Publications

Pseudomonas aeruginosa is an opportunistic human pathogen responsible for a significant proportion of hospital-acquired infections worldwide. P. aeruginosa infections are especially difficult to treat due to its numerous intrinsic and acquired resistance mechanisms. Even though P. aeruginosa was consequently recognised by the World Health Organization as a critical pathogen requiring urgent novel therapeutic agents, the current pipeline for drug discovery has been unable to meet this pressing demand. By rewiring its central metabolism, P. aeruginosa can thrive in diverse infection scenarios. For example, P. aeruginosa can colonize the cystic fibrosis lung by metabolizing long- and short-chain fatty acids, cholesterol and amino acids generated by degradation and fermentation of lung mucin. The metabolism of the above-mentioned nutrients requires a functional 2-methylcitrate cycle (2-MCC) to metabolize propionyl coenzyme A (PrCoA), a metabolic by-product. In addition, the 2-MCC also allows the utilization of propionate as a carbon source. As propionate and its derived catabolites, including PrCoA, are lethally toxic to cells, the 2-MCC is often viewed as a propionate detoxifying pathway. Generally, the 2-MCC is comprised of three core enzymes: 2-methylcitrate synthase (PrpC), 2-methylcitrate dehydratase (PrpD) and 2-methylisocitrate lyase (PrpB). The 2-MCC presents itself as a potential target for therapeutic intervention in P. aeruginosa; in addition to its role in carbon fixation, it is also involved in virulence and establishing infection. In this dissertation, core enzymes in the 2-MCC were characterized: PrpC, PrpD, and PrpB. The biochemical characterization entailed kinetic analysis of enzymes for the subsequent drug-design pipeline. Structural characterization of these three enzymes resulted in multiple crystal structures solved at near-atomic resolution. PrpC and PrpB were targeted for the development of novel antipseudomonal agents using high throughput fragment-based screening (HT-FBS) and X-ray crystallography. This was followed by fragment optimization, leading to one of the initial hits inhibiting PrpC activity in vitro, and preventing growth of P. aeruginosa in the presence of propionate. Putative mechanisms of inhibition are proposed based on the structural data. PrpD, on the other hand, was not included in the HT-FBS due to its non-essential nature in P. aeruginosa. However, NMR spectroscopy identified an unexpected PrpD substrate and product stereochemistry. The crystal structure of P. aeruginosa PrpD shed light on the likely active site and revealed the residues that are critical for activity. Together, these data provide an excellent foundation for targeting this class of enzymes and offers strategies aimed at further improving the inhibitory activity of the fragment hits. Less |Related Solutions: Rock Maker^®

Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens. Less |Related Solutions: Rock Imager^®

Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation. Less |Related Solutions: NT8^®

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) testing is one component of a multilayered mitigation strategy to enable safe in-person school attendance for the K–12 school population. However, costs, logistics, and uncertainty about effectiveness are potential barriers to implementation. We assessed early data from the Massachusetts K–12 public school pooled SARS-CoV2 testing program, which incorporates two novel design elements: in-school “pod pooling” for assembling pools of dry anterior nasal swabs from 5 to 10 individuals and positive pool deconvolution using the BinaxNOW antigen rapid diagnostic test (Ag RDT), to assess the operational and analytical feasibility of this approach. Over 3 months, 187,597 individual swabs were tested across 39,297 pools from 738 schools. The pool positivity rate was 0.8%; 98.2% of pools tested negative and 0.2% inconclusive, and 0.8% of pools submitted could not be tested. Of 310 positive pools, 70.6% had an N1 or N2 probe cycle threshold (CT) value of ≤30. In reflex testing (performed on specimens newly collected from members of the positive pool), 92.5% of fully deconvoluted pools with an N1 or N2 target CT of ≤30 identified a positive individual using the BinaxNOW test performed 1 to 3 days later. However, of 124 positive pools with full reflex testing data available for analysis, 32 (25.8%) of BinaxNOW pool deconvolution testing attempts did not identify a positive individual, requiring additional reflex testing. With sufficient staffing support and low pool positivity rates, pooled sample collection and reflex testing were feasible for schools. These early program findings confirm that screening for K–12 students and staff is achievable at scale with a scheme that incorporates in-school pooling, primary testing by reverse transcription-PCR (RT-PCR), and Ag RDT reflex/deconvolution testing. Less |Related Solutions: Tempest^®

BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia, a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence. Less |Related Solutions: Formulator^®

