Reply to the 'Comment on "Penicillin's catalytic mechanism revealed by inelastic neutrons and quantum chemical theory"' by S. A. Glover, Phys. Chem. Chem. Phys., 2019, 21, 18012.

Herein we comparatively comment on the molecular metric 'amidicity', a descriptor of amide reactivity, and differing methods to determining it; with focus on lactam-rings. Specifically, our established amidicity percentage (AM%) approach is quantitatively contrasted with the transamidation (TA) method. This comment is organised into two sections, firstly addressing the differing methods in context of the computational bases of amidicity. This is followed by the quantitative demonstration that although both the AM% and HRS methods provide estimates of resonance enthalpy (ΔHRE), the former is more reliable across a wider set of systems. The robustness of the AM% approach is affirmed by quantitative matching of experimental NMR and kinetics measurements tracking changes in amide reactivities, including in Penicillin arising from modulation of its amide group and environmental effects.

Formation Mechanism of Benzo(a)pyrene: One of the Most Carcinogenic Polycyclic Aromatic Hydrocarbons (PAH).

The formation of polycyclic aromatic hydrocarbons (PAHs) is a strong global concern due to their harmful effects. To help the reduction of their emissions, a crucial understanding of their formation and a deep exploration of their growth mechanism is required. In the present work, the formation of benzo(a)pyrene was investigated computationally employing chrysene and benz(a)anthracene as starting materials. It was assumed a type of methyl addition/cyclization (MAC) was the valid growth mechanism in this case. Consequently, the reactions implied addition reactions, ring closures, hydrogen abstractions and intramolecular hydrogen shifts. These steps of the mechanism were computed to explore benzo(a)pyene formation. The corresponding energies of the chemical species were determined via hybrid density funcional theory (DFT), B3LYP/6-31+G(d,p) and M06-2X/6-311++G(d,p). Results showed that the two reaction routes had very similar trends energetically, the difference between the energy levels of the corresponding molecules was just 6.13 kJ/mol on average. The most stable structure was obtained in the benzo(a)anthracene pathway.

High efficiency two-photon uncaging coupled by the correction of spontaneous hydrolysis.

Two-photon (TP) uncaging of neurotransmitter molecules is the method of choice to mimic and study the subtleties of neuronal communication either in the intact brain or in slice preparations. However, the currently available caged materials are just at the limit of their usability and have several drawbacks. The local and focal nature of their use may for example be jeopardized by a high spontaneous hydrolysis rate of the commercially available compounds with increased photochemical release rate. Here, using quantum chemical modelling we show the mechanisms of hydrolysis and two-photon activation, and synthesized more effective caged compounds. Furthermore, we have developed a new enzymatic elimination method removing neurotransmitters inadvertently escaping from their compound during experiment. This method, usable both in one and two-photon experiments, allows for the use of materials with an increased rate of photochemical release. The efficiency of the new compound and the enzymatic method and of the new compound are demonstrated in neurophysiological experiments.

Cutting Edge: Role of NK Cells and Surfactant Protein D in Dendritic Cell Lymph Node Homing: Effects of Ozone Exposure.

The roles of NK cells, surfactant protein D (SP-D), and IFN-γ, as well as the effect of ozone (O3) inhalation, were studied on recirculation of pulmonary dendritic cells (DC) to the mediastinal lymph nodes. O3 exposure and lack of SP-D reduced NK cell IFN-γ and lung tissue CCL21 mRNA expression and impaired DC homing to the mediastinal lymph nodes. Notably, addition of recombinant SP-D to naive mononuclear cells stimulated IFN-γ release in vitro. Because NKp46, a glycosylated membrane receptor, was necessary for dose-dependent SP-D binding to NK cells in vitro and DC migration in vivo, we speculate that SP-D may constitutively stimulate IFN-γ production by NK cells, possibly via NKp46. This mechanism could then initiate the IFN-γ/IL-12 feedback circuit, a key amplifier of DC lymph node homing. Inhibition of this process during an acute inflammatory response causes DC retention in the peripheral lung tissue and contributes to injury.

Amide Activation in Ground and Excited States.

Not all amide bonds are created equally. The purpose of the present paper is the reinterpretation of the amide group by means of two concepts: amidicity and carbonylicity. These concepts are meant to provide a new viewpoint in defining the stability and reactivity of amides. With the help of simple quantum-chemical calculations, practicing chemists can easily predict the outcome of a desired process. The main benefit of the concepts is their simplicity. They provide intuitive, but quasi-thermodynamic data, making them a practical rule of thumb for routine use. In the current paper we demonstrate the performance of our methods to describe the chemical character of an amide bond strength and the way of its activation methods. Examples include transamidation, acyl transfer and amide reductions. Also, the method is highly capable for simple interpretation of mechanisms for biological processes, such as protein splicing and drug mechanisms. Finally, we demonstrate how these methods can provide information about photo-activation of amides, through the examples of two caged neurotransmitter derivatives.

Properdin Contributes to Allergic Airway Inflammation through Local C3a Generation.

Complement is implicated in asthma pathogenesis, but its mechanism of action in this disease remains incompletely understood. In this study, we investigated the role of properdin (P), a positive alternative pathway complement regulator, in allergen-induced airway inflammation. Allergen challenge stimulated P release into the airways of asthmatic patients, and P levels positively correlated with proinflammatory cytokines in human bronchoalveolar lavage (BAL). High levels of P were also detected in the BAL of OVA-sensitized and challenged but not naive mice. Compared with wild-type (WT) mice, P-deficient (P(-/-)) mice had markedly reduced total and eosinophil cell counts in BAL and significantly attenuated airway hyperresponsiveness to methacholine. Ab blocking of P at both sensitization and challenge phases or at challenge phase alone, but not at sensitization phase alone, reduced airway inflammation. Conversely, intranasal reconstitution of P to P(-/-) mice at the challenge phase restored airway inflammation to wild-type levels. Notably, C3a levels in the BAL of OVA-challenged P(-/-) mice were significantly lower than in wild-type mice, and intranasal coadministration of an anti-C3a mAb with P to P(-/-) mice prevented restoration of airway inflammation. These results show that P plays a key role in allergen-induced airway inflammation and represents a potential therapeutic target for human asthma.

Group 2 innate lymphoid cells mediate ozone-induced airway inflammation and hyperresponsiveness in mice.

BACKGROUND: Asthmatic patients are highly susceptible to air pollution and in particular to the effects of ozone (O3) inhalation, but the underlying mechanisms remain unclear. OBJECTIVE: Using mouse models of O3-induced airway inflammation and airway hyperresponsiveness (AHR), we sought to investigate the role of the recently discovered group 2 innate lymphoid cells (ILC2s). METHODS: C57BL/6 and BALB/c mice were exposed to Aspergillus fumigatus, O3, or both (3 ppm for 2 hours). ILC2s were isolated by means of fluorescence-activated cell sorting and studied for Il5 and Il13 mRNA expression. ILC2s were depleted with anti-Thy1.2 mAb and replaced by means of intratracheal transfer of ex vivo expanded Thy1.1 ILC2s. Cytokine levels (ELISA and quantitative PCR), inflammatory cell profile, and AHR (flexiVent) were assessed in the mice. RESULTS: In addition to neutrophil influx, O3 inhalation elicited the appearance of eosinophils and IL-5 in the airways of BALB/c but not C57BL/6 mice. Although O3-induced expression of IL-33, a known activator of ILC2s, in the lung was similar between these strains, isolated pulmonary ILC2s from O3-exposed BALB/c mice had significantly greater Il5 and Il13 mRNA expression than C57BL/6 mice. This suggested that an altered ILC2 function in BALB/c mice might mediate the increased O3 responsiveness. Indeed, anti-Thy1.2 treatment abolished but ILC2s added back dramatically enhanced O3-induced AHR. CONCLUSIONS: O3-induced activation of pulmonary ILC2s was necessary and sufficient to mediate asthma-like changes in BALB/c mice. This previously unrecognized role of ILC2s might help explain the heightened susceptibility of human asthmatic airways to O3 exposure.

Protein Stability and Unfolding Following Glycine Radical Formation.

Glycine (Gly) residues are particularly susceptible to hydrogen abstraction; which results in the formation of the capto-dative stabilized Cα-centered Gly radical (GLR) on the protein backbone. We examined the effect of GLR formation on the structure of the Trp cage; tryptophan zipper; and the villin headpiece; three fast-folding and stable miniproteins; using all-atom (OPLS-AA) molecular dynamics simulations. Radicalization changes the conformation of the GLR residue and affects both neighboring residues but did not affect the stability of the Trp zipper. The stability of helices away from the radical center in villin were also affected by radicalization; and GLR in place of Gly15 caused the Trp cage to unfold within 1 µs. These results provide new evidence on the destabilizing effects of protein oxidation by reactive oxygen species.

Molecular ageing: free radical initiated epimerization of thymopentin--a case study.

The epimerization of amino acid residues increases with age in living organisms. In the present study, the structural consequences and thermodynamic functions of the epimerization of thymopentin (TP-5), the active site of the thymic hormone thymopoietin, were studied using molecular dynamics and density functional theory methods. The results show that free radical-initiated D-amino acid formation is energetically favoured (-130 kJmol(-1)) for each residue and induces significant changes to the peptide structure. In comparison to the wild-type (each residue in the L-configuration), the radius of gyration of the D-Asp(3) epimer of the peptide decreased by 0.5 Å, and disrupted the intramolecular hydrogen bonding of the native peptide. Beyond establishing important structural, energetic and thermodynamic benchmarks and reference data for the structure of TP-5, these results disseminate the understanding of molecular ageing, the epimerization of amino acid residues.

Penicillin's catalytic mechanism revealed by inelastic neutrons and quantum chemical theory.

Penicillin, travels through bodily fluids, targeting and acylatively inactivating enzymes responsible for cell-wall synthesis in gram-positive bacteria. Somehow, it avoids metabolic degradation remaining inactive en route. To resolve this ability to switch from a non-active, to a highly reactive form, we investigated the dynamic structure-activity relationship of penicillin by inelastic neutron spectroscopy, reaction kinetics, NMR and multi-scale theoretical modelling (QM/MM and post-HF ab initio). Results show that by a self-activating physiological pH-dependent two-step proton-mediated process, penicillin changes geometry to activate its irreversibly reactive acylation, facilitated by systemic intramolecular energy management and cooperative vibrations. This dynamic mechanism is confirmed by the first ever reported characterisation of an antibiotic by neutrons, achieved on the TOSCA instrument (ISIS facility, RAL, UK).

The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in Aspergillus fumigatus sensitized mice.

BACKGROUND: Lipoprotein-associated phospholipase A2 (Lp-PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) has been implicated in the pathogenesis of cardiovascular disease. A therapeutic targeting of this enzyme was challenged by the concern that increased circulating platelet activating factor (PAF) may predispose to or increase the severity of the allergic airway response. The aim of this study was to investigate whether Lp-PLA2 gene deficiency increases the risk of PAF and IgE-mediated inflammatory responses in vitro and in vivo using mouse models. METHODS: Lp-PLA2-/- mice were generated and back crossed to the C57BL/6 background. PAF-AH activity was measured using a hydrolysis assay in serum and bronchoalveolar lavage (BAL) samples obtained from mice. Aspergillus fumigatus (Af)-specific serum was prepared for passive allergic sensitization of mice in vivo and mast cells in vitro. ß- hexosaminidase release was studied in bone marrow derived mast cells sensitized with Af-specific serum or DNP-IgE and challenged with Af or DNP, respectively. Mice were treated with lipopolysaccharide (LPS) and PAF intratracheally and studied 24 hours later. Mice were sensitized either passively or actively against Af and were studied 48 hours after a single intranasal Af challenge. Airway responsiveness to methacholine, inflammatory cell influx in the lung tissue and BAL, immunoglobulin (ELISA) and cytokine (Luminex) profiles were compared between the wild type (WT) and Lp-PLA2-/- mice. RESULTS: PAF-AH activity was reduced but not completely abolished in Lp-PLA2-/- serum or by in vitro treatment of serum samples with a high saturating concentration of the selective Lp-PLA2 inhibitor, SB-435495. PAF inhalation significantly enhanced airway inflammation of LPS treated WT and Lp-PLA2-/- mice to a similar extent. Sensitized WT and Lp-PLA2-/- bone-marrow derived mast cells released ß-hexosaminidase following stimulation by allergen or IgE crosslinking to equivalent levels. Wild type and Lp-PLA2-/- mice responded to passive or active allergic sensitization by significant IgE production, airway inflammation and hyperresponsiveness after Af challenge. BAL cell influx was not different between these strains while IL-4, IL-5, IL-6 and eotaxin release was attenuated in Lp-PLA2-/- mice. There were no differences in the amount of total IgE levels in the Af sensitized WT and Lp-PLA2-/- mice. CONCLUSIONS: We conclude that Lp-PLA2 deficiency in C57BL/6 mice did not result in a heightened airway inflammation or hyperresponsiveness after PAF/LPS treatment or passive or active allergic sensitization and challenge.

Exploiting diverse stereochemistry of ß-amino acids: toward a rational design of sheet-forming ß-peptide systems.

Due to the two methylene groups in their backbone, ß-amino acids can adopt numerous secondary structures, including helices, sheets and nanotubes. Chirality introduced by the additional side chains can significantly influence the folding preference of ß-peptides composed of chiral ß-amino acids. However, only conceptual suggestions are present in the literature about the effect of chirality on folding preferences. Summarizing both the experimental and computational results, Seebach (Chem Biodivers 1:1111-1240, 2004) has proposed the first selection rule on the effect of side chain chirality, on the folding preference of ß-peptides. In order to extend and fine-tune the aforementioned predictions of Seebach, we have investigated its validity to the novel type of apolar sheet proposed recently (Pohl et al. in J Phys Chem B 114:9338-9348, 2010). In order to facilitate the rational design of sheet-like structures, a systematic study on the effect of chirality on "apolar" sheet stability is presented on disubstituted [HCO-ß-Ala-ß(2,3)-hAla-ß-Ala-NH(2)](2) model peptides calculated at the M05-2X/6-311++G(d,p)//M05-2X/6-31G(d) and B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) levels of theory both in vacuum and in polar and apolar solvents. In addition, both types of "apolar" sheets were investigated; the one with two strands of identical (AA) and enantiomeric (AB) backbone structure. Our results show that heterochirally disubstituted sheets have the greatest preference for sheet formation (ΔG ~ -11 kcal mol(-1)). However, in contrast to Seebach's predictions, "homochiral disubstitution" itself does not necessarily disrupt the sheet structure, rather it could result stable fold (ΔG ~ -5 kcal mol(-1)). Results indicate that both the methyl group orientation and the local conformational effect of substitution affects sheet stability, as point chirality was found to have influence only on the backbone torsional angles. These results enabled us to extend and generalize Seebach's predictions and to propose a more general and accurate "rule of thumb" describing the effect of chirality on sheet stability. This offers an easy-to-use summary on how to design ß-peptide sheet structures. We conclude that heterochirally disubstituted models are the best candidates for sheet formation, if the two strands are substituted in a way to create identical torsional angle sets on the two backbones for ideal hydrogen-bonding pattern. With adequately selected side chains, homochirally disubtituted derivatives may also form sheet structures, and the position of methyl groups would prevent assembly of more than two strands making it ideal to create hairpins.

Application of the systems chemistry approach on the ammonolysis of 1-ethoxycarbonyl- and 1-phenoxycarbonyl-3-(2-thienyl)oxindoles. A method to predict reactivity.

The routine prediction of the reactivity of a complex, multifunctional molecule is a challenging and time-consuming procedure. In the last step of the synthesis of the well-known drug substance tenidap, a nonexpected difference was observed between the reactivities of two closely related carbamate moieties, the N-ethoxycarbonyl and the N-phenoxycarbonyl group. A detailed kinetic study, necessitating a significant computational effort, is described in the present paper for this reaction step. On the other hand, the systems chemistry concept, by analyzing the details of the electronic structure and the connections between functional groups in a fast and simple way, is also able to answer this question using various "-icity" parameters (aromaticity, carbonylicity, olefinicity). The complete systems chemistry approach involves all these conjugativicity parameters, while its further simplified version is based on only one key parameter, which is carbonylicity in the present case. The above methods were compared in terms of their predictive power. The results show that the systems chemistry concept, even its one-parameter version, is applicable for the characterization of this challenging reactivity issue.

The role of entropy in initializing the aggregation of peptides: a first principle study on oligopeptide oligomerization.

The initiation and progression of Alzheimer's disease is coupled to the oligo- and polymerization of amyloid peptides in the brain. Amyloid like aggregates of protein domains were found practically independent of their primary sequences. Thus, the driving force of the transformation from the original to a disordered amyloid fold is expected to lie in the protein backbone common to all proteins. In order to investigate the thermodynamics of oligomerization, full geometry optimizations and frequency calculations were performed both on parallel and antiparallel ß-pleated sheet model structures of [HCO-(Ala)(1-6)-NH(2)](2) and (For-Ala(1-2)-NH(2))(1-6) peptides, both at the B3LYP and M05-2X/6-311++G(d,p)//M05-2X/6-31G(d) levels of theory, both in vacuum and in water. Our results show that relative entropy and enthalpy both show a hyperbolic decrease with increasing residue number and with increasing number of strands as well. Thus, di- and oligomerization are always thermodynamically favored. Antiparallel arrangements were found to have greater stability than parallel arrangements of the polypeptide backbones. During our study the relative changes in thermodynamic functions are found to be constant for long enough peptides, indicating that stability and entropy terms are predictable. All thermodynamic functions of antiparallel di- and oligomers show a staggered nature along the increasing residue number. By identifying and analyzing the 6 newly emerging dimer vibrational modes of the 10- and 14-membered building units, the staggered nature of the entropy function can be rationalized. Thus, the vanishing rotational and translational modes with respect to single strands are converted into entropy terms "holding tight" the dimers and oligomers formed, rationalizing the intrinsic adherence of natural polypeptide backbones to aggregate.

Qualitative assessment of microstructure and Hertzian indentation failure in biocompatible glass ionomer cements.

Discs of biocompatible glass ionomer cements were prepared for Hertzian indentation and subsequent fracture analyses. Specifically, 2 × 10 mm samples for reproducing bottom-initiated radial fracture, complemented by 0.2 × 1 mm samples for optimal resolution with X-ray micro tomography (µCT), maintaining dimensional ratio. The latter allowed for accurate determination of volumetric-porosity of the fully cured material, fracture-branching through three Cartesian axes and incomplete bottom-initiated cracking. Nanocomputed tomography analyses supported the reliability of the µCT results. Complementary 2-dimensional fractographic investigation was carried out by optical and scanning electron microscopies on the larger samples, identifying fracture characteristics. The combined 3-D qualitative assessment of microstructure and fractures, complemented by 2-D methods, provided an increased understanding of the mechanism of mechanical failure in these cements. Specifically, cracks grew to link pores while propagating along glass-matrix interfaces. The methodological development herein is exploitable on related biomaterials and represents a new tool for the rational characterisation, optimisation and design of novel materials for clinical service.

Why Do some Labour Alliances Succeed in Politicizing Europe across Borders? A Comparison of the Right2Water and Fair Transport European Citizens' Initiatives.

Under what conditions can organized labour successfully politicize the European integration process across borders? To answer this question, we compare the European Citizens' Initiatives (ECIs) of two European trade union federations: EPSU's successful Right2Water ECI and ETF's unsuccessful Fair Transport ECI. Our comparison reveals that actor-centred factors matter - namely, unions' ability to create broad coalitions. Successful transnational labour campaigns, however, also depend on structural conditions, namely, the prevailing mode of EU integration pressures faced by unions at a given time. Whereas the Right2Water ECI pre-emptively countered commodification attempts by the European Commission in water services, the Fair Transport ECI attempted to ensure fair working conditions after most of the transport sector had been liberalized. Vertical EU integration attempts that commodify public services are thus more likely to generate successful transnational counter-movements than the horizontal integration pressures on wages and working conditions that followed earlier successful EU liberalization drives.

Chemical evolution of biomolecule building blocks. Can thermodynamics explain the accumulation of glycine in the prebiotic ocean?

It has always been a question of considerable scientific interest why amino acids (and other biomolecule building blocks) formed and accumulated in the prebiotic ocean. In this study, we suggest an answer to this question for the simplest amino acid, glycine. We have shown for the first time that classical equilibrium thermodynamics can explain the most likely selection of glycine (and the derivative of its dipeptide) in aqueous media, although glycine is not the lowest free energy structure among all (404) possible constitutional isomers. Species preceding glycine in the free energy order are either supramolecular complexes of small molecules or such molecules likely to dissociate and thus get back to the gas phase. Then, 2-hydroxyacetamide condensates yielding a thermodynamically favored derivative of glycine dipeptide providing an alternative way for peptide formation. It is remarkable that a simple equilibrium thermodynamic model can explain the accumulation of glycine and provide a reason for the importance of water in the formation process.

Quantum chemical analysis of the unfolding of a penta-glycyl 3(10)-helix initiated by HO(●), HO2(●), and O2(-●).

In this study, the thermodynamic functions of hydrogen abstraction from the C(α) and amide nitrogen of Gly(3) in a homo-pentapeptide (N-Ac-GGGGG-NH(2); G5) by HO(●), HO(2)(●), and O(2)(-●) were computed using the Becke three-parameter Lee-Yang-Parr (B3LYP) density functional. The thermodynamic functions, standard enthalpy (ΔH°), Gibbs free energy (ΔG°), and entropy (ΔS°), of these reactions were computed with G5 in the 3(10)-helical (G5(Hel)) and fully-extended (G5(Ext)) conformations at the B3LYP/6-31G(d) and B3LYP/6-311+G(d,p) levels of theory, both in the gas phase and using the conductor-like polarizable continuum model implicit water model. H abstraction is more favorable at the C(α) than at the amide nitrogen. The secondary structure of G5 affects the bond dissociation energy of the H-C(α), but has a negligible effect on the dissociation energy of the H-N bond. The HO(●) radical is the strongest hydrogen abstractor, followed by HO(2)(●), and finally O(2)(-●). The secondary structure elements, such as H-bonds in the 3(10)-helix, protect the peptide from radical attack by disabling the potential electron delocalization at the C(α), which is possible when G5 is in the extended conformation. The unfolding of the peptide radicals is more favorable than the unfolding of G5(Hel); however, only the HO(●) can initiate the unfolding of G5(Hel) and the formation of G5(Ext)(●). These results are relevant to peptides that are prone to undergoing transitions from helical structures to ß-sheets in the cellular condition known as "oxidative stress" and the results are discussed in this context.

Theoretical Design, Synthesis, and In Vitro Neurobiological Applications of a Highly Efficient Two-Photon Caged GABA Validated on an Epileptic Case.

In this paper, we present an additional, new cage-GABA compound, called 4-amino-1-(4'-dimethylaminoisopropoxy-5',7'-dinitro-2',3'-dihydro-indol-1-yl)-1-oxobutane-γ-aminobutyric acid (iDMPO-DNI-GABA), and currently, this compound is the only photoreagent, which can be applied for GABA uncaging without experimental compromises. By a systematic theoretical design and successful synthesis of several compounds, the best reagent exhibits a high two-photon efficiency within the 700-760 nm range with excellent pharmacological behavior, which proved to be suitable for a complex epileptic study. Quantum chemical design showed that the optimal length of the cationic side chain enhances the two-photon absorption by 1 order of magnitude due to the cooperating internal hydrogen bonding to the extra nitro group on the core. This feature increased solubility while suppressing membrane permeability. The efficiency was demonstrated in a systematic, wide range of in vitro single-cell neurophysiological experiments by electrophysiological as well as calcium imaging techniques. Scalable inhibitory ion currents were elicited by iDMPO-DNI-GABA with appropriate spatial-temporal precision, blocking both spontaneous and evoked cell activity with excellent efficiency. Additionally, to demonstrate its applicability in a real neurobiological study, we could smoothly and selectively modulate neuronal activities during artificial epileptic rhythms first time in a neural network of GCaMP6f transgenic mouse brain slices.