Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

Many dsDNA viruses encode DNA-packaging terminases, each containing a nuclease domain that resolves concatemeric DNA into genome-length units. Terminase nucleases resemble the RNase H-superfamily nucleotidyltransferases in folds, and share a two-metal-ion catalytic mechanism. Here we show that residue K428 of a bacteriophage terminase gp2 nuclease domain mediates binding of the metal cofactor Mg2+. A K428A mutation allows visualization, at high resolution, of a metal ion binding mode with a coupled-octahedral configuration at the active site, exhibiting an unusually short metal-metal distance of 2.42 A° . Such proximity of the two metal ions may play an essential role in catalysis by generating a highly positive electrostatic niche to enable formation of the negatively charged pentacovalent phosphate transition state, and provides the structural basis for distinguishing Mg2+ from Ca2+. Using a metal ion chelator -thujaplicinol as a molecular probe, we observed a second mode of metal ion binding at the active site, mimicking the DNA binding state. Arrangement of the active site residues differs drastically from those in RNase H-like nucleases, suggesting a drifting of the active site configuration during evolution. The two distinct metal ion binding modes unveiledmechanistic details of the two-metalion catalysis at atomic resolution

TLR2 expression is increased in rosacea and stimulates enhanced serine protease production by keratinocytes.

A diverse environment challenges skin to maintain temperature, hydration, and electrolyte balance while also maintaining normal immunological function. Rosacea is a common skin disease that manifests unique inflammatory responses to normal environmental stimuli. We hypothesized that abnormal function of innate immune pattern recognition could explain the enhanced sensitivity of patients with rosacea, and observed that the epidermis of patients with rosacea expressed higher amounts of Toll-like receptor 2 (TLR2) than normal patients. Increased expression of TLR2 was not seen in other inflammatory skin disorders such as atopic dermatitis or psoriasis. Overexpression of TLR2 on keratinocytes, treatment with TLR2 ligands, and analysis of TLR2-deficient mice resulted in a calcium-dependent release of kallikrein 5 from keratinocytes, a critical protease involved in the pathogenesis of rosacea. These observations show that abnormal TLR2 function may explain enhanced inflammatory responses to environmental stimuli and can act as a critical element in the pathogenesis of rosacea

Word-level prominence and “stress deafness” in Maltese English bilinguals

This study investigates “stress deafness” in bilingual speakers of Maltese and Maltese English. Although both reportedly have lexical stress, the acoustic cues to prominence appear to be relatively weak. Further, word-initial pitch peaks make pitch an unreliable cue to lexical stress, which can be elsewhere in the word. In a sequence recall task, we show that speakers dominant in Maltese exhibit a classic “stress deafness” effect, similar to speakers of French. Speakers who identify as balanced or Maltese English dominant have more diverse results and do not show such a strong tendency towards “stress deafness”. These speakers may rely on their exposure to other varieties of English to identify (and recall) word prominences. This study suggests that the nature of stress in Maltese might need to be revisited.peer-reviewe

The acquisition of asserted, presupposed, and pragmatically implied exhaustivity in Hungarian

The paper reports on three experiments in which the exhaustive interpretation of sentences containing the focus particle csak ‘only’, structural focus constructions, and sentences with neutral intonation and word order were investigated. The results obtained not only reveal the developmental trajectory of the adult-like comprehension of each sentence type, but also contribute to the discussion concerning the semantic or pragmatic nature of their exhaustive meaning component. As the three construction types were judged in different ways on a three-point scale, the findings appear to support the hypothesis according to which exhaustivity is part of the asserted content of sentences with csak ‘only’, it is context-independently presupposed in the case of structural focus, and in certain contexts it can arise as an implicature in the case of neutral utterances, as well

Must . . . stay . . . strong!

