Search

Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance, and haemostasis but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, FA assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of FA sites, with the cytoplasmic tails of the integrin β1 and β3 subunits. The binding interface resides in paxillin's LIM3 domain, where based on the NMR structure and functional analyses, a flexible, 7-amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin β3 compromises cell adhesion and motility of murine fibroblasts. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin β3 function.

Vaccines containing tetanus-diphtheria antigens have been postulated to induce cross-reactive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which could protect against coronavirus disease (COVID-19). In this work, we investigated the capacity of Tetanus-diphtheria (Td) vaccine to prime existing T cell immunity to SARS-CoV-2. To that end, we first collected known SARS-CoV-2 specific CD8+ T cell epitopes targeted during the course of SARS-CoV-2 infection in humans and identified as potentially cross-reactive with Td vaccine those sharing similarity with tetanus-diphtheria vaccine antigens, as judged by Levenshtein edit distances (≤ 20% edits per epitope sequence). As a result, we selected 25 potentially cross-reactive SARS-CoV-2 specific CD8+ T cell epitopes with high population coverage that were assembled into a synthetic peptide pool (TDX pool). Using peripheral blood mononuclear cells, we first determined by intracellular IFNγ staining assays existing CD8+ T cell recall responses to the TDX pool and to other peptide pools, including overlapping peptide pools covering SARS-CoV-2 Spike protein and Nucleocapsid phosphoprotein (NP). In the studied subjects, CD8+ T cell recall responses to Spike and TDX peptide pools were dominant and comparable, while recall responses to NP peptide pool were less frequent and weaker. Subsequently, we studied responses to the same peptides using antigen-inexperienced naive T cells primed/stimulated in vitro with Td vaccine. Priming stimulations were carried out by co-culturing naive T cells with autologous irradiated peripheral mononuclear cells in the presence of Td vaccine, IL-2, IL-7 and IL-15. Interestingly, naive CD8+ T cells stimulated/primed with Td vaccine responded strongly and specifically to the TDX pool, not to other SARS-CoV-2 peptide pools. Finally, we show that Td-immunization of C57BL/6J mice elicited T cells cross-reactive with the TDX pool. Collectively, our findings support that tetanus-diphtheria vaccines can prime SARS-CoV-2 cross-reactive T cells and likely contribute to shape the T cell responses to the virus.

Abstract Background The aim of this study is to assess and evaluate the capacities for understanding, appreciation and reasoning of legal minors with psychiatric disorders and their parents and their competence to consent or assent to participation in clinical trials. The beliefs, fears, motivation and influencing factors for decision-making of legal minors and parents were also examined. Methods Using the MacArthur Competence Assessment Tool for Clinical Research (MacCAT-CR), an instrument developed for adults whose capacities to consent are unclear, we provided information about clinical trials and assessed understanding, appreciation and reasoning. We adapted this tool for legal minors and examined 19 children and adolescents between the ages of 7 and 15 with attention deficit/hyperactivity disorder (ADHD) or ADHD combined with oppositional defiant disorder (DSM-IV 314.00/314.01/312.8) enrolled in clinical trials. Parents were also examined using the MacCAT-CR. Results Facts such as the procedures involved in trials or their duration were well understood by legal minors, but more abstract issues like the primary purpose of the trial were not understood by children and adolescents or by many parents. Legal minors also had difficulties understanding the nature of placebo and the probability of receiving placebo. Children's and adolescents' decisions were influenced by fears about their disorder worsening and by problems in their relationship with their parents. Parents wanted the best therapy for their children in order to minimize problems in school. Conclusion Legal minors and parents need to be informed more precisely about specific issues like placebo and the primary purpose of trials. In general, the reasoning of children and adolescents was influenced by their experience with their disorder and decision making was based on reasonable arguments. Their fears were based on everyday experiences such as school performance or family relationships.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a public health concern due to infections with new SARS-CoV-2 variants. Therefore, finding effective preventive and therapeutic treatments against all SARS-CoV-2 variants is of great interest. In this study, we examined the capacity of eucalyptus essential oil (EEO) and eucalyptol (EOL) to prevent SARS-CoV-2 infection, using as a model SARS-CoV-2 Spike pseudotyped lentivirus (SARS-CoV-2 pseudovirus) and 293T cells transfected with human angiotensin-converting enzyme 2 (hACE2-293T cells). First, we determined the cytotoxicity of EEO and EOL using the MTT colorimetric assay, selecting non-cytotoxic concentrations ≤ 0.1% (v/v) for further analysis. Subsequently, we evaluated the capacity of EEO and EOL in cell cultures to preclude infection of hACE2-293T cells by SARS-CoV-2 pseudovirus, using a luciferase-based assay. We found that EEO and EOL significantly reduced SARS-CoV-2 pseudovirus infection, obtaining IC50 values of 0.00895% and 0.0042% (v/v), respectively. Likewise, EEO and EOL also reduced infection by vesicular stomatitis virus (VSV) pseudovirus, although higher concentrations were required. Hence, EEO and EOL may be able to inhibit SARS-CoV-2 infection, at least partially, through a Spike-independent pathway, supporting the implementation of aromatherapy with these agents as a cost-effective antiviral measure.

We provide an overview of the status of Monte-Carlo event generators for high-energy particle physics. Guided by the experimental needs and requirements, we highlight areas of active development, and opportunities for future improvements. Particular emphasis is given to physics models and algorithms that are employed across a variety of experiments. These common themes in event generator development lead to a more comprehensive understanding of physics at the highest energies and intensities, and allow models to be tested against a wealth of data that have been accumulated over the past decades. A cohesive approach to event generator development will allow these models to be further improved and systematic uncertainties to be reduced, directly contributing to future experimental success. Event generators are part of a much larger ecosystem of computational tools. They typically involve a number of unknown model parameters that must be tuned to experimental data, while maintaining the integrity of the underlying physics models. Making both these data, and the analyses with which they have been obtained accessible to future users is an essential aspect of open science and data preservation. It ensures the consistency of physics models across a variety of experiments.

Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disorder mainly driven by mutations in the NPC1 gene, causing lipid accumulation within late endosomes/lysosomes and resulting in progressive neurodegeneration. Although microglial activation precedes neuronal loss, it remains elusive whether loss of the membrane protein NPC1 in microglia actively contributes to NPC pathology. In a mouse model with depletion of NPC1 in myeloid cells, we report severe alterations in microglial lipidomic profiles, including the enrichment of bis(monoacylglycero)phosphate, increased cholesterol, and a decrease in cholesteryl esters. Lipid dyshomeostasis was associated with microglial hyperactivity, marked by an increase in translocator protein 18 kDa (TSPO). These hyperactive microglia initiated a pathological cascade resembling NPC-like phenotypes, including a shortened life span, motor impairments, astrogliosis, neuroaxonal pathology, and increased neurofilament light chain (NF-L), a neuronal injury biomarker. As observed in the mouse model, patients with NPC showed increased NF-L in the blood and microglial hyperactivity, as visualized by TSPO-PET imaging. Reduced TSPO expression in blood-derived macrophages of patients with NPC was measured after N-acetyl-l-leucine treatment, which has been recently shown to have beneficial effects in patients with NPC, suggesting that TSPO is a potential marker to monitor therapeutic interventions for NPC. Conclusively, these results demonstrate that myeloid dysfunction, driven by the loss of NPC1, contributes to NPC disease and should be further investigated for therapeutic targeting and disease monitoring. Editor's summary: Niemann-Pick type C (NPC) disease, a fatal neurological disorder caused by mutations in NPC1, is associated with neuroinflammation and microglial activation. To investigate how microglia are affected by loss of NPC1 and contribute to disease, Dinkel et al. used a mouse model with Npc1 deleted from myeloid cells. These conditional KO mice recapitulated many of the pathological hallmarks seen in patients, such as lipid dyshomeostasis, neurodegeneration, and microgliosis. PET imaging in patients with NPC confirmed the increased microglial activity, and treatment of patients with NALL, an FDA-approved drug for NPC, normalized the phagocytic activity of patient-derived macrophages, suggesting a potential strategy for treatment monitoring. —Daniela Neuhofer [ABSTRACT FROM AUTHOR]

Background: Regarding animal welfare and reversible suppression of reproduction the need for alternatives to surgical castration like slow-release gonadotropin-releasing hormone implants is increasing. Aim: In this study, we evaluated whether the onset of puberty can be suppressed by implantation of a slow-release gonadotropin-releasing hormone implant (4.7 mg deslorelin) in juvenile rams. Methods: Seven juvenile rams (3-5.5 months) were treated with the gonadotropin-releasing hormone analogue deslorelin to analyse the effects on testicular development, sonographic findings of testicular tissue, testosterone concentration in the blood, spermatogenesis and sperm parameters from the epididymis after castration. Seven rams of the same age without an implant served as controls. Results: Follow-up examinations were performed over five months, after which four rams per group were castrated. No significant group differences were found in the andrological parameters on the group level, but testicular development was suppressed in three rams in the treated group. Histological examination revealed spermatogenesis in the testicular tissue in three of four animals treated with a gonadotropin-releasing hormone analogue. Conclusion: The onset of puberty in juvenile rams cannot be reliably suppressed by using the slow-release gonadotropin-releasing hormone implant Suprelorin®.

Abstract The azimuthal correlation, $$\Delta \phi _{12}$$ Δ ϕ 12 , of high transverse momentum jets in pp collisions at $$\sqrt{s}=13$$ s = 13 TeV is studied by applying PB-TMD distributions to NLO calculations via MCatNLO together with the PB-TMD parton shower. A very good description of the cross section as a function of $$\Delta \phi _{12}$$ Δ ϕ 12 is observed. In the back-to-back region of $${\Delta \phi _{12}}\rightarrow \pi $$ Δ ϕ 12 → π , a very good agreement is observed with the PB-TMD Set 2 distributions while significant deviations are obtained with the PB-TMD Set 1 distributions. Set 1 uses the evolution scale while Set 2 uses transverse momentum as an argument in $$\alpha _\mathrm {s}$$ α s , and the above observation therefore confirms the importance of an appropriate soft-gluon coupling in angular ordered parton evolution. The total uncertainties of the predictions are dominated by the scale uncertainties of the matrix element, while the uncertainties coming from the PB-TMDs and the corresponding PB-TMD shower are very small. The $$\Delta \phi _{12}$$ Δ ϕ 12 measurements are also compared with predictions using MCatNLO together Pythia8, illustrating the importance of details of the parton shower evolution.

Abstract The search for new interactions and particles in high-energy collider physics relies on precise background predictions. This has led to many advances in combining precise fixed-order cross-section calculations with detailed event generator simulations. In recent years, fixed-order qcd calculations of inclusive cross sections at n3lo precision have emerged, followed by an impressive progress at producing differential results. Once differential results become publicly available, it would be prudent to embed these into event generators to allow the community to leverage these advances. This note offers some concrete thoughts on me+ps matching at third order in qcd. As a method for testing these thoughts, a toy calculation of e + e − → u u ¯ $$ \overline{u} $$ at O $$ \mathcal{O} $$ ( α s 3 $$ {\alpha}_s^3 $$ ) is constructed, and combined with an event generator through unitary matching. The toy implementation may serve also as blueprint for high-precision qcd predictions at future lepton colliders. As a byproduct of the n3lo matching formula, a new nnlo+ps formula for processes with “additional” jets is obtained.

Rossby‐wave packets (RWPs) along the midlatitude jet are a fundamental ingredient of extratropical dynamics. RWPs have been linked to enhanced atmospheric predictability and the occurrence of extreme weather events. We here investigate the predictability of Northern Hemispheric RWPs as physical entities, defined by enhanced values of the Rossby‐wave envelope field. Using a catalogue of RWPs identified and tracked in reanalysis data, we analyze RWP predictability in a 19‐year period of NOAA GEFSv12 reforecasts. Our analysis adopts the so‐called distance and amplitude score (DAS), a verification metric that avoids both the double‐penalty issue of field‐based verification of coherent features and the complications of an object‐based approach. Applied to the envelope field, forecast errors defined by this metric asymptote towards saturation, but do not completely reach saturation within the 10 days lead time available to this study. The growth rate of the median DAS is highest initially and decreases with lead time. This is a nontrivial result, because the underlying envelope field largely de‐emphasizes phase information, but still exhibits very similar error‐growth characteristics to fields that contain the full phase information. Variations in RWP predictability are dominated by the stage of the RWP life cycle, with higher predictability found for the propagation stage than the onset and decay stages. In addition, RWP predictability exhibits a seasonal cycle, with higher predictability in winter than in summer. Controlling for seasonality and the stage of the life cycle, we find that (i) high‐amplitude RWPs exhibit higher predictability than low‐amplitude RWPs up to 6 days lead time and (ii) there is a general pattern of relatively high predictability over Eurasia. Finally, predictability of the propagating stage is higher if forecasts are initialized after RWP onset than if initialized before onset. In this sense, RWP onset acts as a predictability barrier to the subsequent propagation stage. [ABSTRACT FROM AUTHOR]

