A Mechanism Study on the (+)-ESI-TOF/HRMS Fragmentation of Some PPI Prazoles and Their Related Substances.

Fragmentation mechanisms of some prazoles and their related substances were newly investigated in this paper via positive mode ESI-TOF HRMS1 and HRMS2. Some novel fragmentation rules or ions were found or detected in the research. The pyridine and the benzoimidazole ring remained in most cases during the ionization, and heterolytic fragmentations often occurred near the -S(O)nCH2- linker to give the [1,3]-H migration ion or [1,7]-H migration ion rearranging across the benzoimdazole ring. Smiles rearrangement ionizations also frequently occurred, initiated by the attack of the lone pair electrons from the pyridine ring, and the sulfones gave special N-(2-benzoimdazolyl) pyridine ions (11b and 12c) by a direct extraction from SO2, and the thioethers gave similar framework ions (8c, 9c and 10c) via the rearrangement and a further homolytic cleavage of SH radicals. However, the sulfoxides were seldom detected in the corresponding Smiles rearrangement ions during our measurement, and the N'-oxides of the pyridines did not undergo the Smiles rearrangement ionization due to the absence of the lone pair electrons. The 5/6-membered chelating ions with Na+ or K+ were frequently detected as the molecular and further fragment ions. Some novel and interesting fragment ions containing bivalent (8b and 9b), tetravalent (4b, 5c and 6c) or hexavalent (15b and 16b) sulfurs were first reported here.

Autistic traits predict social avoidance and distress: The chain mediating role of perceived stress and interpersonal alienation.

Social avoidance and distress are the primary aspects of social anxiety. Nonautistic people with high levels of autistic traits are more likely to exhibit social avoidance and distress. However, research has yet to reveal how autistic traits induce social avoidance and distress. To fill this gap, the present study recruited 708 participants to complete the 25-item Autism Spectrum Quotient, Social Avoidance and Distress Scale, Chinese Perceived Stress Scale, and Interpersonal Alienation Subscale. The results indicated that autistic traits significantly predicted social avoidance and distress in nonautistic people. In addition, autistic traits induced social avoidance and distress through perceived stress and interpersonal alienation, respectively. Importantly, perceived stress and interpersonal alienation (including the subdimensions of interpersonal alienation: sense of loneliness, sense of social isolation, and alienation between family members) partially mediated the relationships between autistic traits and social avoidance and distress. Overall, autistic traits predict social avoidance and distress via perceived stress and interpersonal alienation. This finding extends the hypothetical model of clinical anxiety in autism spectrum disorders. Furthermore, reducing perceived stress and interpersonal alienation in nonautistic people with high levels of autistic traits may be a valid intervention method to prevent and eliminate their social avoidance and distress.

Long non-coding RNA LINC00607 silencing exerts antioncogenic effects on thyroid cancer through the CASP9 Promoter methylation.

Thyroid cancer (TC) was the most frequent thyroid malignant tumour, accounting for about 1% of all malignant tumours. Some long non-coding RNAs (lncRNAs) have been reported to exert essential tumour promotion effects, while caspase-9 (CASP9) gene could play a promotive role in the cell apoptosis in TC. However, whether they have a specific effect on TC remains unclear. Hence, this study aims to explore the relationship between LINC00607 and CASP9, and its effect in TC. LINC00607 expression in the TC tissues and cell lines was determined. Then, we explored the combination effect between a LINC00607 and a methylation inhibitor 5-Aza-dc in doxorubicin-resistant ARO cells using colony formation assay, flow cytometry, WST-1 and EdU assay, as well as in vivo tumour growth assay. Besides, the dual-luciferase reporter gene assay, RIP, ChIP, methylation-specific PCR and BSP method were employed to detect the relationship between LINC00607 and CASP9 and its methylation. LINC00607 expression was up-regulated in the doxorubicin-resistant TC cell lines and tissues and negatively correlated to the poor prognosis of TC patients. Knockdown of LINC00607 suppressed doxorubicin resistance, proliferation and colony formation, and promoted cell apoptosis of TC cells in vitro, as well as suppressed tumour growth in vivo, whereas LINC00607 overexpression was observed to exercise the opposite effects. Notably, it was also revealed that LINC00607 down-regulated the CASP9 expression by promoting CASP9 promoter methylation. In conclusion, LINC00607 could inhibit the apoptosis and augment the doxorubicin resistance of TC cells by decreasing CASP9 expression, which might provide a novel therapeutic target for TC treatment.

RNA-splicing factor SART3 regulates translesion DNA synthesis.

Translesion DNA synthesis (TLS) is one mode of DNA damage tolerance that uses specialized DNA polymerases to replicate damaged DNA. DNA polymerase η (Polη) is well known to facilitate TLS across ultraviolet (UV) irradiation and mutations in POLH are implicated in skin carcinogenesis. However, the basis for recruitment of Polη to stalled replication forks is not completely understood. In this study, we used an affinity purification approach to isolate a Polη-containing complex and have identified SART3, a pre-mRNA splicing factor, as a critical regulator to modulate the recruitment of Polη and its partner RAD18 after UV exposure. We show that SART3 interacts with Polη and RAD18 via its C-terminus. Moreover, SART3 can form homodimers to promote the Polη/RAD18 interaction and PCNA monoubiquitination, a key event in TLS. Depletion of SART3 also impairs UV-induced single-stranded DNA (ssDNA) generation and RPA focus formation, resulting in an impaired Polη recruitment and a higher mutation frequency and hypersensitivity after UV treatment. Notably, we found that several SART3 missense mutations in cancer samples lessen its stimulatory effect on PCNA monoubiquitination. Collectively, our findings establish SART3 as a novel Polη/RAD18 association regulator that protects cells from UV-induced DNA damage, which functions in a RNA binding-independent fashion.

Ataxin-3 promotes genome integrity by stabilizing Chk1.

The Chk1 protein is essential for genome integrity maintenance and cell survival in eukaryotic cells. After prolonged replication stress, Chk1 can be targeted for proteasomal degradation to terminate checkpoint signaling after DNA repair finishes. To ensure proper activation of DNA damage checkpoint and DNA repair signaling, a steady-state level of Chk1 needs to be retained under physiological conditions. Here, we report a dynamic signaling pathway that tightly regulates Chk1 stability. Under unperturbed conditions and upon DNA damage, ataxin-3 (ATX3) interacts with Chk1 and protects it from DDB1/CUL4A- and FBXO6/CUL1-mediated polyubiquitination and subsequent degradation, thereby promoting DNA repair and checkpoint signaling. Under prolonged replication stress, ATX3 dissociates from Chk1, concomitant with a stronger binding between Chk1 and its E3 ligase, which causes Chk1 proteasomal degradation. ATX3 deficiency results in pronounced reduction of Chk1 abundance, compromised DNA damage response, G2/M checkpoint defect and decreased cell survival after replication stress, which can all be rescued by ectopic expression of ATX3. Taken together, these findings reveal ATX3 to be a novel deubiquitinase of Chk1, providing a new mechanism of Chk1 stabilization in genome integrity maintenance.

Quantitative proteomics analysis reveals alterations of lysine acetylation in mouse testis in response to heat shock and X-ray exposure.

Environmental stresses are important factors causing male infertility which attracts broad attention. Protein acetylation is a pivotal post-translational modification and modulates diverse physiological processes including spermatogenesis. In this study, we employed quantitative proteomic techniques and bioinformatics tools to analyze the alterations of acetylome profile of mouse testis after heat shock and X-irradiation. Overall, we identified 1139 lysine acetylation sites in 587 proteins in which 1020 lysine acetylation sites were quantified. The Gene Ontology analysis showed that the major acetylated protein groups were involved in generation of precursor metabolites and metabolic processes, and were localized predominantly in cytosolic and mitochondrial. Compared to the control group, 36 sites of 28 acetylated proteins have changed after heat shock, and 49 sites of 43 acetylated proteins for X-ray exposure. Some of the differentially acetylated proteins have been reported to be associated with the progression of spermatogenesis and male fertility. We observed the up-regulated acetylation level change on testis specific histone 2B and heat shock protein upon heat treatment and a sharp decline of acetylation level on histone H2AX under X-ray treatment, suggesting their roles in male germ cells. Notably, the acetylation level on K279 of histone acetyltransferase (Kat7) was down-regulated in both heat and X-ray treatments, indicating that K279 may be a key acetylated site and affect its functions in spermatogenesis. Our results reveal that protein acetylation might add another layer of complexity to the regulation for spermatogenesis, and further functional studies of these proteins will help us elucidate the mechanisms of abnormal spermatogenesis.

Quantitative Phosphoproteomic Study Reveals that Protein Kinase A Regulates Neural Stem Cell Differentiation Through Phosphorylation of Catenin Beta-1 and Glycogen Synthase Kinase 3ß.

Protein phosphorylation is central to the understanding of multiple cellular signaling pathways responsible for regulating the self-renewal and differentiation of neural stem cells (NSCs). Here we performed a large-scale phosphoproteomic analysis of rat fetal NSCs using strong cation exchange chromatography prefractionation and citric acid-assisted two-step enrichment with TiO2 strategy followed by nanoLC-MS/MS analysis. Totally we identified 32,546 phosphosites on 5,091 phosphoproteins, among which 23,945 were class I phosphosites, and quantified 16,000 sites during NSC differentiation. More than 65% of class I phosphosites were novel when compared with PhosphoSitePlus database. Quantification results showed that the early and late stage of NSC differentiation differ greatly. We mapped 69 changed phosphosites on 20 proteins involved in Wnt signaling pathway, including S552 on catenin beta-1 (Ctnnb1) and S9 on glycogen synthase kinase 3ß (Gsk3ß). Western blotting and real-time PCR results proved that Wnt signaling pathway plays critical roles in NSC fate determination. Furthermore, inhibition and activation of PKA dramatically affected the phosphorylation state of Ctnnb1 and Gsk3ß, which regulates the differentiation of NSCs. Our data provides a valuable resource for studying the self-renewal and differentiation of NSCs. Stem Cells 2016;34:2090-2101.

Engineering 1,3-Alternate Calixcarbazole for Recognition and Sensing of Bisphenol F in Water.

Herein, we report a carbazolyl tubular macrocycle (3), which possesses a "π-tube" capable of encapsulating and sensing bisphenol F (BPF), which is a toxic industrial material. In this work, the synthesis of 3, its conformation in solution as well as its binding property to BPF have been investigated. Nuclear magnetic resonance (NMR), ultraviolet-visible light (UV-vis), fluorescence spectra, and molecular modeling studies reveal that the "π-tube" of 3 in 1,3-alternate conformation could encapsulate BPF with 1:1 binding stoichiometry in water, with its orthogonal tweezers sandwiching the two phenol units of BPF. Moreover, 3 could serve as a sensitive fluorescent probe for BPF (limitation of detection = 157 nM).

iTRAQ-based chromatin proteomic screen reveals CHD4-dependent recruitment of MBD2 to sites of DNA damage.

Many DNA repair proteins can be recruited to DNA damage sites upon genotoxic stress. In order to search potential DNA repair proteins involved in cellular response to mitomycin C treatment, we utilized a quantitative proteome to uncover proteins that manifest differentially enrichment in the chromatin fraction after DNA damage. 397 proteins were identified, among which many factors were shown to be involved in chromatin modification and DNA repair by GO analysis. Specifically, methyl-CpG-binding domain protein 2 (MBD2) is revealed to be recruited to DNA damage sites after laser microirradiation, which was mediated through MBD domain and MBD2 C-terminus. Additionally, the recruitment of MBD2 is dependent on poly (ADP-ribose) and chromodomain helicase DNA-binding protein 4 (CHD4). Moreover, knockdown of MBD2 by CRISPR-Cas9 technique results in MMC sensitivity in mammalian cells.

The alteration of H4-K16ac and H3-K27met influences the differentiation of neural stem cells.

The neural stem cell therapy provides a promising future for patients with central nerve system damage, thus an insight into its differentiation mechanism is urgently needed. Herein, we aimed to identify various histone modifications and reveal their impact on the differentiation of neural stem cells (NSCs) toward neurons. Firstly, we labeled primary NSCs using the stable isotope labeling with amino acids in cell culture (SILAC) technique. Then we induced these NSCs to differentiate by all-trans retinoic acid (atRA) or SB216763. Next, we identified the alteration of histone modifications in early-differentiated NSCs by mass spectrometry and verified them by Western blot. Interestingly, these modification alterations and phenotype changes were found similar in NSCs induced by the two different drugs. More interestingly, during the differentiation process H3-K27met was significantly up-regulated while H4-K16ac was not altered at the global level but down-regulated in some low-abundance combinatorial codes. We inhibited the methyltransferase of H3-K27 and deacetylase of H4-K16 simultaneously and found the differentiation procedure was obviously delayed. The function of H4-K16ac and H3-K27met in NSCs differentiation would be useful to reveal the differentiation mechanism and valuable for further neural stem cell therapy.

Calix[3]carbazole: One-Step Synthesis and Host-Guest Binding.

The one-step synthetic strategy for the preparation of the hitherto unknown calix[3]carbazole from readily available starting materials is described. Calix[3]carbazole is obtained in 20% yield, and it could selectively bind the N(C2H5)4(+) cation (tetraethylammonium, TEA) via cation-π interactions. The experimental and modeling results indicate that calix[3]carbazole possesses a larger π-cavity as well as a better chromophoric property than the traditional phenol-based macrocycles, and thus is capable of binding to and optically responding to the relatively large guest TEA.

Evaluation of the accuracy of protein quantification using isotope TMPP-labeled peptides.

N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) reacts rapidly, mildly, and specifically with the N-terminals of proteins and peptides. Thus, it can be developed as an ideal isotope-coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP-based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on-tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP-labeled peptides were combined at different ratios and subsequently analyzed by LC-MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope-coded TMPP method achieved accurate quantification and compatibility with IEF-based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis.

Activated cyclin-dependent kinase 5 promotes microglial phagocytosis of fibrillar ß-amyloid by up-regulating lipoprotein lipase expression.

Amyloid plaques are crucial for the pathogenesis of Alzheimer disease (AD). Phagocytosis of fibrillar ß-amyloid (Aß) by activated microglia is essential for Aß clearance in Alzheimer disease. However, the mechanism underlying Aß clearance in the microglia remains unclear. In this study, we performed stable isotope labeling of amino acids in cultured cells for quantitative proteomics analysis to determine the changes in protein expression in BV2 microglia treated with or without Aß. Among 2742 proteins identified, six were significantly up-regulated and seven were down-regulated by Aß treatment. Bioinformatic analysis revealed strong over-representation of membrane proteins, including lipoprotein lipase (LPL), among proteins regulated by the Aß stimulus. We verified that LPL expression increased at both mRNA and protein levels in response to Aß treatment in BV2 microglia and primary microglial cells. Silencing of LPL reduced microglial phagocytosis of Aß, but did not affect degradation of internalized Aß. Importantly, we found that enhanced cyclin-dependent kinase 5 (CDK5) activity by increasing p35-to-p25 conversion contributed to LPL up-regulation and promoted Aß phagocytosis in microglia, whereas inhibition of CDK5 reduced LPL expression and Aß internalization. Furthermore, Aß plaques was increased with reducing p25 and LPL level in APP/PS1 mouse brains, suggesting that CDK5/p25 signaling plays a crucial role in microglial phagocytosis of Aß. In summary, our findings reveal a potential role of the CDK5/p25-LPL signaling pathway in Aß phagocytosis by microglia and provide a new insight into the molecular pathogenesis of Alzheimer disease.

Polymers for the stabilization and delivery of proteins topically and per os to the insect hemocoel through conjugation with aliphatic polyethylene glycol.

Co-feeding of aliphatic polyethylene glycol (PEG), phospholipase A2, anionic and ionic detergents, and amphipathic glycoside with bovine serum albumin (BSA) as a model protein to fourth stadium tobacco budworms, Heliothis virescens, did not affect the levels of BSA in the hemolymph. Covalent conjugation of small proteins like the decapeptide trypsin modulating oostatic factor (TMOF) to polyethylene glycol was previously shown to protect the peptide from protease attack and enhance its accumulation in the insect hemocoel. Whether this polymer chemistry could do the same for larger proteins was examined. The chemistry for the synthesis of polydispersed aliphatic PEG350-insulin and monodispersed aliphatic PEG333-insulin are described herein. Insulin was used for this synthesis and not BSA to better control conjugation among the available free amine groups. When PEGylated insulin or free insulin were fed in artificial diet to fifth stadium budworms, greater concentrations of insulin using the PEGylated variants were found in the hemolymph than when free insulin was used (a 6.7 and 7.3-fold increase for the PEG350 and PEG333 conjugates, respectively). When insulin is topically applied to the dorsum of H. virescens, no insulin is found in the hemolymph. However, after topical application of the PEGylated insulins, PEG350-insulin and PEG333-insulin were detected in the hemolymph. After injections of insulin into the hemocoel of fourth stadium H. virescens, insulin is completely cleared from the hemolymph in 120min. In comparison, PEG350-insulin and PEG333-insulin were present in the hemolymph for 300 and 240min after injection, respectively, translating to a 3.3 and 2.7-fold increase in the length of time insulin remains in the hemolymph after injection.

Algorithm for community security risk assessment and influencing factors analysis by back propagation neural network.

This paper aims to accurately assess and effectively manage various security risks in the community and overcome the challenges faced by traditional models in handling large amounts of features and high-dimensional data. Hence, this paper utilizes the back propagation neural network (BPNN) to optimize the security risk assessment model. A key challenge of researching community security risk assessment lies in accurately identifying and predicting a range of potential security threats. These threats may encompass natural disasters, public health crises, accidents, and social security issues. The intricate interplay of these risk factors, combined with the dynamic nature of community environments, presents difficulties for traditional risk assessment methodologies to address effectively. Initially, this paper delves into the factors influencing safety incidents within communities and establishes a comprehensive system of safety risk assessment indicators. Leveraging the adaptable and generalizable nature of the BPNN model, the paper proceeds to optimize the BPNN model, enhancing the security risk assessment model through this optimization. Subsequent comparison experiments with traditional models validate the rationality and effectiveness of the proposed model, with hidden layer nodes set at various levels like 10, 15, 20, 25, 30, and 35. These traditional models include Convolutional Neural Network (CNN), Long Short-Term Memory Network (LSTM), Bidirectional Encoder Representations from Transformers (BERT), Generative Pre-trained Transformer (GPT), and eXtreme Gradient Boosting (XGBOOST). Experimental findings demonstrate that with 20 hidden layer nodes, the optimized model achieves a remarkable final recognition accuracy of 99.1 %. Moreover, the optimized model exhibits significantly lower final function loss compared to models with different node numbers. Increasing the number of hidden layer nodes may diminish the optimized model's fit and accuracy. Comparison with traditional models reveals that the average accuracy of the optimized model in community risk identification reaches 98.5 %, with a maximum accuracy of 99.6 %. This marks an improvement of 9%-11 % in recognition accuracy across various risk factors compared to traditional models. Regarding system response time and resource utilization, the optimized model exhibits a response time ranging from 100 ms to 120 ms and consistently lower resource utilization rates across all scenarios, underscoring its efficiency in community security risk assessment. In conclusion, this experiment sheds light on the underlying mechanisms and patterns of community safety risk formation, offering novel perspectives and methodologies for researching community safety risk assessment. The paper concludes by presenting recommendations and strategies for addressing community safety risks based on experimental analysis.

High-confidence de novo peptide sequencing using positive charge derivatization and tandem MS spectra merging.

De novo peptide sequencing holds great promise in discovering new protein sequences and modifications but has often been hindered by low success rate of mass spectra interpretation, mainly due to the diversity of fragment ion types and insufficient information for each ion series. Here, we describe a novel methodology that combines highly efficient on-tip charge derivatization and tandem MS spectra merging, which greatly boosts the performance of interpretation. TMPP-Ac-OSu (succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl)phosphonium bromide) was used to derivatize peptides at N-termini on tips to reduce mass spectra complexity. Then, a novel approach of spectra merging was adopted to combine the benefits of collision-induced dissociation (CID) and electron transfer dissociation (ETD) fragmentation. We applied this methodology to rat C6 glioma cells and the Cyprinus carpio and searched the resulting peptide sequences against the protein database. Then, we achieved thousands of high-confidence peptide sequences, a level that conventional de novo sequencing methods could not reach. Next, we identified dozens of novel peptide sequences by homology searching of sequences that were fully backbone covered but unmatched during the database search. Furthermore, we randomly chose 34 sequences discovered in rat C6 cells and verified them. Finally, we conclude that this novel methodology that combines on-tip positive charge derivatization and tandem MS spectra merging will greatly facilitate the discovery of novel proteins and the proteome analysis of nonmodel organisms.

N-Embedded Cubarene: A Quadrangular Member of the Macrocycle Family.

Despite the large number of synthetic macrocycles, the cubarenes, the quadrangular-shaped macrocyclic arenes, remain less investigated, possibly due either to synthetic challenges or to the lack of suitable building blocks. In this paper, a N-embedded cubarene (cub[4]indolocarbazole) is facilely synthesized by FeCl3·6H2O-catalyzed cyclization in dichloromethane. The endo cavity of cub[4]indolocarbazole can bury quaternary ammonium salts in an intramolecular manner, whereas the intermolecular interaction between its exo walls with Cu2+ generates two-dimensional supramolecular tessellation.

A 2D Dy-based metal-organic framework derived from benzothiadiazole: structure and photocatalytic properties.

A 2D Dy(III) metal-organic layer (MOL 1) was synthesized under solvothermal conditions. Structural analysis suggests that the Dy(III) ions in each one-dimensional (1D) arrangement are evenly arranged in the form of broken lines. The 1D chains are linked to one another via ligands to form a 2D layer that generates a 2D surface with elongated apertures. The photocatalytic activity study suggests that MOL 1 exhibits good catalytic activity in flavonoids by the formation of an O2˙- radical as an intermediate. This is the first reported method of synthesizing flavonoids using chalcones.

Size- and surface charge-dependent hormetic effects of microplastics on bacterial resistance and their interactive effects with quinolone antibiotic.

The facilitation of microplastics (MPs) on bacterial resistance has attracted wide concern, due to the widespread presence of MPs in environmental media and their ubiquitous contact with bacteria strains. Furthermore, MPs possibly co-exist with antibiotics to trigger combined stress on bacterial survival. Therefore, it is significant to reveal the dose-responses of MPs and MP-antibiotic mixtures on bacterial endogenous and exogenous resistance. In this study, 0.1 and 5 µm polystyrenes with no surface functionalization (PS-NF, no charge), surface functionalized with amino groups (PS-NH2, positive charge) and carboxyl groups (PS-COOH, negative charge) were selected as the test MPs, and norfloxacin (NOR) was set as the representative of antibiotics. It was found that six types of PS all inhibited the growth of Escherichia coli (E. coli) but induced hormetic dose-responses on the mutation frequency (MF) and conjugative transfer frequency (CTF) of RP4 plasmid in E. coli. Moreover, these hormetic effects exhibited size- and surface charge-dependent features, where 0.1 µm PS-NH2 (100 mg/L) triggered the maximum stimulatory rates on MF (363.63 %) and CTF (74.80 %). The hormetic phenomena of MF and CTF were also observed in the treatments of PS-NOR mixtures, which varied with the particle size and surface charge of PS. In addition, the interactive effects between PS and NOR indicated that the co-existence of PS and NOR might trigger greater resistance risk than the single pollutants. Mechanistic exploration demonstrated that the increase of cellular reactive oxygen species and the variation of cell membrane permeability participated in the hormetic effects of PS and PS-NOR mixtures on bacterial resistance. This study provides new insights into the individual effects of MPs and the combined effects of MP-antibiotic mixtures on bacterial resistance, which will promote the development of environmental risk assessment of MPs from the perspective of bacterial resistance.

Dose-dependent joint resistance action of antibacterial mixtures in their hormetic effects on bacterial resistance based on concentration addition model.

The judgment of joint resistance action is significant for evaluating the resistance risk of antibacterial mixture. Using bacterial mutation frequency (MF) and conjugative transfer frequency (CTF) to respectively characterize the bacterial endogenous and exogenous resistance, mutation unit and conjugative transfer unit have been proposed to judge the joint resistance action of antibacterial mixture at a certain dose. However, these methods could not evaluate the antibacterial mixture's joint resistance action at a larger concentration-range. In this study, the concentration addition for bacterial resistance (CA-BR) approach was used to judge the joint resistance actions between kanamycin sulfate (KAN) and some other typical antibacterial agents, including sulfonamides (SAs), sulfonamide potentiators (SAPs), and silver antibacterial compounds (SACs). Through comparing the hormetic dose-response curves of the binary mixtures on the MF (or CTF) in Escherichia coli (E. coli) and the corresponding CA-BR curves calculated from the hormetic dose-responses of the single agents, the joint resistance actions between KAN and other agents were judged to exhibit dose-dependent feature: with the increase of mixture concentration, the joint mutation actions between KAN and SAs (or SAPs) were fixed at synergism, and the joint mutation actions between KAN and SACs varied from antagonism to synergism; the joint conjugative transfer actions between KAN and other agents changed from antagonism to synergism. Mechanistic explanation suggested that the heterogeneous pattern of joint resistance action had a close relationship with the interplays among the agents' modes of action, and meanwhile was significantly influenced by their joint survival pressure on E. coli. This study reveals the dose-dependent feature for the joint resistance action of antibacterial mixture and highlights the importance of exposure concentration, which will benefit clarifying the resistance risk of antibacterial mixture in the environment.