In bacteria, trans-translation is the major quality control system for rescuing stalled ribosomes. It is mediated by tmRNA, a hybrid RNA with properties of both a tRNA and a mRNA, and the small protein SmpB. Because trans-translation is absent in eukaryotes but necessary for bacterial fitness or survival, it is a promising target for the development of novel antibiotics. To facilitate screening of chemical libraries, various reliable in vitro and in vivo systems have been created for assessing trans-translational activity. However, the aim of the current work was to permit the safe and easy in vitro evaluation of trans-translation from pathogenic bacteria, which are obviously the ones we should be targeting. Based on green fluorescent protein (GFP) reassembly during active trans-translation, we have created a cell-free assay adapted to the rapid evaluation of trans-translation in ESKAPE bacteria, with 24 different possible combinations. It can be used for easy high-throughput screening of chemical compounds as well as for exploring the mechanism of trans-translation in these pathogens. Less |Related Solutions: Mantis^®

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19. Less |Related Solutions: Formulator^®

Profiling chromatin accessibility at the single-cell level provides critical information about cell type composition and cell-to-cell variation within a complex tissue. Emerging techniques for the interrogation of chromatin accessibility in individual cells allow investigation of the fundamental mechanisms that lead to the variability of different cells. This protocol describes a fast and robust method for single-cell chromatin accessibility profiling based on the assay for transposase-accessible chromatin using sequencing (ATAC-seq). The method combines up-front bulk Tn5 tagging of chromatin with flow cytometry to isolate single nuclei or cells. Reagents required to generate sequencing libraries are added to the same well in the plate where cells are sorted. The protocol described here generates data of high complexity and excellent signal-to-noise ratio and can be combined with index sorting for in-depth characterization of cell types. The whole experimental procedure can be finished within 1 or 2 d with a throughput of hundreds to thousands of nuclei, and the data can be processed by the provided computational pipeline. The execution of the protocol only requires basic techniques and equipment in a molecular biology laboratory with flow cytometry support. Less |Related Solutions: Mantis^®

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance. Less |Related Solutions: Tempest^®

The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer the most inhibitor resistance, K234R, as well as variant G132N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance (NMR) relaxation experiments to probe structural and dynamic properties. The G132N mutant exists in solution in two almost equally populated conformations that exchange with a rate of ca. 88 s−1. The conformational change affects a broad region of the enzyme. The crystal structure reveals that the Asn132 side chain forces the peptide bond between Ser104 and Ile105 in a cis-conformation. The crystal structure suggests multiple conformations for several side chains (e.g., Ser104 and Ser130) and a short loop (positions 214 to 216). In the K234R mutant, the active-site dynamics are significantly diminished with respect to the wild-type enzyme. These results show that multiple evolutionary routes are available to increase inhibitor resistance in BlaC and that active-site dynamics on the millisecond time scale are not required for catalytic function. Less |Related Solutions: NT8^®

Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general. Less |Related Solutions: Rock Imager^®

The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features. Less |Related Solutions: Rock Imager^®

Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis. Less |Related Solutions: Rock Imager^®

The current rise of antibiotic resistant forms of Mycobacterium tuberculosis is a global health threat that calls for new antibiotics. The β-lactamase BlaC of this pathogen prevents the use of β-lactam antibiotics, except in combination with a β-lactamase inhibitor. To understand if exposure to such inhibitors can easily result in resistance, a BlaC evolution experiment was performed, studying the evolutionary adaptability against the inhibitor sulbactam. Several amino acid substitutions in BlaC were shown to confer reduced sensitivity to sulbactam. The G132S mutation causes a reduction in the rate of nitrocefin and ampicillin hydrolysis and simultaneously reduces the sensitivity for sulbactam inhibition. Introduction of the side chain moiety of Ser132 causes the 104–105 peptide bond to assume the cis conformation and the side chain of Ser104 to be rotated toward the sulbactam adduct with which it forms a hydrogen bond not present in the wild-type enzyme. The gatekeeper residue Ile105 also moves. These changes in the entrance of the active site can explain the decreased affinity of G132S BlaC for both substrates and sulbactam. Our results show that BlaC can easily acquire a reduced sensitivity for sulbactam, with a single-amino acid mutation, which could hinder the use of combination therapies. Less |Related Solutions: NT8^®