Integrins are fundamental for cell adhesion and the formation of focal adhesions (FA). Accordingly, these receptors guide embryonic development, tissue maintenance, and haemostasis but are also involved in cancer invasion and metastasis. A detailed understanding of the molecular interactions that drive integrin activation, FA assembly, and downstream signalling cascades is critical. Here, we reveal a direct association of paxillin, a marker protein of FA sites, with the cytoplasmic tails of the integrin β1 and β3 subunits. The binding interface resides in paxillin's LIM3 domain, where based on the NMR structure and functional analyses, a flexible, 7-amino acid loop engages the unstructured part of the integrin cytoplasmic tail. Genetic manipulation of the involved residues in either paxillin or integrin β3 compromises cell adhesion and motility of murine fibroblasts. This direct interaction between paxillin and the integrin cytoplasmic domain identifies an alternative, kindlin-independent mode of integrin outside-in signalling particularly important for integrin β3 function. How do integrins and paxillin interact at focal adhesions? Here, the authors present a 3D-structure of the LIM3 domain of paxillin which reveals a direct association between paxillin and the cytoplasmic tail of the integrin beta subunit. [ABSTRACT FROM AUTHOR]

Abstract We show how a careful analysis of the behaviour of a parton shower Monte Carlo in the vicinity of the soft and collinear regions allows one to formulate a modified MC@NLO-matching prescription that reduces the number of negative-weight events with respect to that stemming from the standard MC@NLO procedure. As a first practical application of such a prescription, that we dub MC@NLO-∆, we have implemented it in the MadGraph5_aMC@NLO framework, by employing the Pythia8 Monte Carlo. We present selected MC@NLO-∆ results at the 13 TeV LHC, and compare them with MC@NLO ones. We find that the former predictions are consistent with the latter ones within the typical matching systematics, and that the reduction of negative-weight events is significant.

The oral mucosa is constantly exposed to a plethora of stimuli including food antigens, commensal microbiota and pathogens, requiring distinct immune responses. We previously reported that human oral epithelial cells (OECs) suppress immune responses to bacteria, using H413 and TR146 OEC lines and primary OECs in co-culture with dendritic cells (DCs) and T cells (OEC-conditioned cells). OECs reduced DCs expression of CD80/CD86 and IL-12/TNFα release and impaired T cell activation. Here, we further evaluated the immunosuppression by these OECs and investigated the underlying mechanisms. OEC-conditioned DCs did not induce CD4 T cell polarization towards Treg, judging by the absence of FoxP3 expression. OECs also repressed T-bet/IFNγ expression in CD4 and CD8 T cells activated by DCs or anti-CD3/CD28 antibodies. This inhibition depended on OEC:T cell ratio and IFNγ repression occurred at the transcriptional level. Time-lapse experiments showed that OECs inhibited early steps of T cell activation, consistent with OECs inability to suppress T cells stimulated with PMA/ionomycin. Blocking CD40/CD40L, CD58/CD2 and PD-L1/PD-1 interactions with specific antibodies did not disrupt T cell suppression by OECs. However, preventing prostaglandin E2 (PGE2) synthesis or blocking PGE2 binding to the cognate EP2/EP4 receptors, restored IFNγ and TNFα production in OEC-conditioned T cells. Finally, treating OECs with poly(I:C), which simulates viral infections, limited T cell suppression. Overall, these results point to an inherent ability of OECs to suppress immune responses, which can nonetheless be eluded when OECs are under direct assault.

Vaccines containing tetanus-diphtheria antigens have been postulated to induce cross-reactive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which could protect against coronavirus disease (COVID-19). In this work, we investigated the capacity of Tetanus-diphtheria (Td) vaccine to prime existing T cell immunity to SARS-CoV-2. To that end, we first collected known SARS-CoV-2 specific CD8+ T cell epitopes targeted during the course of SARS-CoV-2 infection in humans and identified as potentially cross-reactive with Td vaccine those sharing similarity with tetanus-diphtheria vaccine antigens, as judged by Levenshtein edit distances (= 20% edits per epitope sequence). As a result, we selected 25 potentially cross-reactive SARS-CoV-2 specific CD8+ T cell epitopes with high population coverage that were assembled into a synthetic peptide pool (TDX pool). Using peripheral blood mononuclear cells, we first determined by intracellular IFNg staining assays existing CD8+ T cell recall responses to the TDX pool and to other peptide pools, including overlapping peptide pools covering SARS-CoV-2 Spike protein and Nucleocapsid phosphoprotein (NP). In the studied subjects, CD8+ T cell recall responses to Spike and TDX peptide pools were dominant and comparable, while recall responses to NP peptide pool were less frequent and weaker. Subsequently, we studied responses to the same peptides using antigen-inexperienced naive T cells primed/stimulated in vitro with Td vaccine. Priming stimulations were carried out by co-culturing naive T cells with autologous irradiated peripheral mononuclear cells in the presence of Td vaccine, IL-2, IL-7 and IL-15. Interestingly, naive CD8+ T cells stimulated/primed with Td vaccine responded strongly and specifically to the TDX pool, not to other SARS-CoV-2 peptide pools. Finally, we show that Td-immunization of C57BL/6J mice elicited T cells cross-reactive with the TDX pool. Collectively, our findings support that tetanusdiphtheria vaccines can prime SARS-CoV-2 cross-reactive T cells and likely contribute to shape the T cell responses to the virus. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a public health concern due to infections with new SARS-CoV-2 variants. Therefore, finding effective preventive and therapeutic treatments against all SARS-CoV-2 variants is of great interest. In this study, we examined the capacity of eucalyptus essential oil (EEO) and eucalyptol (EOL) to prevent SARS-CoV-2 infection, using as a model SARS-CoV-2 Spike pseudotyped lentivirus (SARS-CoV-2 pseudovirus) and 293T cells transfected with human angiotensin-converting enzyme 2 (hACE2-293T cells). First, we determined the cytotoxicity of EEO and EOL using the MTT colorimetric assay, selecting non-cytotoxic concentrations ≤ 0.1% (v/v) for further analysis. Subsequently, we evaluated the capacity of EEO and EOL in cell cultures to preclude infection of hACE2-293T cells by SARS-CoV-2 pseudovirus, using a luciferase-based assay. We found that EEO and EOL significantly reduced SARS-CoV-2 pseudovirus infection, obtaining IC50 values of 0.00895% and 0.0042% (v/v), respectively. Likewise, EEO and EOL also reduced infection by vesicular stomatitis virus (VSV) pseudovirus, although higher concentrations were required. Hence, EEO and EOL may be able to inhibit SARS-CoV-2 infection, at least partially, through a Spike-independent pathway, supporting the implementation of aromatherapy with these agents as a cost-effective antiviral measure. [ABSTRACT FROM AUTHOR]

Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate‐binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase. [ABSTRACT FROM AUTHOR]

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences between glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing database of experimentally verified IDPs (DisProt) and show that glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while the Glu/Asp variants support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity, where glutamates can support increased helicity, important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in, e.g., transactivation domains and ion-binding.

Bacteriophage-encoded single strand annealing proteins (SSAPs) are recombinases which can substitute the classical, bacterial RecA and manage the DNA metabolism at different steps of phage propagation. SSAPs have been shown to efficiently promote recombination between short and rather divergent DNA sequences and were exploited for in vivo genetic engineering mainly in Gram-negative bacteria. In opposition to the conserved and almost universal bacterial RecA protein, SSAPs display great sequence diversity. The importance for SSAPs in phage biology and phage-bacteria evolution is underlined by their role as key players in events of horizontal gene transfer (HGT). All of the above provoke a constant interest for the identification and study of new phage recombinase proteins in vivo, in vitro as well as in silico. Despite this, a huge body of putative ssap genes escapes conventional classification, as they are not properly annotated. In this work, we performed a wide-scale identification, classification and analysis of SSAPs encoded by the Firmicutes bacteria and their phages. By using sequence similarity network and gene context analyses, we created a new high quality dataset of phage-related SSAPs, substantially increasing the number of annotated SSAPs. We classified the identified SSAPs into seven distinct families, namely RecA, Gp2.5, RecT/Redβ, Erf, Rad52/22, Sak3, and Sak4, organized into three superfamilies. Analysis of the relationships between the revealed protein clusters led us to recognize Sak3-like proteins as a new distinct SSAP family. Our analysis showed an irregular phylogenetic distribution of ssap genes among different bacterial phyla and specific phages, which can be explained by the high rates of ssap HGT. We propose that the evolution of phage recombinases could be tightly linked to the dissemination of bacterial phage-resistance mechanisms (e.g., abortive infection and CRISPR/Cas systems) targeting ssap genes and be a part of the constant phage-bacteria arms race.