Screening and isolation of quorum sensing inhibitors of Pseudomonas aeruginosa from Phellodendron amurense extracts using bio-affinity chromatography.

Drug-resistant bacterial infections pose a significant challenge in the field of bacterial disease treatment. Finding new antibacterial pathways and targets to combat drug-resistant bacteria is crucial. The bacterial quorum sensing (QS) system regulates the expression of bacterial virulence factors. Inhibiting bacterial QS and reducing bacterial virulence can achieve antibacterial therapeutic effects, making QS inhibition an effective strategy to control bacterial pathogenicity. This article mainly focused on the PqsA protein in the QS system of Pseudomonas aeruginosa. An affinity chromatography medium was developed using the SpyTag/SpyCatcher heteropeptide bond system. Berberine, which can interact with the PqsA target, was screened from Phellodendron amurense by affinity chromatography. We characterized its structure, verified its inhibitory activity on P. aeruginosa, and preliminarily analyzed its mechanism using molecular docking technology. This method can also be widely applied to the immobilization of various protein targets and the effective screening of active substances.

Distinct maturation, glucose metabolism, and inflammatory function of human monocytes-derived IDECs mediated by anti-IgE and Pam3CSK4 alone or in combination.

Background: Cell energy metabolism controls the activation and function of dendritic cells (DCs). Inflammatory dendritic epidermal cells (IDECs) in skin lesions of atopic dermatitis (AD) express high-affinity IgE receptor (FcϵRI) and toll-like receptor 2 (TLR2), which mediate the generation and maintenance of inflammation. However, cellular energy metabolism and effector function of IDECs mediated by FcϵRI and TLR2 have not been fully elucidated. Methods: IDECs in vitro were treated with TLR2 agonist Pam3CSK4 and anti-IgE alone or in combination for 24 h. Further, we analyzed the expression of cell surface activation markers, production of inflammatory factors, and cellular energy metabolism profiles of IDECs by using flow cytometry, multiplex assay, RNA sequencing, targeted energy metabolism, and seahorse assays. Results: Compared to the unstimulated or anti-IgE groups, Pam3CSK4 alone or combined with anti-IgE groups significantly increased the expression of CD80, CD83, and CD86 on IDECs, but did not affect the expression of the above markers in the anti-IgE group. The release of inflammatory cytokines increased in the Pam3CSK4 alone or combined with anti-IgE groups, while there was a weak increasing trend in the anti-IgE group. The glycolysis/gluconeogenesis pathway of carbon metabolism was affected in all treatment groups. Furthermore, compared to the control group, we found a decrease in pyruvic acid, upregulation of PFKM, downregulation of FBP1, and increase in extracellular lactate, glycolysis rate, and glycolysis capacity after all treatments, while there was no difference between each treatment group. However, there was no difference in glycolytic reserve and mitochondrial basic and maximum respiration among all groups. Conclusion: Our results indicate that glycolysis of IDECs may be activated through FcϵRI and TLR2 to upregulate inflammatory factors, suggesting that danger signals from bacteria or allergens might evoke an inflammatory response from AD through the glycolysis pathway.

Non-invasive strategy: Developing a topical IL-4Rα-specific nanobody for the treatment of allergic airway diseases.

Inhibiting IL-4 and IL-13 are critical cytokines that induce the pathogenic responses of allergic airway diseases. Currently, monoclonal antibodies targeting IL-4Rα are administered subcutaneously to treat eosinophilic rhinosinusitis and allergic asthma. However, these treatments have several drawbacks. To address these issues, we have developed a novel IL-4Rα-targeting nanobody designed for non-invasive delivery to local inflammatory sites in allergic airway diseases. H5, selected via the ribosomal display applied screening from synthetic nanobody library, underwent dimerization and in-silico affinity maturation using AlphaFold2 and GROMACS resulting in a substantial/dramatic enhancement of its binding affinity. H5 effectively controlled inflammatory markers such as MUC5AC, CCL26, and FOXJ1 in human nasal epithelial cells (HNECs) by inhibiting IL-4 and IL-13 signaling. The bivalent form of H5 showed efficacy in easily accessible cells, such as multi-ciliated cells, while the monovalent variant targeted hard-to-reach cells, such as basal cells of HNECs. In summary, we developed a nanobody that could effectively inhibit inflammatory signaling in HNECs via intranasal administration, showing promise as a non-invasive rhinitis treatment.