Eukaryotic proliferating cell nuclear antigen (PCNA) plays an essential role in orchestrating the assembly of the replisome complex, stimulating processive DNA synthesis, and recruiting other regulatory proteins during the DNA damage response. PCNA and its binding partner network are relatively conserved in eukaryotes, and it exhibits extraordinary structural similarity across species. However, despite this structural similarity, the PCNA of a given species is rarely functional in heterologous systems. In this report, we determined the X-ray crystal structure of Neurospora crassa PCNA (NcPCNA) and compared its structure–function relationship with other available PCNA studies to understand this cross-species incompatibility. We found two regions, the interdomain connecting loop (IDCL) and J loop structures, vary significantly among PCNAs. In particular, the J loop deviates in NcPCNA from that in Saccharomyces cerevisiae PCNA (ScPCNA) by 7 Å. Differences in the IDCL structures result in varied binding affinities of PCNAs for the subunit Pol32 of DNA polymerase delta and for T2-amino alcohol, a small-molecule inhibitor of human PCNA. To validate that these structural differences are accountable for functional incompatibility in S. cerevisiae, we generated NcPCNA mutants mimicking IDCL and J loop structures of ScPCNA. Our genetic analyses suggested that NcPCNA mutants are fully functional in S. cerevisiae. The susceptibility of the strains harboring ScPCNA mimics of NcPCNA to various genotoxic agents was similar to that in yeast cells expressing ScPCNA. Taken together, we conclude that in addition to the overall architecture of PCNA, structures of the IDCL and J loop of PCNA are critical determinants of interspecies functional compatibility. Less |Related Solutions: NT8^®

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system. Less |Related Solutions: Mantis^®

There is increasing attention focussed on the risks associated with mobile phones possibly serving as ‘Trojan Horse’ fomites for microbial transmission in healthcare settings. However, little is reported on the presence of microbes on community derived mobile phones which in 2021, numbered in the billions in circulation with majority being used on a daily basis. Identify viable microbial organisms swabbed from smartphones on a university campus. Entire surfaces of 5 mobile phones were swabbed and examined for their microbial content using pre-agar-based growths followed by downstream DNA metagenomic next-generation sequencing analysis. All phones were contaminated with viable microbes. 173 bacteria, 8 fungi, 8 protists, 53 bacteriophages, 317 virulence factor genes and 41 distinct antibiotic resistant genes were identified. While this research represents a pilot study, the snapshot metagenomic analysis of samples collected from the surface of mobile phones has revealed the presence of a large population of viable microbes and an array of antimicrobial resistant factors. With billions of phones in circulation, these devices might be responsible for the rise of community acquired infections. These pilot results highlight the importance of public health authorities considering mobile phones as ‘Trojan Horse’ devices for microbial transmission and ensure appropriate decontamination campaigns are implemented. Less |Related Solutions: Mantis^®

The chemokine receptor CCR5 plays a vital role in immune surveillance and inflammation. However, molecular details that govern its endogenous chemokine recognition and receptor activation remain elusive. Here we report three cryo-electron microscopy structures of Gi1 protein-coupled CCR5 in a ligand-free state and in complex with the chemokine MIP-1α or RANTES, as well as the crystal structure of MIP-1α-bound CCR5. These structures reveal distinct binding modes of the two chemokines and a specific accommodate pattern of the chemokine for the distal N terminus of CCR5. Together with functional data, the structures demonstrate that chemokine-induced rearrangement of toggle switch and plasticity of the receptor extracellular region are critical for receptor activation, while a conserved tryptophan residue in helix II acts as a trigger of receptor constitutive activation. Less |Related Solutions: Rock Imager^®

A series of nondeuterated and deuterated dipeptidyl aldehyde and masked aldehyde inhibitors that incorporate in their structure a conformationally constrained cyclohexane moiety was synthesized and found to potently inhibit severe acute respiratory syndrome coronavirus-2 3CL protease in biochemical and cell-based assays. Several of the inhibitors were also found to be nanomolar inhibitors of Middle East respiratory syndrome coronavirus 3CL protease. The corresponding latent aldehyde bisulfite adducts were found to be equipotent to the precursor aldehydes. High-resolution cocrystal structures confirmed the mechanism of action and illuminated the structural determinants involved in binding. The spatial disposition of the compounds disclosed herein provides an effective means of accessing new chemical space and optimizing pharmacological activity. The cellular permeability of the identified inhibitors and lack of cytotoxicity warrant their advancement as potential therapeutics for COVID-19. Less |Related Solutions: NT8^®

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2–treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2. Less |Related Solutions: NT8^®

Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor. Less |Related Solutions: NT8^®