This is the fourth installment in our trilogy of papers on epistemic modality.It is a recurring matra that epistemic must creates a statement that is weaker than the corresponding flat-footed assertion: It must be raining vs. It’s raining. Contrary to classic discussions of the phenomenon such as by Karttunen, Kratzer, and Veltman, we argue that instead of having a weak semantics, must presupposes the presence of an indirect inference or deduction rather than of a direct observation. This is independent of the strength of the claim being made. Epistemic must is therefore quite similar to evidential markers of indirect evidence known from languages with rich evidential systems. We work towards a formalization of the evidential component, relying on a structured model of information states (analogous to some models used in the belief dynamics literature). We explain why in many contexts, one can perceive a lack of confidence on the part of the speaker who uses must

Checkpoint Inhibition Reduces the Threshold for Drug-Specific T-Cell Priming and Increases the Incidence of Sulfasalazine Hypersensitivity

An emerging clinical issue associated with immune-oncology agents is the collateral effects on the tolerability of concomitant medications. One report of this phenomenon was the increased incidence of hypersensitivity reactions observed in patients receiving concurrent immune checkpoint inhibitors (ICIs) and sulfasalazine (SLZ). Thus, the aim of this study was to characterize the T cells involved in the pathogenesis of such reactions, and recapitulate the effects of inhibitory checkpoint blockade on de-novo priming responses to compounds within in vitro platforms. A regulatory competent human dendritic cell/T-cell coculture assay was used to model the effects of ICIs on de novo nitroso sulfamethoxazole- and sulfapyridine (SP) (the sulfonamide component of SLZ) hydroxylamine-specific priming responses. The role of T cells in the pathogenesis of the observed reactions was explored in 3 patients through phenotypic characterization of SP/sulfapyridine hydroxylamine (SPHA)-responsive T-cell clones (TCC), and assessment of cross-reactivity and pathways of T-cell activation. Augmentation of the frequency of responding drug-specific T cells and intensity of the T-cell response was observed with PD-1/PD-L1 blockade. Monoclonal populations of SP- and SPHA-responsive T cells were isolated from all 3 patients. A core secretory effector molecule profile (IFN-γ, IL-13, granzyme B, and perforin) was identified for SP and SPHA-responsive TCC, which proceeded through Pi and hapten mechanisms, respectively. Data presented herein provides evidence that drug-responsive T cells are effectors of hypersensitivity reactions observed in oncology patients administered ICIs and SLZ. Perturbation of drug-specific T-cell priming is a plausible explanation for clinical observations of how an increased incidence of these adverse events is occurring

Binary asteroid (31) Euphrosyne: ice-rich and nearly spherical

Aims. Asteroid (31) Euphrosyne is one of the biggest objects in the asteroid main belt and it is also the largest member of its namesake family. The Euphrosyne family occupies a highly inclined region in the outer main belt and contains a remarkably large number of members, which is interpreted as an outcome of a disruptive cratering event. Methods. The goals of this adaptive-optics imaging study are threefold: to characterize the shape of Euphrosyne, to constrain its density, and to search for the large craters that may be associated with the family formation event. Results. We obtained disk-resolved images of Euphrosyne using SPHERE/ZIMPOL at the ESO 8.2 m VLT as part of our large program (ID: 199.C-0074, PI: Vernazza). We reconstructed its 3D shape via the ADAM shape modeling algorithm based on the SPHERE images and the available light curves of this asteroid. We analyzed the dynamics of the satellite with the Genoid meta-heuristic algorithm. Finally, we studied the shape of Euphrosyne using hydrostatic equilibrium models. Conclusions. Our SPHERE observations show that Euphrosyne has a nearly spherical shape with the sphericity index of 0.9888 and its surface lacks large impact craters. Euphrosyne’s diameter is 268 ± 6 km, making it one of the top ten largest main belt asteroids. We detected a satellite of Euphrosyne – S/2019 (31) 1 – that is about 4 km across, on a circular orbit. The mass determined from the orbit of the satellite together with the volume computed from the shape model imply a density of 1665 ± 242 kg m−3, suggesting that Euphrosyne probably contains a large fraction of water ice in its interior. We find that the spherical shape of Euphrosyne is a result of the reaccumulation process following the impact, as in the case of (10) Hygiea. However, our shape analysis reveals that, contrary to Hygiea, the axis ratios of Euphrosyne significantly differ from those suggested by fluid hydrostatic equilibrium following reaccumulation.This work has been supported by the Czech Science Foundation through grant 18-09470S (J. Hanuš, O. Chrenko, P. Ševeček) and by the Charles University Research program No. UNCE/SCI/023. M.B. was supported by the Czech Science Foundation grant 18-04514J. Computational resources were supplied by the Ministry of Education, Youth and Sports of the Czech Republic under the projects CESNET (LM2015042) and IT4Innovations National Supercomputing Centre (LM2015070). P. Vernazza, A. Drouard, M. Ferrais and B. Carry were supported by CNRS/INSU/PNP. M.M. was supported by the National Aeronautics and Space Administration under grant No. 80NSSC18K0849 issued through the Planetary Astronomy Program. The work of TSR was carried out through grant APOSTD/2019/046 by Generalitat Valenciana (Spain). This work was supported by the MINECO (Spanish Ministry of Economy) through grant RTI2018-095076-B-C21 (MINECO/FEDER, UE). The research leading to these results has received funding from the ARC grant for Concerted Research Actions, financed by the Wallonia-Brussels Federation. TRAPPIST is a project funded by the Belgian Fonds (National) de la Recherche Scientifique (F.R.S.-FNRS) under grant FRFC 2.5.594.09.F. TRAPPIST-North is a project funded by the Université de Liège, and performed in collaboration with Cadi Ayyad University of Marrakesh. E. Jehin is a FNRS Senior Research Associate