Optimizing the affinity selection mass spectrometry workflow for efficient identification and ranking of potent USP1 inhibitors.

An optimized Affinity Selection-Mass Spectrometry (AS-MS) workflow has been developed for the efficient identification of potent USP1 inhibitors. USP1 was immobilized on agarose beads, ensuring low small molecule retention, efficient protein capture, and protein stability. The binding affinity of 49 compounds to USP1 was evaluated using the optimized AS-MS method, calculating binding index (BI) values for each compound. Biochemical inhibition assays validated the AS-MS results, revealing a potential correlation between higher BI values and lower IC50 values. This optimized workflow enables rapid identification of high-quality USP1 inhibitor hits, facilitating structure-activity relationship studies and accelerating the discovery of potential cancer therapeutics.

Peptide ligands for the universal purification of exosomes by affinity chromatography.

Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process-related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up-to 50-fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI-Toyopearl resin was finally employed in a two-step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.

Comparative in silico analysis of CNS-active molecules targeting the blood-brain barrier choline transporter for Alzheimer's disease therapy.

This study investigated the blood‒brain barrier (BBB) permeability of the central nervous system (CNS)-active compounds donepezil (DON), methionine (MET), and memantine (MEM) by employing a comprehensive in silico approach. These compounds are of particular interest for Alzheimer's disease (AD) therapy. Rigid-flexible molecular docking simulations indicated favorable binding affinities of all the compounds with BBB-ChT, with DON exhibiting the highest binding affinity (ΔGbind = -10.26 kcal/mol), predominantly mediated by significant hydrophobic interactions. In silico kinetic profiling suggested the stability of the DON/BBB-ChT complex, with ligand release prompted by conformational changes. 3D molecular alignment corroborated a minor conformational shift for DON in its minimal binding energy pose. Predictions indicated that active transport mechanisms notably enhance the brain distribution of donepezil compared to that of MET and MEM. Additionally, DON and MEM exhibited low mutagenic probabilities, while MET was identified as highly mutagenic. Overall, these findings highlight the potential of donepezil for superior BBB penetration, primarily through active transport mechanisms, underscoring the need for further validation through in vitro and in vivo studies for effective AD treatment. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00245-w.

Technological affinity index for interaction between lactic acid bacteria and Saccharomyces cerevisiae strains to modulate the fruity and loreal aroma of Catarratto wines.

Microbial interactions during the fermentation process influence the sensory characteristics of wines. Alongside alcoholic fermentation, malolactic fermentation also plays a crucial role in determining the aromatic traits of wines. The time (t), rate (m) and volatile organic compounds (VOCs) of malolactic fermentation are linked to the interaction between yeasts and lactic acid bacteria. The study investigated the interactions between Lactiplantibacillus plantarum or Oenococcus oeni with Saccharomyces cerevisiae by using the Technological Affinity Index (TAIndex). The co-inoculation of L. plantarum/S. cerevisiae resulted in a higher TAIndex than the co-inoculation of O. oeni/S. cerevisiae conditions. A low TAIndex led to increased aromaticity of the wines. The time and rate of malolactic fermentation have a strong impact on the synthesis of VOCs with a high olfactory impact. Therefore, knowledge of the TAIndex could play a decisive role in improving winemaking planning to produce wines with higher fruit and floral perceptions.

Fast on-rates of chimeric antigen receptors enhance the sensitivity to peptide MHC via antigen rebinding.

Chimeric antigen receptor (CAR) is a synthetic receptor that induces T cell-mediated lysis of abnormal cells. As cancer driver proteins are present at low levels on the cell surface, they can cause weak CAR reactivity, resulting in antigen sensitivity defects and consequently limited therapeutic efficacy. Although affinity maturation enhances the efficacy of CAR-T cell therapy, it causes off-target cross-reactions resulting in adverse effects. Preferentially expressed antigen in melanoma (PRAME) is an intracellular oncoprotein that is overexpressed in various tumors and restricted in normal tissues, except the testis. Therefore, PRAME could be an ideal target for cancer immunotherapy. In this study, we developed an experimental CAR system comprising six single-chain variable fragments that specifically recognizes the PRAMEp301/HLA-A*24:02 complex. Cell-mediated cytotoxicity was demonstrated using a panel of CARs with a wide range of affinities (KD = 10-10-10-7 M) and affinity modulation. CAR-T cells with fast on-rates enhance antigen sensitivity by accelerating the killing rates of these cells. Alanine scanning data demonstrated the potential of genetically engineered CARs to reduce the risk of cross-reactivity, even among CARs with high affinities. Given the correlation between on-rates and dwell time that occurs in rebinding and cell-mediated cytotoxicity, it is proposed that CAR-binding characteristics, including on-rate, play a pivotal role in the lytic capacity of peptide-major histocompatibility complex-targeting CAR-T cells, thus facilitating the development of strategies whereby genetically engineered CARs target intracellular antigens in cancer cells to lyse the cells.

Customizing Pore Structure and Lithiophilic Sites Dual-Gradient Free-Standing 3D Lithium-Based Anode to Enable Excellent Lithium Metal Batteries.

Developing 3D hosts is one of the most promising strategies for putting forward the practical application of lithium(Li)-based anodes. However, the concentration polarization and uniform electric field of the traditional 3D hosts result in undesirable "top growth" of Li, reduced space utilization, and obnoxious dendrites. Herein, a novel dual-gradient 3D host (GDPL-3DH) simultaneously possessing gradient-distributed pore structure and lithiophilic sites is constructed by an electrospinning route. Under the synergistic effect of the gradient-distributed pore and lithiophilic sites, the GDPL-3DH exhibits the gradient-increased electrical conductivity from top to bottom. Also, Li is preferentially and uniformly deposited at the bottom of the GDPL-3DH with a typical "bottom-top" mode confirmed by the optical and SEM images, without Li dendrites. Consequently, an ultra-long lifespan of 5250 h of a symmetrical cell at 2 mA cm-2 with a fixed capacity of 2 mAh cm-2 is achieved. Also, the full cells based on the LiFePO4, S/C, and LiNi0.8Co0.1Mn0.1O2 cathodes all exhibit excellent performances. Specifically, the LiFePO4-based cell maintains a high capacity of 136.8 mAh g-1 after 700 cycles at 1 C (1 C = 170 mA g-1) with 94.7% capacity retention. The novel dual-gradient strategy broadens the perspective of regulating the mechanism of lithium deposition.

A structural peculiarity of Antarctic fish IgM drives the generation of an engineered mAb by CRISPR/Cas9.

IgM is the major circulating Ig isotype in teleost fish, showing in Antarctic fish unique features such as an extraordinary long hinge region, which plays a crucial role in antibody structure and function. In this work, we describe the replacement of the hinge region of a murine monoclonal antibody (mAb) with the peculiar hinge from Antarctic fish IgM. We use the CRISPR/Cas9 system as a powerful tool for generating the engineered mAb. Then, we assessed its functionality by using an innovative plasmonic substrate based on bimetallic nanoislands (AgAuNIs). The affinity constant of the modified mAb was 2.5-fold higher than that obtained from wild-type mAb against the specific antigen. Here, we show the suitability of the CRISPR/Cas9 method for modifying a precise region in immunoglobulin gene loci. The overall results could open a frontier in further structural modifications of mAbs for biomedical and diagnostic purposes.

Harnessing sulfur-binding domains to separate Sp and Rp isomers of phosphorothioate oligonucleotides.