Selective agonism of the estrogen receptor (ER) subtypes, ERα and ERβ, has historically been difficult to achieve due to the high degree of ligand-binding domain structural similarity. Multiple efforts have focused on the use of classical organic scaffolds to model 17β-estradiol geometry in the design of ERβ selective agonists, with several proceeding to various stages of clinical development. Carborane scaffolds offer many unique advantages including the potential for novel ligand/receptor interactions but remain relatively unexplored. We synthesized a series of para-carborane estrogen receptor agonists revealing an ERβ selective structure–activity relationship. We report ERβ agonists with low nanomolar potency, greater than 200-fold selectivity for ERβ over ERα, limited off-target activity against other nuclear receptors, and only sparse CYP450 inhibition at very high micromolar concentrations. The pharmacological properties of our para-carborane ERβ selective agonists measure favorably against clinically developed ERβ agonists and support further evaluation of carborane-based selective estrogen receptor modulators. Less |Related Solutions: Tempest^®

Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites. Less |Related Solutions: Rock Imager^®

Aerogels are ultralight porous materials whose matrix structure can be formed by interlinking 880 nm long M13 phage particles. In theory, changing the phage properties would alter the aerogel matrix, but attempting this using the current production system leads to heterogeneous lengths. A phagemid system that yields a narrow length distribution that can be tuned in 0.3 nm increments from 50 to 2500 nm is designed and, independently, the persistence length varies from 14 to 68 nm by mutating the coat protein. A robotic workflow that automates each step from DNA construction to aerogel synthesis is used to build 1200 aerogels. This is applied to compare Ni–MnOx cathodes built using different matrixes, revealing a pareto-optimal relationship between performance metrics. This work demonstrates the application of genetic engineering to create “tuning knobs” to sweep through material parameter space; in this case, toward creating a physically strong and high-capacity battery. Less |Related Solutions: Mantis^®

SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies. Less |Related Solutions: NT8^®

Blossom-end rot (BER) is a devastating physiological disorder that affects tomato and other vegetables, resulting in significant crop losses. To date, most studies on BER have focused on the environmental factors that affect calcium translocation to the fruit; however, the genetic basis of this disorder remains unknown. To investigate the genetic basis of BER, two F2 and F3:4 populations along with a BC1 population that segregated for BER occurrence were evaluated in the greenhouse. Using the QTL-seq approach, quantitative trait loci (QTL) associated with BER Incidence were identified at the bottom of chromosome (ch) 3 and ch11. Additionally, linkage-based QTL mapping detected another QTL, BER3.1, on ch3 and BER4.1 on ch4. To fine map the QTLs identified by QTL-seq, recombinant screening was performed. BER3.2, the major BER QTL on ch3, was narrowed down from 5.68 to 1.58 Mbp with a 1.5-LOD support interval (SI) corresponding to 209 candidate genes. BER3.2 colocalizes with the fruit weight gene FW3.2/SlKLUH, an ortholog of cytochrome P450 KLUH in Arabidopsis. Further, BER11.1, the major BER QTL on ch11, was narrowed down from 3.99 to 1.13 Mbp with a 1.5-LOD SI interval comprising of 141 candidate genes. Taken together, our results identified and fine mapped the first loci for BER resistance in tomato that will facilitate marker-assistant breeding not only in tomato but also in many other vegetables suffering for BER. Less |Related Solutions: Mantis^®

Sphingosine-1-phosphate (S1P) regulates numerous important physiological functions, including immune response and vascular integrity, via its cognate receptors (S1PR1 to S1PR5); however, it remains unclear how S1P activates S1PRs upon binding. Here, we determined the crystal structure of the active human S1PR3 in complex with its natural agonist S1P at 3.2-Å resolution. S1P exhibits an unbent conformation in the long tunnel, which penetrates through the receptor obliquely. Compared with the inactive S1PR1 structure, four residues surrounding the alkyl tail of S1P (the “quartet core”) exhibit orchestrating rotamer changes that accommodate the moiety, thereby inducing an active conformation. In addition, we reveal that the quartet core determines G protein selectivity of S1PR3. These results offer insight into the structural basis of activation and biased signaling in G protein–coupled receptors and will help the design of biased ligands for optimized therapeutics. Less |Related Solutions: NT8^®

EMBL Grenoble operates the High Throughput Crystallization Laboratory (HTX Lab), a large-scale user facility offering high throughput crystallography services to users worldwide. The HTX lab has a strong focus in the development of new methods in macromolecular crystallography. Through the combination of a high throughput crystallization platform, the CrystalDirect technology for fully automated crystal mounting and cryocooling and the CRIMS software we have developed fully automated pipelines for macromolecular crystallography that can be remotely operated over the internet. These include a protein-to-structure pipeline for the determination of new structures, a pipeline for the rapid characterization of protein-ligand complexes in support of medicinal chemistry, and a large-scale, automated fragment screening pipeline enabling evaluation of libraries of over 1000 fragments. Here we describe how to access and use these resources. Less |Related Solutions: Formulator^®

