Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques(1). A catalytic mechanism, featuring a long-lived oxo-carbenium-ion intermediate, was proposed on the basis of model-building studies(2). The `Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta -glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta -glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated(3). Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by Xray diffraction. We formulate a general catalytic mechanism for all retaining beta -glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate

The aggregation of cytochrome C may be linked to its flexibility during refolding

Large-scale expression of biopharmaceutical proteins in cellular hosts results in production of large insoluble mass aggregates. In order to generate functional product, these aggregates require further processing through refolding with denaturant, a process in itself that can result in aggregation. Using a model folding protein, cytochrome C, we show how an increase in final denaturant concentration decreases the propensity of the protein to aggregate during refolding. Using polarised fluorescence anisotropy, we show how reduced levels of aggregation can be achieved by increasing the period of time the protein remains flexible during refolding, mediated through dilution ratios. This highlights the relationship between the flexibility of a protein and its propensity to aggregate. We attribute this behaviour to the preferential urea-residue interaction, over self-association between molecules

Bench-to-bedside review : targeting antioxidants to mitochondria in sepsis

Peer reviewedPublisher PD

Electrodeposition of Protocrystalline Germanium from Supercritical Difluoromethane

We report results for the electrochemistry of the germanium(II) tri-halide anions, [GeCl3]?, [GeBr3]? and [GeI3]?, in supercritical difluoromethane containing 60?mm [NnBu4][BF4] at 19.1?MPa and 358?K. The voltammetry shows mass-transport-limited currents for reduction to germanium at gold on the first scan. There is no evidence of a germanium stripping peak and, on subsequent scans, the electrode slowly passivates with the deposition of approximately 0.4??m of material. The redox potentials for the reduction of the three tri-halides are in the order [GeCl3]? <[GeBr3]? <[GeI3]?, with the iodide being the most easily reduced complex. Electrodeposition of germanium onto TiN electrodes from supercritical difluoromethane at 19.1?MPa and 358?K, using either 16?mm [EMIM][GeI3] with 60?mm [EMIM][BF4] or 16?mm [NnBu4][GeI3] with 60?mm [NnBu4][BF4], gave deposition rates of 2–3??m?h?1. Raman spectroscopy and transmission electron microscopy showed that the resulting germanium films were protocrystalline, containing nanocrystals of germanium embedded in an amorphous germanium matri

Guiding structures with multiply connected cross-sections: evolution of propagation in external fields at complex Robin parameters

Properties of the two-dimensional ring and three-dimensional infinitely long straight hollow waveguide with unit width and inner radius $\rho_0$ in the superposition of the longitudinal uniform magnetic field $\bf B$ and Aharonov-Bohm flux are analyzed within the framework of the scalar Helmholtz equation under the assumption that the Robin boundary conditions at the inner and outer confining walls contain extrapolation lengths $\Lambda_{in}$ and $\Lambda_{out}$, respectively, with nonzero imaginary parts. It is shown that, compared to the disk geometry, the annulus opens up additional possibilities of varying magnetization and currents by tuning imaginary components of the Robin parameters on each confining circumference; in particular, the possibility of restoring a lossless longitudinal flux by zeroing imaginary part $E_i$ of the total transverse energy $E$ is discussed. The energy $E$ turns real under special correlation between the imaginary parts of $\Lambda_{in}$ and $\Lambda_{out}$ with the opposite signs what physically corresponds to the equal transverse fluxes through the inner and outer interfaces of the annulus. In the asymptotic case of the very large radius, simple expressions are derived and applied to the analysis of the dependence of the real energy $E$ on $\Lambda_{in}$ and $\Lambda_{out}$. New features also emerge in the magnetic field influence; for example, if, for the quantum disk, the imaginary energy $E_i$ is quenched by the strong intensities $B$, then for the annulus this takes place only when the inner Robin distance $\Lambda_{in}$ is real; otherwise, it almost quadratically depends on $B$ with the corresponding enhancement of the reactive scattering. Closely related problem of the hole in the otherwise uniform medium is also addressed for real and complex extrapolation lengths with the emphasis on the comparative analysis with its dot counterpart.Comment: 37 pages, 9 figure

The Curse Of An Aching Heart

https://digitalcommons.library.umaine.edu/mmb-vp/1257/thumbnail.jp

The role of the right temporoparietal junction in perceptual conflict: detection or resolution?

The right temporoparietal junction (rTPJ) is a polysensory cortical area that plays a key role in perception and awareness. Neuroimaging evidence shows activation of rTPJ in intersensory and sensorimotor conflict situations, but it remains unclear whether this activity reflects detection or resolution of such conflicts. To address this question, we manipulated the relationship between touch and vision using the so-called mirror-box illusion. Participants' hands lay on either side of a mirror, which occluded their left hand and reflected their right hand, but created the illusion that they were looking directly at their left hand. The experimenter simultaneously touched either the middle (D3) or the ring finger (D4) of each hand. Participants judged, which finger was touched on their occluded left hand. The visual stimulus corresponding to the touch on the right hand was therefore either congruent (same finger as touch) or incongruent (different finger from touch) with the task-relevant touch on the left hand. Single-pulse transcranial magnetic stimulation (TMS) was delivered to the rTPJ immediately after touch. Accuracy in localizing the left touch was worse for D4 than for D3, particularly when visual stimulation was incongruent. However, following TMS, accuracy improved selectively for D4 in incongruent trials, suggesting that the effects of the conflicting visual information were reduced. These findings suggest a role of rTPJ in detecting, rather than resolving, intersensory conflict