Chemical synthesis of phosphoromonothioate oligonucleotides (PS-ONs) is not stereo-specific and produces a mixture of Rp and Sp diastereomers, whose disparate reactivity can complicate applications. Although the current methods to separate these diastereomers which rely on chromatography are constantly improving, many Rp and Sp diastereomers are still co-eluted. Here, based on sulfur-binding domains that specifically recognize phosphorothioated DNA and RNA in Rp configuration, we developed a universal separation system for phosphorothioate oligonucleotide isomers using immobilized SBD (SPOIS). With the scalable SPOIS, His-tagged SBD is immobilized onto Ni-nitrilotriacetic acid-coated magnetic beads to form a beads/SBD complex, Rp isomers of the mixture can be completely bound by SBD and separated from Sp isomers unbound in liquid phase, then recovered through suitable elution approach. Using the phosphoromonothioate single-stranded DNA as a model, SPOIS separated PS-ON diastereomers of 4 nt to 50 nt in length at yields of 60-90% of the starting Rp isomers, with PS linkage not locating at 5' or 3' end. Within this length range, PS-ON diastereomers that co-eluted in HPLC could be separated by SPOIS at yields of 84% and 89% for Rp and Sp stereoisomers, respectively. Furthermore, as each Rp phosphorothioate linkage can be bound by SBD, SPOIS allowed the separation of stereoisomers with multiple uniform Sp configurations for multiple phosphorothioate modifications. A second generation of SPOIS was developed using the thermolabile and non-sequence-specific SBDPed, enabling fast and high-yield recovery of PS substrate stereoisomers for the DNAzyme Cd16 and further demonstrating the efficiency of this method. KEY POINTS: • SPOIS allows isomer separations of the Rp and Sp isomers co-eluted on HPLC. • SPOIS can obtain Sp isomers with 5 min and Rp in 20 min from PS-ON diastereomers. • SPOIS was successfully applied to separate isomers of PS substrates of DNAzyme.

Virtual Screening and Validation of Affinity DNA Functional Ligands for IgG Fc Segment.

The effective attachment of antibodies to the immune sensing interface is a crucial factor that determines the detection performance of immunosensors. Therefore, this study aims to investigate a novel antibody immobilization material with low molecular weight, high stability, and excellent directional immobilization effect. In this study, we employed molecular docking technology based on the ZDOCK algorithm to virtually screen DNA functional ligands (DNAFL) for the Fc segment of antibodies. Through a comprehensive analysis of the key binding sites and contact propensities at the interface between DNAFL and IgG antibody, we have gained valuable insights into the affinity relationship, as well as the principles governing amino acid and nucleotide interactions at this interface. Furthermore, molecular affinity experiments and competitive binding experiments were conducted to validate both the binding ability of DNAFL to IgG antibody and its actual binding site. Through affinity experiments using multi-base sequences, we identified bases that significantly influence antibody-DNAFL binding and successfully obtained DNAFL with an enhanced affinity towards the IgG Fc segment. These findings provide a theoretical foundation for the targeted design of higher-affinity DNAFLs while also presenting a new technical approach for immunosensor preparation with potential applications in biodetection.

Functional analysis on the role of HvHKT1.4 in barley (Hordeum vulgare L.) salinity tolerance.

High-affinity potassium transporters (HKTs) are well known proteins that govern the partitioning of Na+ between roots and shoots. Six HvHKTs were identified in barley and designated as HvHKT1.1, HvHKT1.3, HvHKT1.4, HvHKT1.5, HvHKT2.1 and HvHKT2.2 according to their similarity to previously reported OsHKTs. Among these HvHKTs, HvHKT1.4 was highly up-regulated under salinity stress in both leaves and roots of Golden Promise. Subcellular localization analysis showed that HvHKT1.4 is a plasma-membrane-localized protein. The knockout mutants of HvHKT1.4 showed greater salinity sensitivity and higher Na+ concentration in leaves than wild-type plants. Haplotype analysis of HvHKT1.4 in 344 barley accessions showed 15 single nucleotide substitutions in the CDS region, belonging to five haplotypes. Significant differences in mean salinity damage scores, leaf Na+ contents and Na+/K+ were found between Hap5 and other haplotypes with Hap5 showing better salinity tolerance. The results indicated that HvHKT1.4 can be an effective target in improving salinity tolerance through ion homeostasis.

Continuous Biosensor Based on Particle Motion: How Does the Concentration Measurement Precision Depend on Time Scale?

Continuous biosensors measure concentration-time profiles of biomolecular substances in order to allow for comparisons of measurement data over long periods of time. To make meaningful comparisons of time-dependent data, it is essential to understand how measurement imprecision depends on the time interval between two evaluation points, as the applicable imprecision determines the significance of measured concentration differences. Here, we define a set of measurement imprecisions that relate to different sources of variation and different time scales, ranging from minutes to weeks, and study these using statistical analyses of measurement data. The methodology is exemplified for Biosensing by Particle Motion (BPM), a continuous, affinity-based sensing technology with single-particle and single-molecule resolution. The studied BPM sensor measures specific small molecules (glycoalkaloids) in an industrial food matrix (potato fruit juice). Measurements were performed over several months at two different locations, on nearly 50 sensor cartridges with in total more than 1000 fluid injections. Statistical analyses of the measured signals and concentrations show that the relative residuals are normally distributed, allowing extraction and comparisons of the proposed imprecision parameters. The results indicate that sensor noise is the most important source of variation followed by sample pretreatment. Variations caused by fluidic transport, changes of the sensor during use (drift), and variations due to different sensor cartridges and cartridge replacements appear to be small. The imprecision due to sensor noise is recorded over few-minute time scales and is attributed to stochastic fluctuations of the single-molecule measurement principle, false-positive signals in the signal processing, and nonspecific interactions. The developed methodology elucidates both time-dependent and time-independent factors in the measurement imprecision, providing essential knowledge for interpreting concentration-time profiles as well as for further development of continuous biosensing technologies.

Affinity-independent memory B cell origin of the early antibody-secreting cell response in naive individuals upon SARS-CoV-2 vaccination.

Memory B cells (MBCs) formed over the individual's lifetime constitute nearly half of the circulating B cell repertoire in humans. These pre-existing MBCs dominate recall responses to their cognate antigens, but how they respond to recognition of novel antigens is not well understood. Here, we tracked the origin and followed the differentiation paths of MBCs in the early anti-spike (S) response to mRNA vaccination in SARS-CoV-2-naive individuals on single-cell and monoclonal antibody levels. Pre-existing, highly mutated MBCs showed no signs of germinal center re-entry and rapidly developed into mature antibody-secreting cells (ASCs). By contrast, and despite similar levels of S reactivity, naive B cells showed strong signs of antibody affinity maturation before differentiating into MBCs and ASCs. Thus, pre-existing human MBCs differentiate into ASCs in response to novel antigens, but the quality of the humoral and cellular anti-S response improved through the clonal selection and affinity maturation of naive precursors.

BCR signaling in germinal center B cell selection.

When mature B cells are activated by antigens, the selection of these activated B cells takes place particularly during T cell-dependent immune responses in which an improved antibody repertoire is generated through somatic hypermutation in germinal centers (GCs). In this process the importance of antigen presentation by GC B cells, and subsequent T follicular helper (Tfh) cell help in positive selection of GC B cells, has been well appreciated. By contrast, the role of B cell receptor (BCR) signaling per se remains unclear. Strong experimental support for the involvement of BCR signaling in GC B cell selection has now been provided. Interestingly, these studies suggest that several checkpoints operating through the BCR ensure affinity maturation.

Joint Screening and Identification of Potential Targets of Nitazoxanide by Affinity Chromatography and Label-Free Techniques.