Mammalian medial and lateral hippocampal networks preferentially process spatial- and object-related information, respectively. However, the mechanisms underlying the assembly of such parallel networks during development remain largely unknown. Our study shows that, in mice, complementary expression of cell surface molecules teneurin-3 (Ten3) and latrophilin-2 (Lphn2) in the medial and lateral hippocampal networks, respectively, guides the precise assembly of CA1-to-subiculum connections in both networks. In the medial network, Ten3-expressing (Ten3+) CA1 axons are repelled by target-derived Lphn2, revealing that Lphn2- and Ten3-mediated heterophilic repulsion and Ten3-mediated homophilic attraction cooperate to control precise target selection of CA1 axons. In the lateral network, Lphn2-expressing (Lphn2+) CA1 axons are confined to Lphn2+ targets via repulsion from Ten3+ targets. Our findings demonstrate that assembly of parallel hippocampal networks follows a "Ten3→Ten3, Lphn2→Lphn2" rule instructed by reciprocal repulsions. Less |Related Solutions: Mantis^®

Background Psoriasis is an inflammatory, IL-17–driven skin disease in which autoantigen-induced CD8+ T cells have been identified as pathogenic drivers. Objective Our study focused on comprehensively characterizing the phenotypic variation of CD8+ T cells in psoriatic lesions. Methods We used single-cell RNA sequencing to compare CD8+ T-cell transcriptomic heterogeneity between psoriatic and healthy skin. Results We identified 11 transcriptionally diverse CD8+ T-cell subsets in psoriatic and healthy skin. Among several inflammatory subsets enriched in psoriatic skin, we observed 2 Tc17 cell subsets that were metabolically divergent, were developmentally related, and expressed CXCL13, which we found to be a biomarker of psoriasis severity and which achieved comparable or greater accuracy than IL17A in a support vector machine classifier of psoriasis and healthy transcriptomes. Despite high coinhibitory receptor expression in the Tc17 cell clusters, a comparison of these cells with melanoma-infiltrating CD8+ T cells revealed upregulated cytokine, cytolytic, and metabolic transcriptional activity in the psoriatic cells that differed from an exhaustion program. Conclusion Using high-resolution single-cell profiling in tissue, we have uncovered the diverse landscape of CD8+ T cells in psoriatic and healthy skin, including 2 nonexhausted Tc17 cell subsets associated with disease severity. Less |Related Solutions: Mantis^®

The Parkin co-regulated gene protein (PACRG) binds at the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG binds to the adaptor protein meiosis expressed gene 1 (MEIG1), but how they bind to microtubules is unknown. Here, we report the crystal structure of human PACRG in complex with MEIG1. PACRG adopts a helical repeat fold with a loop that interacts with MEIG1. Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy, we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation of the inner junction. We show that the homologous PACRG-like protein also mediates dual tubulin interactions but does not bind MEIG1. Our findings establish a framework to assess the function of the PACRG family of proteins and MEIG1 in regulating axoneme assembly. Less |Related Solutions: NT8^®

Plant pathogens cause disease through secreted effector proteins, which act to modulate host physiology and promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilised a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3. We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination. We show this approach can be successfully applied to effectors from other pathogenic fungi. Complementing this, an in-silico study uncovered the prevalence of an expanded subclass of effectors from fungi. The β-barrel fold of SnTox3 is a novel fold among fungal effectors. We demonstrate that SnTox3 is a pre-pro-protein and that the protease Kex2 removes the pro-domain. Our in-silico studies suggest that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi, and we demonstrate this experimentally for effectors from Fusarium oxysporum f sp. lycopersici. We propose that K2PP effectors are highly prevalent among fungal effectors. The identification and classification of K2PP effectors has broad implications for the approaches used to study their function in fungal virulence. Less |Related Solutions: Rock Imager^®

The zinc-finger transcription factor Helios is critical for maintaining the identity, anergic phenotype and suppressive activity of regulatory T (Treg) cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small-molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity. Less |Related Solutions: NT8^®

Biological macromolecules, such as proteins and nucleic acids, are composed of linked monomers and play an important role in biological functions based on their three-dimensional (3D) structures. Proteins are composed of one or more polypeptide chains of different amino acid residues. These polypeptide chains fold into a 3D structure to constitute a functional protein. The 3D structure information of proteins can be applied to analyze protein-ligand processes and interactions. Furthermore, the 3D structure information of proteins can serve as the basis for structure-based target selection for drug discovery research. As it is not possible for protein 3D structures to be seen even under the most advanced light microscope, other methods are employed to determine their 3D structures. Since proteins can form crystals, X-ray crystallography can be used to solve the 3D structures of these proteins. In the deposited protein data bank (PDB), nearly 90% of protein structures are solved through X-ray crystallography. As a result, X-ray crystallography is the fundamental method for characterizing the atomic structure of proteins.
Notably, the primary and oldest method of X-ray crystallography is single-crystal X-ray diffraction. The major challenge of using this method is obtaining well-ordered crystals with a suitable size for crystallographic data collection. The demand for larger and well-ordered protein crystals has introduced difficulties for those proteins which cannot grow to larger dimensions.
With the development of synchrotron radiation, the brilliant beams achieved through synchrotron radiation have decreased the necessary protein crystal size for conventional X-ray diffraction crystallography. A free-electron laser (FEL) uses a much brighter beam, which decreases the dimensions of protein crystals that are required for diffraction data collection. Consequently, today micro-sized and nano-sized protein crystals are preferred. This preference for small crystals creates a strong demand to develop and establish new methods and instrumentation to identify, detect and analyze protein nano- and micro-crystals.
Current methods to detect micro-sized and nano-sized protein crystals mainly include bright-field imaging, ultraviolet fluorescence (UV) imaging, second harmonic generation (SHG) imaging and X-ray powder diffraction. However, each of these imaging methods has its own limitations. Because of this, a reliable and advanced imaging method is required.
The present work describes an in-house developed multi-modalities multiphoton instrument that is composed of three imaging methods, which are third-harmonic generation (THG), second-harmonic generation (SHG) and three-photon excited ultraviolet fluorescence (3PEUVF). To analyze the feasibility and detection sensitivity of the multimodal MPM system, different protein crystals and salt crystals were prepared with different symmetries. The combined effect of THG, SHG and 3PEUVF imaging is precise, as the system is able to identify nano- or micro-sized protein crystals and can distinguish between protein crystals, salt crystals and amorphous aggregates.
During the testing process, a detailed study of the angular-dependent SHG polarization response was conducted. The results demonstrated that the SHG polarization response of the crystal is highly sensitive to the lattice orientation of crystals. As a result, SHG polarization can extend its potential for protein crystal detection and characterization.
To better compare the differences between commercial imaging instruments and MPM system instruments, the in vitro nanocrystal samples were simultaneously tested with dynamic light scattering (DLS), depolarized dynamic light scattering (DDLS), transmission electron microscopy (TEM) and X-ray powder diffraction. For second-order nonlinear optical imaging of chiral crystals (SONICC) and MPM imaging instrument, the experimental results illustrate that the MPM imaging instrument processes a non-invasive detection method and high detection sensitivity to detect in vitro and in vivo protein nanocrystals. Notably, the nano-sized or sub-micro-sized protein crystals can be detected efficiently through the MPM system. For in vitro protein crystals, the MPM system reduces the risk of obtaining false-negative and false-positive results in crystal detection through providing a higher signal sensitivity. Moreover, the MPM imaging system offers the possibility for in vivo crystals to be detected. Furthermore, weak SHG signals from centrosymmetric crystals are also observed with the MPM system. Less |Related Solutions: SONICC^®

The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design. Less |Related Solutions: NT8^®

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment. Less |Related Solutions: Mantis^®

Existing protocols for full-length single-cell RNA sequencing produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single-nucleotide polymorphisms and allow assembly of the VDJ region of the T- and B-cell-receptor sequences. Since full-length protocols are mostly plate-based at present, they are also suited to profiling cell types where cell numbers are limiting, such as rare cell types during development. A disadvantage of these methods has been the scalability and cost of the experiments, which has limited their popularity as compared with droplet-based and nanowell approaches. Here, we describe an automated protocol for full-length single-cell RNA sequencing, including both an in-house automated Smart-seq2 protocol and a commercial kit–based workflow. The protocols take 3–5 d to complete, depending on the number of plates processed in a batch. We discuss these two protocols in terms of ease of use, equipment requirements, running time, cost per sample and sequencing quality. By benchmarking the lysis buffers, reverse transcription enzymes and their combinations, we have optimized the in-house automated protocol to dramatically reduce its cost. An automated setup can be adopted easily by a competent researcher with basic laboratory skills and no prior automation experience. These pipelines have been employed successfully for several research projects allied with the Human Cell Atlas initiative (www.humancellatlas.org). Less |Related Solutions: Mantis^®

