GprC of the nematode-trapping fungus Arthrobotrys flagrans activates mitochondria and reprograms fungal cells for nematode hunting.

Initiation of development requires differential gene expression and metabolic adaptations. Here we show in the nematode-trapping fungus, Arthrobotrys flagrans, that both are achieved through a dual-function G-protein-coupled receptor (GPCR). A. flagrans develops adhesive traps and recognizes its prey, Caenorhabditis elegans, through nematode-specific pheromones (ascarosides). Gene-expression analyses revealed that ascarosides activate the fungal GPCR, GprC, at the plasma membrane and together with the G-protein alpha subunit GasA, reprograms the cell. However, GprC and GasA also reside in mitochondria and boost respiration. This dual localization of GprC in A. flagrans resembles the localization of the cannabinoid receptor CB1 in humans. The C. elegans ascaroside-sensing GPCR, SRBC66 and GPCRs of many fungi are also predicted for dual localization, suggesting broad evolutionary conservation. An SRBC64/66-GprC chimaeric protein was functional in A. flagrans, and C. elegans SRBC64/66 and DAF38 share ascaroside-binding sites with the fungal GprC receptor, suggesting 400-million-year convergent evolution.

Pharmacological modulation of developmental and synaptic phenotypes in human SHANK3 deficient stem cell-derived neuronal models.

Phelan-McDermid syndrome (PMDS) arises from mutations in the terminal region of chromosome 22q13, impacting the SHANK3 gene. The resulting deficiency of the postsynaptic density scaffolding protein SHANK3 is associated with autism spectrum disorder (ASD). We examined 12 different PMDS patient and CRISPR-engineered stem cell-derived neuronal models and controls and found that reduced expression of SHANK3 leads to neuronal hyperdifferentiation, increased synapse formation, and decreased neuronal activity. We performed automated imaging-based screening of 7,120 target-annotated small molecules and identified three compounds that rescued SHANK3-dependent neuronal hyperdifferentiation. One compound, Benproperine, rescued the decreased colocalization of Actin Related Protein 2/3 Complex Subunit 2 (ARPC2) with ß-actin and rescued increased synapse formation in SHANK3 deficient neurons when administered early during differentiation. Neuronal activity was only mildly affected, highlighting Benproperine's effects as a neurodevelopmental modulator. This study demonstrates that small molecular compounds that reverse developmental phenotypes can be identified in human neuronal PMDS models.

Predicting deamidation and isomerization sites in therapeutic antibodies using structure-based in silico approaches.

Asparagine (Asn) deamidation and aspartic acid (Asp) isomerization are common degradation pathways that affect the stability of therapeutic antibodies. These modifications can pose a significant challenge in the development of biopharmaceuticals. As such, the early engineering and selection of chemically stable monoclonal antibodies (mAbs) can substantially mitigate the risk of subsequent failure. In this study, we introduce a novel in silico approach for predicting deamidation and isomerization sites in therapeutic antibodies by analyzing the structural environment surrounding asparagine and aspartate residues. The resulting quantitative structure-activity relationship (QSAR) model was trained using previously published forced degradation data from 57 clinical-stage mAbs. The predictive accuracy of the model was evaluated for four different states of the protein structure: (1) static homology models, (2) enhancing low-frequency vibrational modes during short molecular dynamics (MD) runs, (3) a combination of (2) with a protonation state reassignment, and (4) conventional full-atomistic MD simulations. The most effective QSAR model considered the accessible surface area (ASA) of the residue, the pKa value of the backbone amide, and the root mean square deviations of both the alpha carbon and the side chain. The accuracy was further enhanced by incorporating the QSAR model into a decision tree, which also includes empirical information about the sequential successor and the position in the protein. The resulting model has been implemented as a plugin named "Forecasting Reactivity of Isomerization and Deamidation in Antibodies" in MOE software, completed with a user-friendly graphical interface to facilitate its use.

Dynamics of Citrate Coordination on Gold Nanoparticles Under Low Specific Power Laser-Induced Heating.

SERS evolution recorded over a drop-coated coffee-ring pattern of citrate-capped gold colloids was investigated as a function of time under low-specific laser power. Spectral changes caused by plasmon-induced reaction could not be detected, but a long-term transient original spectral profile showing additional lines was observed. We performed deep qualitative and quantitative SERS intensity variation analysis based on the complementary use of extreme deviation and cross-correlation statistics, which provided further insights on the behavior of citrate-capping layers of gold nanoparticles upon laser illumination. More precisely, the cross-correlation analysis made possible to follow the so-called individual events denoting particular resonance structures, in which groups of modes were assigned to an evolution of citrate coordination on gold surface driven by photo-activation. As a consequence, the detection limit was increased and new lines were related to the presence of a very low amount of dicarboxy-acetone (DCA), which was already present in the system.

ATR represents a therapeutic vulnerability in clear cell renal cell carcinoma.

Metastatic clear cell renal cell carcinomas (ccRCCs) are resistant to DNA-damaging chemotherapies, limiting therapeutic options for patients whose tumors are resistant to tyrosine kinase inhibitors and/or immune checkpoint therapies. Here we show that mouse and human ccRCCs were frequently characterized by high levels of endogenous DNA damage and that cultured ccRCC cells exhibited intact cellular responses to chemotherapy-induced DNA damage. We identify that pharmacological inhibition of the DNA damage-sensing kinase ataxia telangiectasia and Rad3-related protein (ATR) with the orally administered, potent, and selective drug M4344 (gartisertib) induced antiproliferative effects in ccRCC cells. This effect was due to replication stress and accumulation of DNA damage in S phase. In some cells, DNA damage persisted into subsequent G2/M and G1 phases, leading to the frequent accumulation of micronuclei. Daily single-agent treatment with M4344 inhibited the growth of ccRCC xenograft tumors. M4344 synergized with chemotherapeutic drugs including cisplatin and carboplatin and the poly(ADP-ribose) polymerase inhibitor olaparib in mouse and human ccRCC cells. Weekly M4344 plus cisplatin treatment showed therapeutic synergy in ccRCC xenografts and was efficacious in an autochthonous mouse ccRCC model. These studies identify ATR inhibition as a potential novel therapeutic option for ccRCC.

Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites.

New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.

A crucial role for Jagunal homolog 1 in humoral immunity and antibody glycosylation in mice and humans.

Jagunal homolog 1 (JAGN1) has been identified as a critical regulator of neutrophil biology in mutant mice and rare-disease patients carrying JAGN1 mutations. Here, we report that Jagn1 deficiency results in alterations in the endoplasmic reticulum (ER) of antibody-producing cells as well as decreased antibody production and secretion. Consequently, mice lacking Jagn1 in B cells exhibit reduced serum immunoglobulin (Ig) levels at steady state and fail to mount an efficient humoral immune response upon immunization with specific antigens or when challenged with viral infections. We also demonstrate that Jagn1 deficiency in B cells results in aberrant IgG N-glycosylation leading to enhanced Fc receptor binding. Jagn1 deficiency in particular affects fucosylation of IgG subtypes in mice as well as rare-disease patients with loss-of-function mutations in JAGN1. Moreover, we show that ER stress affects antibody glycosylation. Our data uncover a novel and key role for JAGN1 and ER stress in antibody glycosylation and humoral immunity in mice and humans.

Acquired resistance to anti-MAPK targeted therapy confers an immune-evasive tumor microenvironment and cross-resistance to immunotherapy in melanoma.

How targeted therapies and immunotherapies shape tumors, and thereby influence subsequent therapeutic responses, is poorly understood. In the present study, we show, in melanoma patients and mouse models, that when tumors relapse after targeted therapy with MAPK pathway inhibitors, they are cross-resistant to immunotherapies, despite the different modes of action of these therapies. We find that cross-resistance is mediated by a cancer cell-instructed, immunosuppressive tumor microenvironment that lacks functional CD103+ dendritic cells, precluding an effective T cell response. Restoring the numbers and functionality of CD103+ dendritic cells can re-sensitize cross-resistant tumors to immunotherapy. Cross-resistance does not arise from selective pressure of an immune response during evolution of resistance, but from the MAPK pathway, which not only is reactivated, but also exhibits an increased transcriptional output that drives immune evasion. Our work provides mechanistic evidence for cross-resistance between two unrelated therapies, and a scientific rationale for treating patients with immunotherapy before they acquire resistance to targeted therapy.

Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling.

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling. A HOIP K784R mutant is catalytically active but shows reduced induction of an NF-κB reporter relative to wild-type HOIP. HOIP K784 is evolutionarily conserved, equivalent to HOIP K778 in mice. We generated HoipK778R/K778R knock-in mice, which show no overt developmental phenotypes; however, in response to TNF, HoipK778R/K778R mouse embryonic fibroblasts display mildly suppressed NF-κB activation and increased apoptotic markers. On the other hand, HOIP K778R enhances the TNF-induced formation of TNFR complex II and an interaction between TNFR complex II and LUBAC. Loss of the LUBAC component SHARPIN leads to embryonic lethality in HoipK778R/K778R mice, which is rescued by knockout of TNFR1. We propose that site-specific ubiquitination of HOIP regulates a LUBAC-dependent switch between survival and apoptosis in TNF signaling.

Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids.

BACKGROUND: Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production. OBJECTIVE: The aim of this study was to investigate how megakaryocytes interact and progress through the endothelial cells of mouse bone marrow sinusoids. METHODS: We used a combination of fluorescence, electron, and three-dimensional microscopy to characterize the cellular events between megakaryocytes and endothelial cells. RESULTS: We identified protrusive, F-actin-based podosome-like structures, called in vivo-MK podosomes, which initiate the formation of pores through endothelial cells. These structures present a collective and spatial organization through their interconnection via a contractile network of actomyosin, essential to regulate the endothelial openings. This ensures proper passage of megakaryocyte-derived processes into the blood circulation to promote thrombopoiesis. CONCLUSION: This study provides novel insight into the in vivo function of podosomes of megakaryocytes with critical importance to platelet production.

Matrix Decomposition of Carbon-Fiber-Reinforced Plastics via the Activation of Semiconductors.

The present study proposed a novel process for the matrix decomposition of carbon-fiber-reinforced plastics (CFRPs). For this purpose, the influence of ultraviolet (UV) radiation paired with semiconductors on CFRP was analyzed. Then, suitable process parameters for superficial and in-depth matrix decomposition in CFRP were evaluated. The epoxy resin was decomposed most effectively without damaging the embedded carbon fiber by using a UV light-emitting diode (LED) spotlight (395 nm, Semray 4103 by Heraeus Noblelight) at a power level of 66% compared to the maximum power of the spotlight. Using a distance of 10 mm and a treatment duration of only 35-40 s achieved a depth of two layers with an area of 750 mm2, which is suitable for technological CFRP repair procedures. In addition to the characterization of the process, the treated CFRP samples were analyzed based on several analytical methods, namely, light microscopy (LM), scanning electron microscopy (SEM), and atomic force microscopy (AFM). Subsequently, the prepared carbon fibers (CFs) were tested using filament tensiometry, single filament tensile tests, and thermogravimetric measurements. All analyses showed the power level of 66% to be superior to the use of 96% power. The gentle ("fiber friendly") matrix destruction reduced the damage to the surface of the fibers and maintained their properties, such as maximum elongation and maximum tensile strength, at the level of the reference materials.

HACE1 Prevents Lung Carcinogenesis via Inhibition of RAC-Family GTPases.

HACE1 is an E3 ubiquitin ligase with important roles in tumor biology and tissue homeostasis. Loss or mutation of HACE1 has been associated with the occurrence of a variety of neoplasms, but the underlying mechanisms have not been defined yet. Here, we report that HACE1 is frequently mutated in human lung cancer. In mice, loss of Hace1 led to enhanced progression of KRasG12D -driven lung tumors. Additional ablation of the oncogenic GTPase Rac1 partially reduced progression of Hace1-/- lung tumors. RAC2, a novel ubiquitylation target of HACE1, could compensate for the absence of its homolog RAC1 in Hace1-deficient, but not in HACE1-sufficient tumors. Accordingly, ablation of both Rac1 and Rac2 fully averted the increased progression of KRasG12D -driven lung tumors in Hace1-/- mice. In patients with lung cancer, increased expression of HACE1 correlated with reduced levels of RAC1 and RAC2 and prolonged survival, whereas elevated expression of RAC1 and RAC2 was associated with poor prognosis. This work defines HACE1 as a crucial regulator of the oncogenic activity of RAC-family GTPases in lung cancer development. SIGNIFICANCE: These findings reveal that mutation of the tumor suppressor HACE1 disrupts its role as a regulator of the oncogenic activity of RAC-family GTPases in human and murine lung cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/3009/F1.large.jpg.

Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Apelin inhibition prevents resistance and metastasis associated with anti-angiogenic therapy.

Angiogenesis is a hallmark of cancer, promoting growth and metastasis. Anti-angiogenic treatment has limited efficacy due to therapy-induced blood vessel alterations, often followed by local hypoxia, tumor adaptation, progression, and metastasis. It is therefore paramount to overcome therapy-induced resistance. We show that Apelin inhibition potently remodels the tumor microenvironment, reducing angiogenesis, and effectively blunting tumor growth. Functionally, targeting Apelin improves vessel function and reduces polymorphonuclear myeloid-derived suppressor cell infiltration. Importantly, in mammary and lung cancer, Apelin prevents resistance to anti-angiogenic receptor tyrosine kinase (RTK) inhibitor therapy, reducing growth and angiogenesis in lung and breast cancer models without increased hypoxia in the tumor microenvironment. Apelin blockage also prevents RTK inhibitor-induced metastases, and high Apelin levels correlate with poor prognosis of anti-angiogenic therapy patients. These data identify a druggable anti-angiogenic drug target that reduces tumor blood vessel densities and normalizes the tumor vasculature to decrease metastases.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

SynAggreg: A Multifunctional High-Throughput Technology for Precision Study of Amyloid Aggregation and Systematic Discovery of Synergistic Inhibitor Compounds.

Numerous proteins can coalesce into amyloid self-assemblies, which are responsible for a class of diseases called amyloidoses, but which can also fulfill important biological functions and are of great interest for biotechnology. Amyloid aggregation is a complex multi-step process, poorly prone to detailed structural studies. Therefore, small molecules interacting with amyloids are often used as tools to probe the amyloid aggregation pathway and in some cases to treat amyloidoses as they prevent pathogenic protein aggregation. Here, we report on SynAggreg, an in vitro high-throughput (HT) platform dedicated to the precision study of amyloid aggregation and the effect of modulator compounds. SynAggreg relies on an accurate bi-fluorescent amyloid-tracer readout that overcomes some limitations of existing HT methods. It allows addressing diverse aspects of aggregation modulation that are critical for pathomechanistic studies, such as the specificity of compounds toward various amyloids and their effects on aggregation kinetics, as well as the co-assembly propensity of distinct amyloids and the influence of prion-like seeding on self-assembly. Furthermore, SynAggreg is the first HT technology that integrates tailored methodology to systematically identify synergistic compound combinations-an emerging strategy to improve fatal amyloidoses by targeting multiple steps of the aggregation pathway. To this end, we apply analytical combinatorial scores to rank the inhibition efficiency of couples of compounds and to readily detect synergism. Finally, the SynAggreg platform should be suited for the characterization of a broad class of amyloids, whether of interest for drug development purposes, for fundamental research on amyloid functions, or for biotechnological applications.

Triple bonds of niobium with silicon, germaniun and tin: the tetrylidyne complexes [(κ3-tmps)(CO)2Nb[triple bond, length as m-dash]E-R] (E = Si, Ge, Sn; tmps = MeSi(CH2PMe2)3; R = aryl).

A systematic, efficient approach to first complexes containing a triple bond between niobium and the elements silicon, germanium or tin is reported. The approach involves a metathetical exchange of the niobium-centered nucleophile (NMe4)[Nb(CO)4(κ2-tmps)] (1) (tmps = MeSi(CH2PMe2)3) with a suitable organotetrel(ii)halide. Compound 1 was obtained from (NMe4)[Nb(CO)6] and the triphosphane tmps by photodecarbonylation. Reaction of 1 with the disilene E-Tbb(Br)Si[double bond, length as m-dash]Si(Br)Tbb in the presence of 4-dimethylaminopyridine afforded selectively the red-brown silylidyne complex [(κ3-tmps)(CO)2Nb[triple bond, length as m-dash]Si-Tbb] (2-Si, Tbb = 4-tert-butyl-2,6-bis(bis(trimethylsilyl)methyl)phenyl). Similarly, treatment of 1 with E(ArMes)Cl (E = Ge, Sn; ArMes = 2,6-mesitylphenyl) afforded after elimination of (NMe4)Cl and two CO ligands the deep magenta colored germylidyne complex [(κ3-tmps)(CO)2Nb[triple bond, length as m-dash]Ge-ArMes] (3-Ge), and the deep violet, light-sensitive stannylidyne complex [(κ3-tmps)(CO)2Nb[triple bond, length as m-dash]Sn-ArMes] (3-Sn), respectively. Formation of 3-Sn proceeds via the niobiastannylene [(κ3-tmps)(CO)3Nb-SnArMes] (4-Sn), which was detected by IR and NMR spectroscopy. The niobium tetrylidyne complexes 2-Si, 3-Ge and 3-Sn were fully characterized and their solid-state structures determined by single-crystal X-ray diffraction studies. All complexes feature an almost linear tetrel coordination and the shortest Nb-E bond lengths (d(Nb-Si) = 232.7(2) pm; d(Nb-Ge) = 235.79(4) pm; d(Nb-Sn) = 253.3(1) pm) reported to date. Reaction of 3-Ge with a large excess of H2O afforded upon cleavage of the Nb-Ge triple bond the hydridogermanediol Ge(ArMes)H(OH)2. Photodecarbonylation of [CpNb(CO)4] (Cp = η5-C5H5) in the presence of Ge(ArMes)Cl afforded the red-orange chlorogermylidene complex [Cp(CO)3Nb[double bond, length as m-dash]Ge(ArMes)Cl] (5-Ge). The molecular structure of 5-Ge features an upright conformation of the germylidene ligand, a trigonal-planar coordinated Ge atom, and a Nb-Ge double bond length of 251.78(6) pm, which lies in-between the Nb-Ge triple bond length of 3-Ge (235.79(4) pm) and a Nb-Ge single bond length (267.3 pm). Cyclic voltammetric studies of 2-Si, 3-Ge, and 3-Sn reveal several electron-transfer steps. One-electron oxidation and reduction of the germylidyne complex of 3-Ge in THF are electrochemically reversible suggesting that both the radical cation and radical anion of 3-Ge are accessible species in solution.

Trajectory selection in high harmonic generation by controlling the phase between orthogonal two-color fields.

We demonstrate control of short and long quantum trajectories in high harmonic emission through the use of an orthogonally polarized two-color field. By controlling the relative phase ϕ between the two fields we show via classical and quantum calculations that we can steer the two-dimensional trajectories to return, or not, to the core and so control the relative strength of the short or long quantum trajectory contribution. In experiments, we demonstrate that this leads to robust control over the trajectory contributions using a drive field from a femtosecond laser composed of the fundamental ω at 800 nm (intensity ∼1.2×10(14) W cm(-2)) and its weaker orthogonally polarized second harmonic 2ω (intensity ∼0.3×10(14) W cm(-2)) with the relative phase between the ω and 2ω fields varied simply by tilting a fused silica plate. This is the first demonstration of short and long quantum trajectory control at the single-atom level.

Enhancement of high harmonics generated by field steering of electrons in a two-color orthogonally polarized laser field.

We demonstrate enhancement by 1 order of magnitude of the high-order harmonics generated in argon by combining a fundamental field at 1300 nm (10(14) W cm(-2)) and its orthogonally polarized second harmonic at 650 nm (2 × 10(13) W cm(-2)) and by controlling the relative phase between them. This extends earlier work by ensuring that the main effect is the combined field steering the electron trajectory with negligible contribution from multiphoton effects compared to the previous schemes with 800/400 nm fields. We access a broad energy range of harmonics (from 20 eV to 80 eV) at a low laser intensity (far below the ionization saturation limit) and observe deep modulation of the harmonic yield with a period of π in the relative phase. Strong field theoretical analysis reveals that this is principally due to the steering of the recolliding electron wave packet by the two-color field. Our modeling also shows that the atto chirp can be controlled, leading to production of shorter pulses.