BACKGROUND: Nitazoxanide not only exhibits a broad spectrum of activities against various pathogens infecting animals and humans but also induces cellular autophagy. Currently, the pattern of action and subcellular targets of nitazoxanide-induced cellular autophagy are still unclear. METHODS: To identify potential targets of nitazoxanide in mammalian cells, we developed an af-finity chromatography system using tizoxanide, a deacetyl derivative of nitazoxanide, as a ligand. Affinity chromatography was performed using VERO cell extracts on tizoxanide-biotin, and the isolated binding proteins were identified by mass spectrometry. Candidate target proteins ob-tained using affinity chromatography were co-analysed with the drug affinity response target sta-bility method. Fluorescent probes obtained by coupling rhodamine B to nitazoxanide were used for intracellular localisation of the binding targets. Solvent-induced protein precipitation profiling and thermal proteome profiling were used to further validate the binding proteins. RESULTS: The joint analysis of the drug affinity response target stability method and affinity chro-matography resulted in the screening of six possible candidate target proteins. Fluorescent probes localised the nitazoxanide-binding protein around the nuclear membrane. Molecular docking re-vealed that the binding proteins mainly formed hydrogen bonds with the nitro group of nitazoxa-nide. Solvent-induced protein precipitation profiling and thermal proteome profiling further vali-dated SEC61A, PSMD12, and PRKAG1 as potential target proteins of nitazoxanide. CONCLUSION: The data supports the idea that nitazoxanide is a multifunctional compound with multiple targets.

2D Metal-Oxide Nanosheets as a Homogeneous Li-Ion Flux Regulator for High-Performance Anodeless Lithium Metal Batteries.

The anodeless battery design has recently gained significant interest by eliminating the direct use of a thick lithium (Li) foil. However, it suffers from inhomogeneous Li+ flux, resulting in dendrite growth and a short cycling life. To address this, the exfoliation of layered-structure titanium oxide to 2D nanosheets (2DTiOx) is proposed to precisely control Li+ flux at the atomic scale by maximizing Li+ affinitive Ti sites. Compared to cells without these nanosheets, the Li|2DTiOx|Cu half-cell demonstrates stable cyclability over 900 cycles, with a Coulombic efficiency (CE) over 99% at 0.5 mA cm-2 and 0.5 mAh cm-2. Similarly, a long stable cycling life over 1500 h at 1.0-3.0 mA cm-2 is observed for a 2DTiOx-based symmetric cell containing a limited Li amount from electrodeposited Li metal (e-Li|2DTiOx|e-Li). The full cells (e-Li|2DTiOx||NCM811 and e-Li|2DTiOx||LFP) coupled with NCM811 and LFP cathodes showed a long cycle life of 400 cycles at 1.0 C and 0.5 C, respectively. The exceptional battery performance is attributed to the uniform Li disposition on the 2DTiOx electrode, emphasizing the crucial role of the exposed basal plane in 2DTiOx as an efficient atomic scale Li+ flux regulator. This strategy is expected to advance next-generation lithium metal batteries (LMBs) by highlighting the significance of Li+ affinity at the Ti sites of 2DTiOx nanosheets.

Secretome analysis using Affinity Proteomics and Immunoassays: a focus on Tumor Biology.

The study of the cellular secretome using proteomic techniques continues to capture the attention of the research community across a broad range of topics in biomedical research. Due to their untargeted nature, independence from the model system employed, historically superior depth of analysis, as well as comparative affordability, mass spectrometry-based approaches traditionally dominate such analyses. More recently, however, affinity-based proteomic assays have massively gained in analytical depth, which together with their high sensitivity, dynamic range coverage as well as high throughput capabilities render them exquisitely suited to secretome analysis. In this review, we revisit the analytical challenges implied by secretomics and provide an overview of affinity-based proteomic platforms currently available for such analyses, using the study of the tumor secretome as an example for basic and translational research.

Carbon Suboxide, C2O3: A hidden σ0π2 Carbene.

Electronic structure of carbon suboxide, C3O2, has been recently described as OC→C←CO having two lone pairs at the central carbon atom, thereby called as "Carbones". Although it has a linear geometry, the presence of two lone pairs comes to fore when its reactivity is analyzed with two protons. However, no attention had been paid on its alternating reactivity with hydride ions. Herein, detailed quantum chemical calculations predict that carbon suboxide can also have significant hydride ion affinity. This reactivity is in tune with σ0π2 carbene character of carbon suboxide. This study also shows that such a σ0π2 carbene character is also prevalent in carbodiphosphorane, C(PH3)2. This bonding situation has been hitherto unexplored in "Carbone" chemistry.