A rapid cell-free expression and screening platform for antibody discovery

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly

Encephalopathy, Hypoglycemia, and Flailing Extremities: A Case of Bilateral Chorea““Ballism Associated with Diabetic Ketoacidosis

Background: Hypo/hyperglycemia is a known cause of chorea and hemiballism. The temporallobes, hippocampus, basal ganglia, and substantia nigra are most susceptible to hypoglycemic changes. Methods: We present a caseof bilateral chorea and bi-ballism accompanied by encephalopathyin the setting of severe hypoglycemia and diabetic ketoacidosis. The patient had brain MRI changes involving both caudate nuclei, temporal lobes, and hippocampi. Discussion: This case demonstrates the basal ganglia's vulnerability to hypoglycemia and the need for cautious evaluation of involuntary movements when they occur in the setting of encephalopathy

Cytomegalovirus Viral Load and Virus-Specific Immune Reconstitution after Peripheral Blood Stem Cell versus Bone Marrow Transplantation

Peripheral blood stem cell (PBSC) products contain more T cells and monocytes when compared with bone marrow (BM), leading to fewer bacterial and fungal infections. Cytomegelovirus (CMV) viral load and disease as well as CMV-specific immune reconstitution were compared in patients enrolled in a randomized trial comparing PSBC and BM transplantation. There was a higher rate of CMV infection and disease during the first 100 days after transplantation among PBSC recipients (any antigenemia/DNAemia: PBSC, 63% vs BM, 42%, P = .04; CMV disease: PBSC, 17% vs BM, 4%, P = .03). By 2 years, CMV disease rates were similar. The early increase in CMV events correlated temporarily with lower CMV-specific CD4+ T helper and CD8+ cytotoxic T lymphocyte function at 30 days after transplantation in PBSC recipients. By 3 months after transplantation and thereafter, CMV-specific immune responses were similar between BM and PBSC recipients. In conclusion, higher CMV infection and disease rates occurred in PBSC transplant recipients early after transplantation. These differences may be because of a transient delay in CMV-specific immune reconstitution following PBSC transplantation

Shigella sonnei infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence.

Funder: Lister Institute of Preventive Medicine; funder-id: http://dx.doi.org/10.13039/501100001255Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries

Janus II: a new generation application-driven computer for spin-system simulations

This paper describes the architecture, the development and the implementation of Janus II, a new generation application-driven number cruncher optimized for Monte Carlo simulations of spin systems (mainly spin glasses). This domain of computational physics is a recognized grand challenge of high-performance computing: the resources necessary to study in detail theoretical models that can make contact with experimental data are by far beyond those available using commodity computer systems. On the other hand, several specific features of the associated algorithms suggest that unconventional computer architectures, which can be implemented with available electronics technologies, may lead to order of magnitude increases in performance, reducing to acceptable values on human scales the time needed to carry out simulation campaigns that would take centuries on commercially available machines. Janus II is one such machine, recently developed and commissioned, that builds upon and improves on the successful JANUS machine, which has been used for physics since 2008 and is still in operation today. This paper describes in detail the motivations behind the project, the computational requirements, the architecture and the implementation of this new machine and compares its expected performances with those of currently available commercial systems.Comment: 28 pages, 6 figure

Binding of Soluble Yeast β-Glucan to Human Neutrophils and Monocytes is Complement-Dependent

The immunomodulatory properties of yeast β-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate β-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble β-glucans. Using a well-characterized, pharmaceutical grade, soluble yeast β-glucan, this study evaluated and characterized the binding of soluble β-glucan to human neutrophils and monocytes. The results demonstrated that soluble β-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble β-glucan in these cells. Binding of soluble β-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble β-glucan was demonstrated by detection of iC3b, the complement opsonin on β-glucan-bound cells, as well as by the direct binding of iC3b to β-glucan in the absence of cells. Binding of β-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding

Analysis of meteorology-chemistry interactions during air pollution episodes using online coupled models within AQMEII Phase-2

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).This study reviews the top ranked meteorology and chemistry interactions in online coupled models recommended by an experts’ survey conducted in COST Action EuMetChem and examines the sensitivity of those interactions during two pollution episodes: the Russian forest fires 25 Jul -15 Aug 2010 and a Saharan dust transport event from 1 Oct -31 Oct 2010 as a part of the AQMEII phase-2 exercise. Three WRF-Chem model simulations were performed for the forest fire case for a baseline without any aerosol feedback on meteorology, a simulation with aerosol direct effects only and a simulation including both direct and indirect effects. For the dust case study, eight WRF-Chem and one WRF-CMAQ simulations were selected from the set of simulations conducted in the framework of AQMEII. Of these two simulations considered no feedbacks, two included direct effects only and five simulations included both direct and indirect effects. The results from both episodes demonstrate that it is important to include the meteorology and chemistry interactions in online-coupled models. Model evaluations using routine observations collected in AQMEII phase-2 and observations from a station in Moscow show that for the fire case the simulation including only aerosol direct effects has better performance than the simulations with no aerosol feedbacks or including both direct and indirect effects. The normalized mean biases are significantly reduced by 10-20% for PM10 when including aerosol direct effects. The analysis for the dust case confirms that models perform better when including aerosol direct effects, but worse when including both aerosol direct and indirect effects, which suggests that the representation of aerosol indirect effects needs to be improved in the model.Peer reviewedFinal Published versio

IKK phosphorylates Huntingtin and targets it for degradation by the proteasome and lysosome

Expansion of the polyglutamine repeat within the protein Huntingtin (Htt) causes Huntington's disease, a neurodegenerative disease associated with aging and the accumulation of mutant Htt in diseased neurons. Understanding the mechanisms that influence Htt cellular degradation may target treatments designed to activate mutant Htt clearance pathways. We find that Htt is phosphorylated by the inflammatory kinase IKK, enhancing its normal clearance by the proteasome and lysosome. Phosphorylation of Htt regulates additional post-translational modifications, including Htt ubiquitination, SUMOylation, and acetylation, and increases Htt nuclear localization, cleavage, and clearance mediated by lysosomal-associated membrane protein 2A and Hsc70. We propose that IKK activates mutant Htt clearance until an age-related loss of proteasome/lysosome function promotes accumulation of toxic post-translationally modified mutant Htt. Thus, IKK activation may modulate mutant Htt neurotoxicity depending on the cell's ability to degrade the modified species

MtMTP2-Facilitated Zinc Transport Into Intracellular Compartments Is Essential for Nodule Development in Medicago truncatula

Zinc (Zn) is an essential nutrient for plants that is involved in almost every biological process. This includes symbiotic nitrogen fixation, a process carried out by endosymbiotic bacteria (rhizobia) living within differentiated plant cells of legume root nodules. Zn transport in nodules involves delivery from the root, via the vasculature, release into the apoplast and uptake into nodule cells. Once in the cytosol, Zn can be used directly by cytosolic proteins or delivered into organelles, including symbiosomes of infected cells, by Zn efflux transporters. Medicago truncatula MtMTP2 (Medtr4g064893) is a nodule-induced Zn-efflux protein that was localized to an intracellular compartment in root epidermal and endodermal cells, as well as in nodule cells. Although the MtMTP2 gene is expressed in roots, shoots, and nodules, mtp2 mutants exhibited growth defects only under symbiotic, nitrogen-fixing conditions. Loss of MtMTP2 function resulted in altered nodule development, defects in bacteroid differentiation, and severe reduction of nitrogenase activity. The results presented here support a role of MtMTP2 in intracellular compartmentation of Zn, which is required for effective symbiotic nitrogen fixation in M. truncatula

Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases