Mechanism Of Neutralization Research Articles

Strategy-proof and envy-free mechanisms for house allocation

We consider the problem of allocating indivisible objects to agents when agents have strict preferences over objects. There are inherent trade-offs between competing notions of efficiency, fairness and incentives in such assignment mechanisms. It is, therefore, natural to consider mechanisms that satisfy two of these three properties in their strongest notions, while trying to improve on the third dimension. In this paper, we are motivated by the following question: Is there a strategy-proof and envy-free random assignment mechanism more efficient than equal division?Our contributions in this paper are twofold. First, we further explore the incompatibility between efficiency, fairness, and strategy-proofness within random assignment mechanisms. We define a new notion of efficiency that is weaker than ex-post efficiency and prove that any strategy-proof and envy-free mechanism must sacrifice efficiency even in this very weak sense. Next, we introduce a new family of mechanisms called Rank Exchange mechanisms that are strategy-proof and envy-free and stochastically dominate equal division. Further, we show that Rank Exchange mechanisms characterize the set of neutral, strategy-proof, and envy-free mechanisms that also satisfy a natural separability axiom that may be of independent interest.

Development and characterization of phage display-derived anti-toxin antibodies neutralizing TcdA and TcdB of Clostridioides difficile.

TcdA and TcdB are known as the major virulence attributes of Clostridioides difficile. Hence, neutralizing the TcdA and TcdB activities can be considered as an efficient therapeutic approach against C. difficile infection (CDI). In this work, we utilized phage display technique to select single-chain fragment variable (scFv) fragments as recombinant antibodies displayed on the surface of phages, which specifically target native TcdA, or TcdB (nTcdA and nTcdB), and their recombinant C-terminal combined repetitive oligopeptide (CROP) domains (rTcdA and rTcdB). After three rounds of biopanning, abundance of phage clones displaying high reactivity with TcdA or TcdB was quantified through enzyme-linked immunosorbent assay (ELISA). Furthermore, selected scFvs were characterized by cell viability and neutralization assays. The gene expression of immunological markers, IL-8 and TNF-α, was examined in treated Caco-2 cells by RT-qPCR. The epitopes of neutralizing scFvs were also identified by molecular docking. Totally, 18 scFv antibodies (seven for TcdA and 11 for TcdB) were identified by ELISA. Among selected scFvs, two clones for TcdA (rA-C2, A-C9) and three clones for TcdB (rB-B4, B-F5, B-F11) exhibited the highest neutralizing activity in Caco-2 and Vero cells. Moreover, the cocktail of anti-TcdA and anti-TcdB antibodies notably decreased the mRNA expression of TNF-α and IL-8 in Caco-2 cells. Molecular docking revealed that the interaction between scFv and toxin was mostly restricted to CROP domain of TcdA or TcdB. Our results collectively provided more insights for the development of neutralizing scFvs against C. difficile toxins using phage display. Further research is needed to meticulously evaluate the potential of scFvs as an alternative treatment for CDI using animal models and clinical trials.IMPORTANCETargeting the major toxins of Clostridioides difficile by neutralizing antibodies is a novel therapeutic approach for CDI. Here, we report a panel of new anti-TcdA (rA-C2, A-C9) and anti-TcdB (rB-B4, B-F5, and B-F11) recombinant antibody fragments (scFvs) isolated from Tomlinson I and J libraries using phage display technique. These scFv antibodies were capable of neutralizing their respective toxin and showed promise as potential therapeutics against TcdA and TcdB of C. difficile in different in vitro models. In addition, in silico analysis showed that at least two neutralization mechanisms, including inhibiting cell surface binding of toxins and inhibiting toxin internalization can be proposed for the isolated scFvs in this work. These findings provide more insights for the applicability of specific scFvs toward C. difficile toxins at in vitro level. However, further research is required to evaluate the potential application of these scFvs as therapeutic agents for CDI treatment in clinical setting.

Structure-based design of a strain transcending AMA1-RON2L malaria vaccine

Apical membrane antigen 1 (AMA1) is a key malaria vaccine candidate and target of neutralizing antibodies. AMA1 binds to a loop in rhoptry neck protein 2 (RON2L) to form the moving junction during parasite invasion of host cells, and this complex is conserved among apicomplexan parasites. AMA1-RON2L complex immunization achieves higher growth inhibitory activity than AMA1 alone and protects mice against Plasmodium yoelii challenge. Here, three single-component AMA1-RON2L immunogens were designed that retain the structure of the two-component AMA1-RON2L complex: one structure-based design (SBD1) and two insertion fusions. All immunogens elicited high antibody titers with potent growth inhibitory activity, yet these antibodies did not block RON2L binding to AMA1. The SBD1 immunogen induced significantly more potent strain-transcending neutralizing antibody responses against diverse strains of Plasmodium falciparum than AMA1 or AMA1-RON2L complex vaccination. This indicates that SBD1 directs neutralizing antibody responses to strain-transcending epitopes in AMA1 that are independent of RON2L binding. This work underscores the importance of neutralization mechanisms that are distinct from RON2 blockade. The stable single-component SBD1 immunogen elicits potent strain-transcending protection that may drive the development of next-generation vaccines for improved malaria and apicomplexan parasite control.

Inhalable Nanofitin demonstrates high neutralization of SARS-CoV-2 virus via direct application in respiratory tract

Nanofitins are small and hyperthermostable alternative protein scaffolds that display physicochemical properties making them suitable for the development of topical therapeutics, notably for the treatment of pulmonary infectious diseases. Local administration of biologics to the lungs involves a particularly stressful step of nebulization that is poorly tolerated by most antibodies, which limits their application by this delivery route. During the COVID-19 pandemic, we generated anti-SARS-CoV-2 monomeric Nanofitins of high specificity for the spike protein. Hit Nanofitin candidates were identified based on their binding properties with punctual spike mutants and assembled into a linear multimeric construction constituting of four different Nanofitins, allowing the generation of a highly potent anti-SARS-CoV-2 molecule. The therapeutic efficacy of the multimeric assembly was demonstrated both in invitro and invivo models. Interestingly, the neutralization mechanism of the multimeric construction seems to involve a particular conformation switch of the spike trimer. In addition, we reported the stability and the conserved activity of the tetrameric construction after nebulization. This advantageous developability feature for pulmonary administration associated with the ease of assembly, as well as the fast generation process position the Nanofitin technology as a potential therapeutic solution for emerging infectious diseases.

A quaternary ammonium salt grafted tannin-based flocculant boosts the conjugative transfer of plasmid-born antibiotic resistance genes: The nonnegligible side of their flocculation-sterilization properties

This study developed dual-function tannin-based flocculants, namely tannin-graft-acrylamide-diallyl dimethyl ammonium chloride (TGCC-A/TGCC-C), endowed with enhanced flocculation-sterilization properties. The impacts of these flocculants on proliferation and transformation of antibiotic resistance genes (ARGs) among bacteria during the flocculation-deposition process were examined. TGCC-A/TGCC-C exhibited remarkable flocculation capacities towards both Escherichia coli and Staphylococcus aureus, encompassing a logarithmic range of initial cell density (108–109 CFU/mL) and a broad pH spectrum (pH 2–11). The grafted quaternary ammonium salt groups played pivotal parts in flocculation through charge neutralization and bridging mechanisms, concurrently contributing to sterilization by disrupting cellular membranes. The correlation between flocculation and sterilization entails a sequential progression, where an excess of TGCC, initially employed for flocculation, is subsequently consumed for sterilization purposes. The frequencies of ARGs conjugative transfer were enhanced in bacterial flocs across all TGCC treatments, stemming from augmented bacterial aggregation and cell membrane permeability, elicited stress response, and up-regulated genes encoding plasmid transfer. These findings underscore the indispensable role of flocculation-sterilization effects in mediating the propagation of ARGs, consequently providing substantial support for the scientific evaluation of the environmental risks associated with flocculants in the context of ARGs dissemination during the treatment of raw water featuring high bacterial density.

The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin–antitoxin and related phage defense systems

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.

Structures and immune recognition of Env trimers from two Asia prevalent HIV-1 CRFs

Structure-guided immunofocusing HIV-1 vaccine design entails a comprehensive understanding of Envs from diverse HIV-1 subtypes, including circulating recombinant forms (CRFs). Here, we present the cryo-EM structures of Envs from two Asia prevalent CRFs (CRF01_AE and CRF07_BC) at 3.0 and 3.5 Å. We compare the structures and glycosylation patterns of Envs from different subtypes and perform cross-clade statistical analyses to reveal the unique features of CRF01_AE V1 region, which are associated with the resistance to certain bNAbs. We also solve a 4.1 Å cryo-EM structure of CRF01_AE Env in complex with F6, the first bNAb from CRF01_AE-infected individuals. F6 recognizes a gp120-gp41 spanning epitope to allosterically destabilize the Env trimer apex and weaken inter-protomer packing, which in turn hinders the receptor binding and induces Env trimer disassembly, demonstrating a dual mechanism of neutralization. These findings broaden our understanding of CRF Envs and shed lights on immunofocusing HIV-1 vaccine design.

Potent cross-neutralization of respiratory syncytial virus and human metapneumovirus through a structurally conserved antibody recognition mode

Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) infections pose a significant health burden. Using pre-fusion conformation fusion (F)proteins, we isolated a panel of anti-F antibodies from a human donor. One antibody (RSV-199) potently cross-neutralized 8 RSV and hMPV strains by recognizing antigenic site III, which is partially conserved in RSV and hMPV F. Next, we determined the cryoelectron microscopy (cryo-EM) structures of RSV-199 bound to RSV F trimers, hMPV F monomers, and an unexpected dimeric form of hMPV F. These structures revealed how RSV-199 engages both RSV and hMPV F proteins through conserved interactions of the antibody heavy-chain variable region and how variability within heavy-chain complementarity-determining region 3 (HCDR3) can be accommodated at the F protein interface in site-III-directed antibodies. Furthermore, RSV-199 offered enhanced protection against RSV A and B strains and hMPV in cotton rats. These findings highlight the mechanisms of broad neutralization and therapeutic potential of RSV-199.

Exploring the acid neutralizing effect in rainwater collected at a tropical urban area: Central Valley, Costa Rica

We report on the chemical and the carbon isotopic composition of dissolved inorganic carbon (DIC) of rainwater collected between May and October 2020 in the Central Valley, Costa Rica. Precipitation samples were collected daily (N = 55) and analyzed for major ions, DIC, and δ13CDIC. Significant correlation (p < 0.05) between main acidic (SO42− and NO3−) and major alkaline (Ca2+ and NH4+) species confirmed a very effective acid neutralization effect in rainwater (average pH: 5.90 ± 0.74). Significant temporal variations (p < 0.05) of δ13CDIC indicated the contribution of carbonate salts in rainwater from May to October but also CO2 dissolution at the beginning of the wet season (May), probably due to increased CO2 emissions from soil degassing. Temporal changes of Ca2+ neutralization factors followed the observed changes in δ13CDIC, which confirmed the high buffer capacity of precipitation in our study. HYSPLIT analysis also revealed long-range contributions of pedogenetic carbonates (e.g., Saharan dust) responsible for the acid neutralization capacity of rainwater (e.g., from July to September). Principal component analysis showed that four main factors explain 65% of the variance are: i) acid neutralization processes (Ca2+ neutralization factor), ii) marine salts (Cl−, Na+), iii) fossil fuels (SO42−, NO3−), and iv) agriculture/fertilizers (NO3−, NH4+, K+). Our study demonstrated that a combined approach of chemical, isotope, and statistical analysis techniques can help unravel the mechanism of acid neutralization of rainwater in tropical urban areas. This information has strong implications for future studies related with the impact of acid deposition on ecosystem functioning, water quality, and infrastructure degradation.

A chart to evaluate the effect of active control on a bridge

To reinforce and improve the function of a light bridge for disaster relief, a neutral equilibrium mechanism (NEM) was developed and applied for controlling the internal force and reducing the vertical deformation of a bridge to form as virtual piers. The control force of the NEM was generated by a proportional–integral–derivative controller with a combination of displacement GP, speed GD and adjusted GI gain coefficients to neutralise the vertical deformation of a bridge that had been created by a moving live load and the dead weight of the bridge. To effectively evaluate the control efficiency of bridge deformation under this mechanism, a multi-dimensional quality evaluation method is proposed to assess the control benefits of bridge deformation under different combinations of control coefficients. The test and analysis results in the control performance analysis chart show clearly that the control performance index MTIi is around 2–3, 3 and 5, respectively, for the combination of: GP = 0.5, GD = 0.0, GI = 0.02; GP = 1.0, GD = 0.0, GI = 0.02; and GP = 2.0, GD = 0.0, GI = 0.02. The analysis results present a clear demonstration of control performance, which can serve as a reference for future practical control safety.

Bottom Hadrochemistry in High-Energy Hadronic Collisions.

The hadrochemistry of bottom quarks (b) produced in hadronic collisions encodes valuable information on the mechanism of color neutralization in these reactions. Since the b-quark mass is much larger than the typical hadronic scale of ∼1 GeV, bb[over ¯] pair production is expected to be well separated from subsequent hadronization processes. A significantly larger fraction of b baryons has been observed in proton-proton (pp) and proton-antiproton (pp[over ¯]) reactions relative to e^{+}e^{-} collisions, challenging theoretical descriptions. We address this problem by employing a statistical hadronization approach with an augmented set of b-hadron states beyond currently measured ones, guided by the relativistic quark model and lattice-QCD computations. Assuming relative chemical equilibrium between different b-hadron yields, thermal densities are used as fragmentation weights of b quarks into various hadron species. With quark model estimates of the decay patterns of excited states, the fragmentation fractions of weakly decaying b hadrons are computed and found to agree with measurements in pp[over ¯] collisions at the Tevatron. By combining transverse-momentum (p_{T}) distributions of b quarks from perturbative QCD with thermal weights and independent fragmentation toward high p_{T}, a fair description of the p_{T}-dependent B[over ¯]_{s}^{0}/B^{-} and Λ_{b}^{0}/B^{-} ratios measured in pp collisions at the LHC is obtained. The observed enhancement of Λ_{b}^{0} production is attributed to the feeddown from thus far unobserved excited b baryons. Finally, we implement the hadrochemistry into a strongly coupled transport approach for b quarks in heavy-ion collisions, utilizing previously determined b-quark transport coefficients in the quark-gluon plasma, to highlight the modifications of hadrochemistry and collective behavior of b hadrons in Pb-Pb collisions at the LHC.

Catalytic spike antibodies: A new paradigm in neutralization

In this issue, McConnell etal.10 demonstrate that COVID-19 convalescent plasma (CCP)-derived antibodies can neutralize SARS-CoV-2 by proteolytically cleaving the spike protein. The CCP antibody-mediated catalysis has broader implications beyond COVID-19 and can be applicable in understanding the mechanism of antibody-based neutralization of different pathogens.

Development of a monoclonal antibody recognizing novel linear neutralizing epitope on H protein of canine distemper virus vaccine strains (America-1 genotype)

Canine distemper virus (CDV) is an economically important virus responsible for canine distemper (CD), a highly contagious disease that afflicts various animal species worldwide. The hemagglutinin (H) protein is the major neutralizing target of virus. Therefore, it is often considered as immunogen to prepare neutralizing antibodies. The accurate identification of neutralizing epitope will provide important antigenic information and extend the knowledge of mechanisms of virus neutralization. In this study, we generated a neutralizing monoclonal antibody (mAb) 4C6 against CDV H protein, and defined the minimal linear epitope 238DIEREFDT245, which was highly conserved in America-1 genotype of CDV strains (vaccines). The mAb 4C6 could not react with a CDV strain that had two substitutions of D238Y and R241G in the epitope, which appeared in most CDV strains of the other genotypes. Besides, a few different amino acid mutations in the epitope were also included. Collectively, the epitope 238DIEREFDT245 was variable in the other genotypes of CDV strains. The epitope 238DIEREFDT245 was exposed to the surface of CDV H protein, showing good antigenicity. These data will provide insights into structure, function and antigenicity of H protein and lay the foundation for the development of diagnostic technologies and vaccine design for CDV.

