant system that interacts with a bilateral anterior temporal network. Neuropsychological deficits in object naming can reflect both the increased demands specific to the task and the more general difficulties in language processing.

Background: Smoking is a learnt behavior during adolescence and understanding the factor/s associated with smoking will assist in identifying suitable measures in combating the rising prevalence of smoking among adolescents. This research aimed to identify the factor/s associated with smoking among form four students in Kota Tinggi, Johor. Multistage sampling was used to select a representative sample of students in 2008 and data were collected using a self-administered validated questionnaire. This study revealed that the overall smoking prevalence was 19.0% with a significantly higher proportion of male smokers (35.8%) as compared to females (3.15%). Adolescents who were male (aOR 6.6, 95%CI 2.61-16.4), those who had peer/s who smoked (aOR 4.03, 95% CI 1.31-12.4), and those who studied in rural areas and Felda Settlements ( aOR 4.59, 95 CI 1.11-18.0; aOR 9.42, 95%CI 3.91-29.1) were more likely to smoke in the past one week. On the other hand, adolescents with better knowledge on the hazards of smoking and negative attitudes towards smoking were less likely to smoke (aOR 0.51, 95%CI 0.37-0.72; aOR 0.67, 95%CI 0.46-0.99). Future promotional and interventional programmes on smoking should be considered and the above identified risk factors integrated to reduce smoking prevalence among students of school-going ages in Kota Tinggi. Johor.

Peptide radical cations A(n)Y(•+) (where n = 3, 4, or 5) and A5W(•+) have been generated by collision-induced dissociation (CID) of [Cu(II)(tpy)(peptide)](•2+) complexes. Apart from the charge-driven fragmentation at the N-Cα bond of the hetero residue producing either [c + 2H](+) or [z - H](•+) ions and radical-driven fragmentation at the Cα-C bond to give a(+) ions, unusual product ions [x + H](•+) and [z + H](•+) are abundant in the CID spectra of the peptides with the hetero residue in the second or third position of the chain. The formation of these ions requires that both the charge and radical be located on the peptide backbone. Energy-resolved spectra established that the [z + H](•+) ion can be produced either directly from the peptide radical cation or via the fragment ion [x + H](•+). Additionally, backbone dissociation by loss of the C-terminal amino acid giving [b(n-1) - H](•+) increases in abundance with the length of the peptides. Mechanisms by which peptide radical cations dissociate have been modeled using density functional theory (B3LYP/6-31++G** level) on tetrapeptides AYAG(•+), AAYG(•+), and AWAG(•+).

Nicotine, the addictive component of cigarettes, promotes lung cancer proliferation via the α7-nicotinic acetylcholine receptor (α7-nAChR) subtype. The present manuscript explores the effect of nicotine exposure on α7-nAChR levels in squamous cell carcinoma of the lung (SCC-L) in vitro and in vivo. Nicotine (at concentrations present in the plasma of average smokers) increased α7-nAChR levels in human SCC-L cell lines. Nicotine-induced up-regulation of α7-nAChR was confirmed in vivo by chicken chorioallantoic membrane models. We also observed that the levels of α7-nAChR in human SCC-L tumors (isolated from patients who are active smokers) correlated with their smoking history. Nicotine increased the levels of α7-nAChR mRNA and α7-nAChR transcription in human SCC-L cell lines and SCC-L tumors. Nicotine-induced up-regulation of α7-nAChR required GATA4 and GATA6. ChIP assays showed that nicotine induced the binding of GATA4 or GATA6 to Sp1 on the α7-nAChR promoter, thereby inducing its transcription and increasing its levels in human SCC-L. Our data are clinically relevant because SCC-L patients smoked for decades before being diagnosed with cancer. It may be envisaged that continuous exposure to nicotine (in such SCC-L patients) causes up-regulation of α7-nAChRs, which facilitates tumor growth and progression. Our results will also be relevant to many SCC-L patients exposed to nicotine via second-hand smoke, electronic cigarettes, and patches or gums to quit smoking.

Gas-phase complexes of five metal ions with the dipeptide HisGly have been characterized by DFT computations and by infrared multiple photon dissociation spectroscopy (IRMPD) using the free electron laser FELIX. Fine agreement is found in all five cases between the predicted IR spectral features of the lowest energy structures and the observed IRMPD spectra in the diagnostic region 1500-1800 cm(-1), and the agreement is largely satisfactory at longer wavelengths from 1000 to 1500 cm(-1). Weak-binding metal ions (K(+), Ba(2+), and Ca(2+)) predominantly adopt the charge-solvated (CS) mode of chelation involving both carbonyl oxygens, an imidazole nitrogen of the histidine side chain, and possibly the amino nitrogen. Complexes with Mg(2+) and Ni(2+) are found to adopt iminol (Im) binding, involving the deprotonated amide nitrogen, with tetradentate chelation. This tetradentate coordination of Ni(II) is the preferred binding mode in the gas phase, against the expectation under condensed-phase conditions that such binding would be sterically unfavorable and overshadowed by other outcomes such as metal ion hydration and formation of dimeric complexes. The HisGly results are compared with corresponding results for the PheAla, PheGly, and PhePhe ligands, and parallel behavior is seen for the dipeptides with N-terminal Phe versus His residues. An exception is the different chelation pattern determined for PhePhe versus HisGly, reflecting the intercalation-type cation binding pocket of the PhePhe ligand. The complexes group into three well-defined spectroscopic patterns: nickel and magnesium, calcium and barium, and potassium. Factors leading to differentiation of these distinct spectroscopic categories are (1) differing propensities for choosing the iminol binding pattern, and (2) single versus double charge on the metal center. Nickel and magnesium ions show similar gas-phase binding behavior, contrasting with their quite different patterns of peptide interaction in condensed phases.

Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by α7-, α3β2-, and β3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II-induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy.

Gene regulation by small RNAs (sRNAs) has been extensively studied in various bacteria. However, the presence and roles of sRNAs in mycobacteria remain largely unclear. Immunoprecipitation of RNA chaperone Hfq to enrich for sRNAs is one of the effective methods to isolate sRNAs. However, the lack of an identified mycobacterial hfq restricts the feasibility of this approach. We developed a novel method that takes advantage of the conserved inherent sRNAs-binding capability of heterologous Hfq from Escherichia coli to enrich sRNAs from Mycobacterium smegmatis, a model organism for studying Mycobacterium tuberculosis. We validated 12 trans-encoded and 12 cis-encoded novel sRNAs in M. smegmatis. Many of these sRNAs are differentially expressed at exponential phase compared with stationary phase, suggesting that sRNAs are involved in the growth of mycobacteria. Intriguingly, five of the cis-encoded novel sRNAs target known transposases. Phylogenetic conservation analysis shows that these sRNAs are pathogenicity dependent. We believe that our findings will serve as an important reference for future analysis of sRNAs regulation in mycobacteria and will contribute significantly to the development of sRNAs prediction programs. Moreover, this novel method of using heterologous Hfq for sRNAs enrichment can be of general use for the discovery of bacterial sRNAs in which no endogenous Hfq is identified.

We contrasted the neuroanatomical substrates of sub-acute and chronic visuospatial deficits associated with different aspects of unilateral neglect using computed tomography scans acquired as part of routine clinical diagnosis. Voxel-wise statistical analyses were conducted on a group of 160 stroke patients scanned at a sub-acute stage. Lesion-deficit relationships were assessed across the whole brain, separately for grey and white matter. We assessed lesions that were associated with behavioural performance (i) at a sub-acute stage (within 3 months of the stroke) and (ii) at a chronic stage (after 9 months post stroke). Allocentric and egocentric neglect symptoms at the sub-acute stage were associated with lesions to dissociated regions within the frontal lobe, amongst other regions. However the frontal lesions were not associated with neglect at the chronic stage. On the other hand, lesions in the angular gyrus were associated with persistent allocentric neglect. In contrast, lesions within the superior temporal gyrus extending into the supramarginal gyrus, as well as lesions within the basal ganglia and insula, were associated with persistent egocentric neglect. Damage within the temporo-parietal junction was associated with both types of neglect at the sub-acute stage and 9 months later. Furthermore, white matter disconnections resulting from damage along the superior longitudinal fasciculus were associated with both types of neglect and critically related to both sub-acute and chronic deficits. Finally, there was a significant difference in the lesion volume between patients who recovered from neglect and patients with chronic deficits. The findings presented provide evidence that (i) the lesion location and lesion size can be used to successfully predict the outcome of neglect based on clinical CT scans, (ii) lesion location alone can serve as a critical predictor for persistent neglect symptoms, (iii) wide spread lesions are associated with neglect symptoms at the sub-acute stage but only some of these are critical for predicting whether neglect will become a chronic disorder and (iv) the severity of behavioural symptoms can be a useful predictor of recovery in the absence of neuroimaging findings on clinical scans. We discuss the implications for understanding the symptoms of the neglect syndrome, the recovery of function and the use of clinical scans to predict outcome.

Purpose: Nicotine, the active component of cigarette smoke, has been found to stimulate angiogenesis in several experimental systems. In this study, the Matrigel duplex assay (Matrigel; BD Biosciences, Franklin Lakes, NJ) and the rat retinal explant assay were used to explore the molecular mechanisms underlying the proangiogenic effects of nicotine in endothelial cells., Methods: Western blot analysis was performed to determine the nicotinic acetylcholine receptor (nAChR) subtypes expressed on primary human retinal microvascular endothelial cells (HRMECs). The angiogenic effect of nicotine in the retina was evaluated with the duplex assay. The results obtained from the assay were confirmed by the rat retinal explant angiogenesis assay. ELISAs were used to measure MMP-2, -9, and -13 levels in HRMEC culture supernatants. The role of α7-nAChRs in nicotine-induced angiogenesis was examined by siRNA techniques., Results: Nicotine-induced angiogenesis required nAChR function and was associated with the upregulation of MMP-2 and -9 in HRMECs. Specifically, α7-nAChRs mediated the stimulatory effects of nicotine on retinal angiogenesis and MMP levels. Treatment of HRMECs with α7-nAChR antagonists ablated nicotine-induced angiogenesis. The inhibitory actions of α7-nAChR antagonists correlated with the suppression of MMP-2 and -9 levels in HRMECs., Conclusions: The α7-nAChR is vital for the proangiogenic activity of nicotine. The α7-nAChRs expressed on HRMECs upregulate levels of MMP-2 and -9, which stimulate retinal angiogenesis. The data also suggest that α7-nAChR antagonists could be useful agents for the therapy of angiogenesis-related retinal diseases.

Phosphodiesterases (PDEs) catalyze the hydrolysis of second messengers cAMP and cGMP in regulating many important cellular signals and have been recognized as important drug targets. Experimentally, a range of specificity/selectivity toward cAMP and cGMP is well-known for the individual PDE families. The study reported here reveals that PDEs might also exhibit selectivity toward conformations of the endogenous substrates cAMP and cGMP. Molecular dynamics simulations and free energy study have been applied to study the binding of the cAMP torsional conformers about the glycosyl bond in PDE10A2. The computational results elucidated that PDE10A2 is energetically more favorable in complex with the syn cAMP conformer (as reported in the crystal structure) and the binding of anti cAMP to PDE10A2 would lead to either a nonreactive configuration or significant perturbation on the catalytic pocket of the enzyme. This experimentally inaccessible information provides important molecular insights for the development of effective PDE10 ligands.

Background: Small cell lung cancer (SCLC) is characterized by rapid progression and low survival rates. Therefore, novel therapeutic agents are urgently needed for this disease. Capsaicin, the active ingredient of chilli peppers, displays anti-proliferative activity in prostate and epidermoid cancer in vitro. However, the anti-proliferative activity of capsaicin has not been studied in human SCLCs. The present manuscript fills this void of knowledge and explores the anti-proliferative effect of capsaicin in SCLC in vitro and in vivo., Methodology/principal Findings: BrdU assays and PCNA ELISAs showed that capsaicin displays robust anti-proliferative activity in four human SCLC cell lines. Furthermore, capsaicin potently suppressed the growth of H69 human SCLC tumors in vivo as ascertained by CAM assays and nude mice models. The second part of our study attempted to provide insight into molecular mechanisms underlying the anti-proliferative activity of capsaicin. We found that the anti-proliferative activity of capsaicin is correlated with a decrease in the expression of E2F-responsive proliferative genes like cyclin E, thymidylate synthase, cdc25A and cdc6, both at mRNA and protein levels. The transcription factor E2F4 mediated the anti-proliferative activity of capsaicin. Ablation of E2F4 levels by siRNA methodology suppressed capsaicin-induced G1 arrest. ChIP assays demonstrated that capsaicin caused the recruitment of E2F4 and p130 on E2F-responsive proliferative promoters, thereby inhibiting cell proliferation., Conclusions/significance: Our findings suggest that the anti-proliferative effects of capsaicin could be useful in the therapy of human SCLCs.

Two hundred two autologous pulmonary valves were transplanted into the aortic position between 1967 and 1982 at the National Heart Hospital in London. The indication for operation was congenital or acquired aortic valve disease, and the patients were followed for periods from 1 to 4 years. The patients were not anti-coagulated, but the entire series has been completely free from thromboembolism or bleeding. The actuarial prediction of freedom from valve-related deaths was 82 +/- 6% at the end of the fourteenth year after operation; deaths were due to reoperations for technical failure and to infective endocarditis. Event-free survival of the autologous pulmonary valve in the aortic position was 73 +/- 6% after 14 years at risk. Valve failure resulted mainly from technical problems encountered during the early years of surgical experience. There was no macroscopic or histological evidence of calcification in any of the failed valves. The right ventricular outflow was reconstructed with an aortic homograft in the majority of patients; 81 +/- 5% of these homografts demonstrated event-free performance over a 12-year follow-up period. It is concluded that the long-term performance of a pulmonary autograft inserted for aortic valve disease is superior to that of any other valve substitute and that the operation offers an almost ideal means of aortic valve replacement in appropriate patients.

This article reports the successful surgical treatment of six patients who presented with prosthetic endocarditis and uncontrolled bacteremia. They were treated by replacement of the aortic root with a preserved homograft aortic valve. With this technique, ventricular-aortic continuity was established with a centrally flowing valve together with its attached aorta. The weakened aortic and the associated root abscess were thereby excluded from the high-anulus pressure systemic circulation. There were no operative deaths, and the longest postoperative survival time is 10 years. Two patients have conduction disturbance and symptoms relating to their malfunctioning aortic valves. The remaining four patients are all well at 5 years, 2 years, 9 months, and 6 months with no further evidence of infection. Homograft aortic root replacement is a suitable treatment for this potentially lethal condition.