Approaches in biotechnological applications of natural polymers

Natural polymers, such as gums and mucilage, are biocompatible, cheap, easily available and non-toxic materials of native origin. These polymers are increasingly preferred over synthetic materials for industrial applications due to their intrinsic properties, as well as they are considered alternative sources of raw materials since they present characteristics of sustainability, biodegradability and biosafety. As definition, gums and mucilages are polysaccharides or complex carbohydrates consisting of one or more monosaccharides or their derivatives linked in bewildering variety of linkages and structures. Natural gums are considered polysaccharides naturally occurring in varieties of plant seeds and exudates, tree or shrub exudates, seaweed extracts, fungi, bacteria, and animal sources. Water-soluble gums, also known as hydrocolloids, are considered exudates and are pathological products; therefore, they do not form a part of cell wall. On the other hand, mucilages are part of cell and physiological products. It is important to highlight that gums represent the largest amounts of polymer materials derived from plants. Gums have enormously large and broad applications in both food and non-food industries, being commonly used as thickening, binding, emulsifying, suspending, stabilizing agents and matrices for drug release in pharmaceutical and cosmetic industries. In the food industry, their gelling properties and the ability to mold edible films and coatings are extensively studied. The use of gums depends on the intrinsic properties that they provide, often at costs below those of synthetic polymers. For upgrading the value of gums, they are being processed into various forms, including the most recent nanomaterials, for various biotechnological applications. Thus, the main natural polymers including galactomannans, cellulose, chitin, agar, carrageenan, alginate, cashew gum, pectin and starch, in addition to the current researches about them are reviewed in this article.. }To the Conselho Nacional de Desenvolvimento Cientfíico e Tecnológico (CNPq) for fellowships (LCBBC and MGCC) and the Coordenação de Aperfeiçoamento de Pessoal de Nvíel Superior (CAPES) (PBSA). This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and COMPETE 2020 (POCI-01-0145-FEDER-006684) (JAT)

Association of blood-based brain injury biomarker concentrations with outcomes after pediatric cardiac arrest

Importance: Families and clinicians have limited validated tools available to assist in estimating long-term outcomes early after pediatric cardiac arrest. Blood-based brain-specific biomarkers may be helpful tools to aid in outcome assessment. Objective: To analyze the association of blood-based brain injury biomarker concentrations with outcomes 1 year after pediatric cardiac arrest. Design, Setting, and Participants: The Personalizing Outcomes After Child Cardiac Arrest multicenter prospective cohort study was conducted in pediatric intensive care units at 14 academic referral centers in the US between May 16, 2017, and August 19, 2020, with the primary investigators blinded to 1-year outcomes. The study included 120 children aged 48 hours to 17 years who were resuscitated after cardiac arrest, had pre-cardiac arrest Pediatric Cerebral Performance Category scores of 1 to 3 points, and were admitted to an intensive care unit after cardiac arrest. Exposure: Cardiac arrest. Main Outcomes and Measures: The primary outcome was an unfavorable outcome (death or survival with a Vineland Adaptive Behavior Scales, third edition, score of \u3c70 points) at 1 year after cardiac arrest. Glial fibrillary acidic protein (GFAP), ubiquitin carboxyl-terminal esterase L1 (UCH-L1), neurofilament light (NfL), and tau concentrations were measured in blood samples from days 1 to 3 after cardiac arrest. Multivariate logistic regression and area under the receiver operating characteristic curve (AUROC) analyses were performed to examine the association of each biomarker with outcomes on days 1 to 3. Results: Among 120 children with primary outcome data available, the median (IQR) age was 1.0 (0-8.5) year; 71 children (59.2%) were male. A total of 5 children (4.2%) were Asian, 19 (15.8%) were Black, 81 (67.5%) were White, and 15 (12.5%) were of unknown race; among 110 children with data on ethnicity, 11 (10.0%) were Hispanic, and 99 (90.0%) were non-Hispanic. Overall, 70 children (58.3%) had a favorable outcome, and 50 children (41.7%) had an unfavorable outcome, including 43 deaths. On days 1 to 3 after cardiac arrest, concentrations of all 4 measured biomarkers were higher in children with an unfavorable vs a favorable outcome at 1 year. After covariate adjustment, NfL concentrations on day 1 (adjusted odds ratio [aOR], 5.91; 95% CI, 1.82-19.19), day 2 (aOR, 11.88; 95% CI, 3.82-36.92), and day 3 (aOR, 10.22; 95% CI, 3.14-33.33); UCH-L1 concentrations on day 2 (aOR, 11.27; 95% CI, 3.00-42.36) and day 3 (aOR, 7.56; 95% CI, 2.11-27.09); GFAP concentrations on day 2 (aOR, 2.31; 95% CI, 1.19-4.48) and day 3 (aOR, 2.19; 95% CI, 1.19-4.03); and tau concentrations on day 1 (aOR, 2.44; 95% CI, 1.14-5.25), day 2 (aOR, 2.28; 95% CI, 1.31-3.97), and day 3 (aOR, 2.04; 95% CI, 1.16-3.57) were associated with an unfavorable outcome. The AUROC models were significantly higher with vs without the addition of NfL on day 2 (AUROC, 0.932 [95% CI, 0.877-0.987] vs 0.871 [95% CI, 0.793-0.949]; P = .02) and day 3 (AUROC, 0.921 [95% CI, 0.857-0.986] vs 0.870 [95% CI, 0.786-0.953]; P = .03). Conclusions and Relevance: In this cohort study, blood-based brain injury biomarkers, especially NfL, were associated with an unfavorable outcome at 1 year after pediatric cardiac arrest. Additional evaluation of the accuracy of the association between biomarkers and neurodevelopmental outcomes beyond 1 year is needed