Spike-protein proteolytic antibodies in COVID-19 convalescent plasma contribute to SARS-CoV-2 neutralization

Understanding the mechanisms of antibody-mediated neutralization of SARS-CoV-2 is critical in combating the COVID-19 pandemic. Based on previous reports of antibody catalysis, we investigated the proteolysis of spike (S)by antibodies in COVID-19 convalescent plasma (CCP) and its contribution to viral neutralization. Quenched fluorescent peptides were designed based on S epitopes to sensitively detect antibody-mediated proteolysis. We observed epitope cleavage by CCP from different donors which persisted when plasma was heat-treated or when IgG was isolated from plasma. Further, purified CCP antibodies proteolyzed recombinant S domains, as well as authentic viral S. Cleavage of S variants suggests CCP antibody-mediated proteolysis is a durable phenomenon despite antigenic drift. We differentiated viral neutralization occurring via direct interference with receptor binding from that occurring by antibody-mediated proteolysis, demonstrating that antibody catalysis enhanced neutralization. These results suggest that antibody-catalyzed damage of S is an immunologically relevant function of neutralizing antibodies against SARS-CoV-2.

The Utilization of Pectin as Natural Coagulant-Aid in Congo Red Dye Removal

Coagulation using inorganic compounds such as aluminum sulfate is commonly used in water-wastewater treatment. However, there are some drawbacks to its utilization, such as a significant decrease in the treated water’s pH, non-biodegradable sludge, and a potential negative impact on human mental health (dementia and Alzheimer's). The use of inorganic coagulants can be minimized with the addition of natural-based coagulant-aid such as pectin. In this study, Congo red solution, a model dye substance, was coagulated by varying the pH (3–7) using alum coagulant to determine the best pH for coagulation. At the best pH, pectin was introduced at various doses (0–30 mg/L), and subsequently various dye concentrations (50–100 mg/L). The effect of pectin as coagulant-aid was compared with aluminum sulfate and pectin only; with a response of %removal and sludge volume. It was found that the Congo red dye coagulation had the best %removal at pH 6 indicating a charge neutralization mechanism. The addition of 15 mg/L pectin at an aluminum sulfate dose of 30 mg/L resulted in 97.7% dye removal with a sludge volume of 14 mL/L at a Congo red concentration of 50 mg/L. This value is higher compared to those of aluminum sulfate and pectin only which gave 75.6 and 3.19% removals, respectively. Furthermore, the addition of pectin as a natural coagulant-aid could halve the sludge volume due to the formation of denser flocs. The results show a promising potential of pectin as a natural coagulant-aid in water-wastewater treatment.

Phenotypic differentiation in populations of a gladiator tree frog: environment, genetic drift and sexual selection

Abstract Phenotypic differentiation among animal populations is common, yet few studies have simultaneously examined the adaptive and neutral mechanisms behind it. Such evolutionary processes become more relevant in species with complex behaviours that undergo global and local selective pressures throughout their geographical range. Here we measured and compared morphological and acoustic variation across the distribution range of a Neotropical gladiator tree frog that shows elaborate reproduction (territoriality, complex courtship and female choice). We then incorporated molecular and landscape data to examine the roles of sexual selection, genetic drift and acoustic adaptation to the environment in call differentiation, i.e. the acoustic adaptation hypothesis (AAH). We found that calls varied more than morphology among populations, but differences in calls or morphological traits were not explained by genetic differentiation. We found no evidence for the AAH, but a significant relationship in the opposite direction regarding call frequencies suggests an indirect role of sexual selection. Differentiation on call traits that are associated with individual discrimination and/or female attraction also corroborated an important role of sexual selection. We show that multitrait and multimechanism approaches can elucidate intricate processes leading to phenotypic variation among individuals and populations. We emphasize that studies of species with complex reproductive behaviours across their range may provide insights into different selective pressures leading to phenotypic differentiation.