Stratified lakes and ponds featuring steep oxygen gradients are significant net sources of greenhouse gases and hotspots in the carbon cycle. Despite their significant biogeochemical roles, the microbial communities, especially in the oxygen depleted compartments, are poorly known. Here, we present a comprehensive dataset including 267 shotgun metagenomes from 41 stratified lakes and ponds mainly located in the boreal and subarctic regions, but also including one tropical reservoir and one temperate lake. For most lakes and ponds, the data includes a vertical sample set spanning from the oxic surface to the anoxic bottom layer. The majority of the samples were collected during the open water period, but also a total of 29 samples were collected from under the ice. In addition to the metagenomic sequences, the dataset includes environmental variables for the samples, such as oxygen, nutrient and organic carbon concentrations. The dataset is ideal for further exploring the microbial taxonomic and functional diversity in freshwater environments and potential climate change impacts on the functioning of these ecosystems. Less |Related Solutions: Mantis^®

The adhesion G protein–coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF1,2,5). Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1–4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1–3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55–CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55–CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein–coupled receptors. Less |Related Solutions: NT8^®

The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide–lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide. Less |Related Solutions: Rock Imager^®

Artemisinin-based combination therapies (ACTs), the World Health Organization-recommended first-line therapy for uncomplicated falciparum malaria, has led to significant decreases in malaria-associated morbidity and mortality in the past two decades. Decreased therapeutic efficacy of artemisinins, the cornerstone of ACTs, is threatening the gains made against this disease. As such, novel therapeutics with uncompromised mechanisms of action are needed to combat parasite-mediated antimalarial resistance. We have previously reported the antimalarial activity of Plasmodium falciparum-specific proteasome inhibitors in conjunction with a variety of antimalarials in clinical use or in preclinical investigations and of proteasome mutants generated in response to these inhibitors. Here, we discover that despite harboring K13C580Y, which has conventionally mediated artemisinin resistance in vitro as measured by increased survival in ring-stage survival assays (RSA), the Cam3.II strain parasites of Cambodian origin that have acquired an additional mutation in the proteasome display increased susceptibility to DHA and OZ439. This discovery implicates the proteasome in peroxide susceptibilities and has favorable implications on the use of peroxide and proteasome inhibitor combination therapy for the treatment of artemisinin-resistant malaria Less |Related Solutions: Mantis^®

The SpinVessel system provides a methodology using pulsed radial flow to gently mix and uniformly suspend particulates (cells, magnetic beads, silica beads, and microcarrier beads) for automated assays. SpinVessels are well suited for aliquoting on robotic liquid handlers and with robotic reagent dispensers, as well as manually. The SpinVessel system combines two critical features: (1) special internal side fins and projections in the bottom of the vessels and (2) an instrument that quickly spins the vessels and repeatedly reverses the spin direction. This rapid reversing motion sends multiple pulses of fluid up the side walls of the SpinVessel, creating a circular radial flow pattern. We tested five different particulates and six different SpinVessels with volume capacities varying from 50 mL to 1200 mL. SpinVessels are compatible with either single-, 8-, 12-, 96-, or 384-channel pipettors or with siphon tubing on robotic reagent dispensers. Experiments have demonstrated high viability of cells and undamaged morphology of microcarrier beads even after hours of constant agitation. The uniformity of aliquots collected at various vertical depths and horizontally across the SpinVessels demonstrated that cells, magnetic beads, and silica beads were uniformly suspended throughout the height and breadth of the SpinVessels, and uniformity of samples was consistent from the beginning to the end of the aliquoting procedure. Only 5 min of mixing is required to resuspend settled particulates. This novel mixing methodology has many applications in laboratory automation where particulate aliquot uniformity and/or particulate integrity are important to automating assays. Less |Related Solutions: Mantis^®

An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs). Broad and potent VRC01-class bNAbs have been isolated from multiple infected individuals, suggesting that they could be reproducibly elicited by vaccination. Several HIV-1 envelope-derived germline-targeting immunogens have been designed to engage naive VRC01-class precursor B cells. However, they also present off-target epitopes that could hinder development of VRC01-class bNAbs. We characterize a panel of anti-idiotypic monoclonal antibodies (ai-mAbs) raised against inferred-germline (iGL) VRC01-class antibodies. By leveraging binding, structural, and B cell sorting data, we engineered a bispecific molecule derived from two ai-mAbs; one specific for VRC01-class heavy chains and one specific for VRC01-class light chains. The bispecific molecule preferentially activates iGL-VRC01 B cells in vitro and induces specific antibody responses in a murine adoptive transfer model with a diverse polyclonal B cell repertoire. This molecule represents an alternative non-envelope-derived germline-targeting immunogen that can selectively activate VRC01-class precursors in vivo. Less |Related Solutions: Rock Imager^®

Branching pattern formation is common in many microbes. Extensive studies have focused on addressing how such patterns emerge from local cell–cell and cell–environment interactions. However, little is known about whether and to what extent these patterns play a physiological role. Here, we consider the colonization of bacteria as an optimization problem to find the colony patterns that maximize colony growth efficiency under different environmental conditions. We demonstrate that Pseudomonas aeruginosa colonies develop branching patterns with characteristics comparable to the prediction of modeling; for example, colonies form thin branches in a nutrient-poor environment. Hence, the formation of branching patterns represents an optimal strategy for the growth of Pseudomonas aeruginosa colonies. The quantitative relationship between colony patterns and growth conditions enables us to develop a coarse-grained model to predict diverse colony patterns under more complex conditions, which we validated experimentally. Our results offer new insights into branching pattern formation as a problem-solving social behavior in microbes and enable fast and accurate predictions of complex spatial patterns in branching colonies. Less |Related Solutions: Mantis^®

Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals. Less |Related Solutions: NT8^®

Rationale: Clinical interest in combining targeted radionuclide therapies (TRT) with immunotherapies is growing. External beam radiation therapy (EBRT) activates a type 1 interferon (IFN1) response mediated via stimulator of interferon genes (STING), and this is critical to its therapeutic interaction with immune checkpoint blockade. However, little is known about the time course of IFN1 activation after EBRT or whether this may be induced by decay of a TRT source. Methods: We examined the IFN1 response and expression of immune susceptibility markers in B78 and B16 melanomas and MOC2 head and neck cancer murine models using qPCR and western blot. For TRT, we used 90Y chelated to NM600, an alkylphosphocholine analog that exhibits selective uptake and retention in tumor cells including B78 and MOC2. Results: We observed significant IFN1 activation in all cell lines, with peak activation in B78, B16, and MOC2 cell lines occurring 7, 7, and 1 days, respectively, following RT for all doses. This effect was STING-dependent. Select IFN response genes remained upregulated at 14 days following RT. IFN1 activation following STING agonist treatment in vitro was identical to RT suggesting time course differences between cell lines were mediated by STING pathway kinetics and not DNA damage susceptibility. In vivo delivery of EBRT and TRT to B78 and MOC2 tumors resulted in a comparable time course and magnitude of IFN1 activation. In the MOC2 model, the combination of 90Y-NM600 and dual checkpoint blockade therapy reduced tumor growth and prolonged survival compared to single agent therapy and cumulative dose equivalent combination EBRT and dual checkpoint blockade therapy. Conclusions: We report the time course of the STING-dependent IFN1 response following radiation in multiple murine tumor models. We show the potential of TRT to stimulate IFN1 activation that is comparable to that observed with EBRT and this may be critical to the therapeutic integration of TRT with immunotherapies. Less |Related Solutions: Tempest^®

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 mg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public Vgenes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models. Less |Related Solutions: Rock Imager^®

Stem cell dysfunction drives many age-related disorders. Identifying mechanisms that initially compromise stem cell behavior represent early targets to promote tissue function later in life. Here, we pinpoint multiple factors that disrupt neural stem cell (NSC) behavior in the adult hippocampus. Clonal tracing showed that NSCs exhibit asynchronous depletion by identifying short-term NSCs (ST-NSCs) and long-term NSCs (LT-NSCs). ST-NSCs divide rapidly to generate neurons and deplete in the young brain. Meanwhile, multipotent LT-NSCs are maintained for months but are pushed out of homeostasis by lengthening quiescence. Single-cell transcriptome analysis of deep NSC quiescence revealed several hallmarks of molecular aging in the mature brain and identified tyrosine-protein kinase Abl1 as an NSC aging factor. Treatment with the Abl inhibitor imatinib increased NSC activation without impairing NSC maintenance in the middle-aged brain. Our study indicates that hippocampal NSCs are particularly vulnerable and adaptable to cellular aging. Less |Related Solutions: Mantis^®

A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling. Less |Related Solutions: Rock Imager^®