A basin-free spherical shape as an outcome of a giant impact on asteroid Hygiea

(10) Hygiea is the fourth largest main belt asteroid and the only known asteroid whose surface composition appears similar to that of the dwarf planet (1) Ceres1,2, suggesting a similar origin for these two objects. Hygiea suffered a giant impact more than 2 Gyr ago3 that is at the origin of one of the largest asteroid families. However, Hygeia has never been observed with sufficiently high resolution to resolve the details of its surface or to constrain its size and shape. Here, we report high-angular-resolution imaging observations of Hygiea with the VLT/SPHERE instrument (~20 mas at 600 nm) that reveal a basin-free nearly spherical shape with a volume-equivalent radius of 217 ± 7 km, implying a density of 1,944 ± 250 kg m−3 to 1σ. In addition, we have determined a new rotation period for Hygiea of ~13.8 h, which is half the currently accepted value. Numerical simulations of the family-forming event show that Hygiea’s spherical shape and family can be explained by a collision with a large projectile (diameter ~75–150 km). By comparing Hygiea’s sphericity with that of other Solar System objects, it appears that Hygiea is nearly as spherical as Ceres, opening up the possibility for this object to be reclassified as a dwarf planet.P.V., A.D. and B.C. were supported by CNRS/INSU/PNP. M.Brož was supported by grant 18-04514J of the Czech Science Foundation. J.H. and J.D. were supported by grant 18-09470S of the Czech Science Foundation and by the Charles University Research Programme no. UNCE/SCI/023. This project has received funding from the European Union’s Horizon 2020 research and innovation programmes under grant agreement nos 730890 and 687378. This material reflects only the authors’ views, and the European Commission is not liable for any use that may be made of the information contained herein. TRAPPIST-North is a project funded by the University of Liège, in collaboration with Cadi Ayyad University of Marrakech (Morocco). TRAPPIST-South is a project funded by the Belgian Fonds (National) de la Recherche Scientifique (F.R.S.-FNRS) under grant FRFC 2.5.594.09.F. E.J. and M.G. are F.R.S.-FNRS Senior Research Associates

Unagreement is an illusion

This is the author accepted manuscript. The final version is available from Springer via http://dx.doi.org/10.1007/s11049-015-9311-yThis paper proposes an analysis of unagreement, a phenomenon involving an apparent mismatch between a definite third person plural subject and first or second person plural subject agreement observed in various null subject languages (e.g. Spanish, Modern Greek and Bulgarian), but notoriously absent in others (e.g. Italian, European Portuguese). A cross-linguistic correlation between unagreement and the structure of adnominal pronoun constructions suggests that the availability of unagreement depends on whether person and definiteness are hosted by separate heads (in languages like Greek) or bundled on a single head (i.e. pronominal determiners in languages like Italian). Null spell-out of the head hosting person features high in the extended nominal projection of the subject leads to unagreement. The lack of unagreement in languages with pronominal determiners results from the interaction of their syntactic structure with the properties of the vocabulary items realising the head encoding both person and definiteness. The analysis provides a principled explanation for the cross-linguistic distribution of unagreement and suggests a unified framework for deriving unagreement, adnominal pronoun constructions, personal pronouns and pro