Neutralization evolution of concrete under acid rain and carbonation erosion: a review

Concrete structures in atmospheric environments are constantly affected by acid rain and carbon dioxide erosion, and the evolution of concrete neutralization is an important factor influencing the long-term performance of the material. To deepen our understanding of the long-term performance evolution mechanisms of concrete, this study reviews the mechanism of acid rain and carbonation neutralization, summarizes studies regarding the evolution of the macroscopic properties and microstructures of concrete, and discusses the internal and external influencing factors and prediction models for concrete neutralization evolution. A thorough analysis of existing studies reveals that the microscopic properties and microstructures of concrete undergo a process of optimization followed by decay in response to acid rain neutralization. The expansion products from acid rain can lead to the deterioration of concrete microstructure, whereas carbonation neutralization products enhance microstructural compactness. Moreover, the study found that the degree of concrete neutralization increases with an increase in the water-cement ratio (W/C). Furthermore, the maximal degree of concrete neutralization occurs within a relative humidity ranging from 45% to 65%. According to the diffusion theory, the process of concrete neutralization is an evolutionary one that operates from the outside to the inside of the material and is influenced by various factors. The aim of this review is to provide scholars with a systematic understanding of the evolution of concrete neutralization under acid rain and carbonation, and to establish a theoretical foundation for future research in this area. Potential areas for future research on concrete neutralization are also described in this review.

Assessing neutral transport mechanisms in aspect ratio dependent etching by means of experiments and multiscale plasma modeling

Since the onset of pattern transfer technologies for chip manufacturing, various strategies have been developed to circumvent or overcome aspect ratio dependent etching (ARDE). These methods have, however, their own limitations in terms of etch non-idealities, throughput or costs. Moreover, they have mainly been optimized for individual in-device features and die-scale patterns, while occasionally ending up with poor patterning of metrology marks, affecting the alignment and overlay in lithography. Obtaining a better understanding of the underlying mechanisms of ARDE and how to mitigate them therefore remains a relevant challenge to date, for both marks and advanced nodes. In this work, we accordingly assessed the neutral transport mechanisms in ARDE by means of experiments and multiscale modeling for SiO2 etching with CHF3/Ar and CF4/Ar plasmas. The experiments revealed a local maximum in the etch rate for an aspect ratio around unity, i.e. the simultaneous occurrence of regular and inverse reactive ion etching lag for a given etch condition. We were able to reproduce this ARDE trend in the simulations without taking into account charging effects and the polymer layer thickness, suggesting shadowing and diffuse reflection of neutrals as the primary underlying mechanisms. Subsequently, we explored four methods with the simulations to regulate ARDE, by varying the incident plasma species fluxes, the amount of polymer deposition, the ion energy and angular distribution and the initial hardmask sidewall angle, for which the latter was found to be promising in particular. Although our study focusses on feature dimensions characteristic to metrology marks and back-end-of-the-line integration, the obtained insights have a broader relevance, e.g. to the patterning of advanced nodes. Additionally, this work supports the insight that physisorption may be more important in plasma etching at room temperature than originally thought, in line with other recent studies, a topic on which we recommend further research.

Phylogeny-Related Variations in Venomics: A Test in a Subset of Habu Snakes (Protobothrops).

We conducted a comparative analysis to unveil the divergence among venoms from a subset of Old World habu snakes (Protobothrops) in terms of venomic profiles and toxicological and enzymatic activities. A total of 14 protein families were identified in the venoms from these habu snakes, and 11 of them were shared among these venoms. The venoms of five adult habu snakes were overwhelmingly dominated by SVMP (32.56 ± 13.94%), PLA2 (22.93 ± 9.26%), and SVSP (16.27 ± 4.79%), with a total abundance of over 65%, while the subadult P. mangshanensis had an extremely low abundance of PLA2 (1.23%) but a high abundance of CTL (51.47%), followed by SVMP (22.06%) and SVSP (10.90%). Apparent interspecific variations in lethality and enzymatic activities were also explored in habu snake venoms, but no variations in myotoxicity were found. Except for SVSP, the resemblance of the relatives within Protobothrops in other venom traits was estimated to deviate from Brownian motion evolution based on phylogenetic signals. A comparative analysis further validated that the degree of covariation between phylogeny and venom variation is evolutionarily labile and varies among clades of closely related snakes. Our findings indicate a high level of interspecific variation in the venom proteomes of habu snakes, both in the presence or absence and the relative abundance of venom protein families, and that these venoms might have evolved under a combination of adaptive and neutral mechanisms.

Structural insights into DarT toxin neutralization by cognate DarG antitoxin: ssDNA mimicry by DarG C-terminal domain keeps the DarT toxin inhibited

In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions.