DFT Study on the Mechanism of the Palladium-Catalyzed [3 + 2] Annulation of Aromatic Amides with Maleimides via Benzylic and meta-C-H Bond Activation: Role of the External Ligand Ac-Gly-OH.

The mechanism of the Ac-Gly-OH-assisted palladium-catalyzed [3 + 2] annulation of aromatic amides with maleimides is investigated using density functional theory calculations. The results show that the reaction undergoes the sequential steps of N-H bond deprotonation, first benzylic C-H bond activation, maleimide insertion, second meta-C-H bond activation, reductive elimination, and oxidation. The external ligand Ac-Gly-OH acts as the internal base for hydrogen abstraction in the first benzylic C-H bond activation. The maleimide insertion step is found to be the rate-determining step. Based on the nearly same energetic span of the two pathways to generate the enantio products, the computational results are consistent with the experimental observation that the terminal [3 + 2] annulation products are racemic when using an achiral ligand. These calculation results disclose the detailed reaction mechanism and shed light on some experimental ambiguities.

Density Functional Theory Study of the Mechanism of Ni-Catalyzed Carboxylation of Aryl C(sp2)-S Bonds with CO2: Computational Evidence for the Multifaceted Role of Additive Zn.

The mechanism of Ni-catalyzed carboxylation of aryl C(sp2)-S bonds with CO2 was studied for the first time by density functional theory calculations. We first proposed another possible reaction pathway in which CO2 insertion occurs prior to reduction. Then, we performed calculations on all proposed reaction pathways, and our calculation results show that the pathway in which reduction occurs prior to CO2 insertion is the favored pathway for this reaction. Additionally, our calculations disclose that additive Zn0 acts in multifaceted roles. (1) Zn0 acts as a reductant to reduce the NiI and NiII intermediates. (2) The simultaneously formed ZnIIBr2 can undergo transmetalation with NiI or NiII intermediates to produce an aryl reservoir by forming arylzinc species. (3) ZnIIBr2 can also coordinate to the CO2 to lower the energy barrier of the CO2 insertion step. Moreover, the calculation results suggest that CO2 insertion is the rate-determining step of the reaction, and CO2 is easier to insert into the NiI-Ph bond rather than into the NiII-Ph bond. These calculation results can improve our understanding of the mechanism of the carboxylation process and the multifaceted roles of metal additive Zn0 and provide theoretical guidance for improving the carboxylation reaction.

Density Functional Theory Study on the Mechanism of Nickel-Catalyzed 3,3-Dialkynylation of 2-Aryl Acrylamides Via Double Vinylic C-H Bond Activation.

The mechanisms of Ni-catalyzed 3,3-dialkynylation of 2-aryl acrylamide have been investigated by using density functional theory calculations. The result shows that this reaction includes double alkynylation, which involves sequential key steps of vinylic C-H bond activation, successive oxidative addition, and reductive elimination, with the second C-H bond activation being the rate-determining step. C-H and N-H bond activation occurs via the concerted metalation-deprotonation mechanism. The calculations show that no transition state exists in the first reductive elimination process, and a negative free energy barrier in the second reductive elimination process though a transition state is identified, indicating that the nickel-catalyzed vinylic C(sp2)-C(sp) bond formation does not require activation energy. Z-E isomerization is the prerequisite for the second alkynylation. In addition, our spin-flip TDDFT (SF-TDDFT) computational result discloses that the actual process of Z-E isomerization occurs on the potential energy surface of the first excited singlet state S1.

DFT Study on Mechanism of Ni-Al Bimetallic-Catalyzed C-H Cyclization to Construct Tricyclic Imidazoles: Roles of NHC Ligand and AlMe3.

The mechanism of the Ni-Al bimetallic-catalyzed C-H cyclization to construct tricyclic imidazoles is investigated using density functional theory calculations. The calculation result shows that the reaction mechanism involves sequential steps of substrate coordination, ligand-to-ligand hydrogen transfer (LLHT), and C-C reductive elimination to produce the final product tricyclic imidazole. The LLHT step is calculated to be the rate-determining step. The oxidative addition of the benzimidazole C-H bond to the Ni center and the insertion of the alkene into the Ni-H bond occur concertedly in the LLHT step. The effects of N-heterocyclic carbene (NHC) ligands and AlMe3 on the reactivity and regioselectivity were also analyzed. These calculation results shed light on some ambiguous suggestions from experiments.

Human Biomonitoring of Environmental Chemicals among Elderly in Wuhan, China: Prioritizing Risks Using EPA's ToxCast Database.

In line with the "healthy aging" principle, we aim to assess the exposure map and health risks of environmental chemicals in the elderly. Blood samples from 918 elderly individuals in Wuhan, China, were analyzed using the combined gas/liquid-mass spectrometry technology to detect levels of 118 environmental chemicals. Cluster analysis identified exposure profiles, while risk indexes and bioanalytical equivalence percentages were calculated using EPA's ToxCast database. The detection rates for 87 compounds exceeded 70%. DEHP, DiBP, naphthalene, phenanthrene, DnBP, pyrene, anthracene, permethrin, fluoranthene, and PFOS showed the highest concentrations. Fat-soluble pollutants varied across lifestyles. In cluster 2, which was characterized by higher concentrations of fat-soluble substances, the proportion of smokers or drinkers was higher than that of nonsmokers or nondrinkers. Pesticides emerged as the most active environmental chemicals in peroxisome proliferator-activated receptor gamma antagonist, thyroid hormone receptor (TR) antagonist, TR agonist, and androgen receptor (AR) agonist activity assays. Additionally, PAEs and polycyclic aromatic hydrocarbons played significant roles as active contaminants for the corresponding targets of AR antagonists and estrogen receptor alpha. We proposed a list of priority pollutants linked to endocrine-disrupting toxic effects in the elderly, which may provide the groundwork for further research into environmental etiology.

LSD1 is a subunit of the NuRD complex and targets the metastasis programs in breast cancer.

Lysine-specific demethylase 1 (LSD1) exerts pathway-specific activity in animal development and has been linked to several high-risk cancers. Here, we report that LSD1 is an integral component of the Mi-2/nucleosome remodeling and deacetylase (NuRD) complex. Transcriptional target analysis revealed that the LSD1/NuRD complexes regulate several cellular signaling pathways including TGFbeta1 signaling pathway that are critically involved in cell proliferation, survival, and epithelial-to-mesenchymal transition. We demonstrated that LSD1 inhibits the invasion of breast cancer cells in vitro and suppresses breast cancer metastatic potential in vivo. We found that LSD1 is downregulated in breast carcinomas and that its level of expression is negatively correlated with that of TGFbeta1. Our data provide a molecular basis for the interplay of histone demethylation and deacetylation in chromatin remodeling. By enlisting LSD1, the NuRD complex expands its chromatin remodeling capacity to include ATPase, histone deacetylase, and histone demethylase.

The Transcriptional Regulator TfmR Directly Regulates Two Pathogenic Pathways in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc) is a notorious plant pathogen. Like most bacterial pathogens, Xoc has evolved a complex regulatory network to modulate the expression of various genes related to pathogenicity. Here, we have identified TfmR, a transcriptional regulator belonging to the TetR family, as a key player in the virulence mechanisms of this phytopathogenic bacterium. We have demonstrated genetically that tfmR is involved in the hypersensitive response (HR), pathogenicity, motility and extracellular polysaccharide production of this phytopathogenic bacterium. Our investigations extended to exploring TfmR's interaction with RpfG and HrpX, two prominent virulence regulators in Xanthomonas species. We found that TfmR directly binds to the promoter region of RpfG, thereby positively regulating its expression. Notably, constitutive expression of RpfG partly reinstates the pathogenicity compromised by TfmR-deletion mutants. Furthermore, our studies revealed that TfmR also exerts direct positive regulation on the expression of the T3SS regulator HrpX. Similar to RpfG, sustained expression of HrpX partially restores the pathogenicity of TfmR-deletion mutants. These findings underscore TfmR's multifaceted role as a central regulator governing key virulence pathways in Xoc. Importantly, our research sheds light on the intricate molecular mechanisms underlying the regulation of pathogenicity in this plant pathogen.

Candida tropicalis oligopeptide transporters assist in the transmembrane transport of the antimicrobial peptide CGA-N9.

Candida tropicalis is often reported as the second or third most common pathogen causing fungal infections. Antimicrobial peptides (AMPs) have attracted increasing attention for their broad-spectrum antimicrobial properties and low cytotoxicity. Our previous studies have shown that CGA-N9, a non-membrane-rupturing AMP, crosses the cell membrane to exert anticandidal activity. We speculate that there are some related transporters that assist in the transmembrane transport of CGA-N9. In this study, the relationship between CGA-N9 lethality kinetics and its real-time transmembrane amount in C. tropicalis cells was investigated. The results demonstrated that there was a positive correlation between its candicidal activity and transmembrane amount. A total of 12 oligopeptide transporter (OPT) coding sequences (CDSs) were cloned from C. tropicalis by using the conservative OPT gene sequences of Candida spp. to design primers and were named C. tropicalis OPTs (CtOPTs). The results of RT‒qPCR demonstrated that the expression levels of CtOPT1, CtOPT9 and CtOPT12 were correlated with the CGA-N9 transmembrane amount in a time-dependent manner. The results of molecular docking demonstrated that CtOPT1, CtOPT9 and CtOPT12 interact strongly with CGA-N9. Therefore, CtOPT1, CtOPT9 and CtOPT12 were predicted to assist in the transmembrane transport of the AMP CGA-N9.

Mild Hypothermia Alleviates Complement C5a-Induced Neuronal Autophagy During Brain Ischemia-Reperfusion Injury After Cardiac Arrest.

After restoration of spontaneous circulation (ROSC) following cardiac arrest, complements can be activated and excessive autophagy can contribute to the brain ischemia-reperfusion (I/R) injury. Mild hypothermia (HT) protects against brain I/R injury after ROSC, but the mechanisms have not been fully elucidated. Here, we found that HT significantly inhibited the increases in serum NSE, S100ß, and C5a, as well as neurologic deficit scores, TUNEL-positive cells, and autophagic vacuoles in the pig brain cortex after ROSC. The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen-glucose deprivation/reoxygenation. HT could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Our findings demonstrated that C5a could bind to C5aR1 to induce neuronal autophagy during the brain I/R injury, which was associated with the inhibited PI3K/Akt/mTOR pathway. HT could inhibit C5a-induced neuronal autophagy by regulating the C5a-C5aR1 interaction and the PI3K/Akt/mTOR pathway, which might be one of the neuroprotective mechanisms underlying I/R injury. The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen-glucose deprivation/reoxygenation. Mild hypothermia (HT) could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Proposed mechanism by which HT protects against brain I/R injury by repressing C5a-C5aR1-induced excessive autophagy. Complement activation in response to brain I/R injury generates C5a that can interact with C5aR1 to inactivate mTOR, probably through the PI3K-AKT pathway, which can finally lead to autophagy activation. The excessively activated autophagy ultimately contributes to cell apoptosis and brain injury. HT may alleviate complement activation and then reduce C5a-induced autophagy to protect against brain I/R injury. HT, mild hypothermia; I/R, ischemia reperfusion.

Computational Study of Iron-Catalyzed Intramolecular [2 + 2] Cycloaddition and Cycloisomerization of Enyne Acetates: Mechanism and Selectivity.

The mechanism of iron-catalyzed intramolecular [2 + 2] cycloaddition and cycloisomerization of enyne acetates has been investigated with DFT computations. Both mechanisms start the catalytic cycle from the stepwise 1,2-acyloxy migration to afford the iron carbene. The [2 + 2] cycloaddition mechanism involves subsequent key steps of [2 + 2] cycloaddition, 1,2-acyloxy migration, and reductive elimination to generate the azabicyclo [3.2.0] heptane product, with the reductive elimination being the rate-determining step. The cycloisomerization mechanism involves subsequent key steps of [2 + 2] cycloaddition, stepwise 1,4-acyloxy migration to produce the allenylpyrrolidine product, with the 1,4-acyloxy migration being the rate-determining step. Reaction potential energy surfaces for two model substrates that have or do not have alkene-terminal substituents have been investigated and the origins of the selectivities have been disclosed. Moreover, energy profiles with three possible spin states (SFe = 0, 1, 2) have been considered. The reaction is suggested to occur mainly on the singlet potential energy surface with a few spin crossovers between singlet and triplet states involved, which indicates that this reaction should have two-state reactivity (TSR).

Activities of a broad-spectrum antimicrobial peptide analogue SAMP-A4-C8 and its combat against pneumonia in Staphylococcus aureus-infected mice.

Antimicrobial peptides and their analogues have become substitutes for antibiotics in recent years. The antimicrobial peptide analogue SAMP-A4-C8 (n-octanoic-VRLLRRRI) with high antimicrobial activity was found in our lab. We speculate that it may kill pathogens by some lethal mechanism of action. In the present investigation, the microbicidal activities of SAMP-A4-C8 and its mechanism of action were investigated. The results demonstrated that SAMP-A4-C8 had lethal activities against Staphylococcus aureus and Candida albicans by cell disruption. Based on its microbicidal activities, we believe that it is worth further research for its potential as drug candidate. The results showed that SAMP-A4-C8, with low propensity to induce the resistance of S. aureus and C. albicans, could kill the persister cells of S. aureus and C. albicans, exhibited biofilm forming inhibition activity and preformed biofilm eradication ability against S. aureus and C. albicans, and displayed therapeutic potential on pneumonia in S. aureus-infected mice by reducing lung inflammation. The present study provided a promising drug candidate in the war against multidrug resistance.

Associations between abdominal obesity indices and pathological features of non-alcoholic fatty liver disease: Chinese visceral adiposity index.

BACKGROUND AND AIM: Many abdominal obesity indices such as waist circumference (WC), lipid accumulation product (LAP), visceral obesity index (VAI), and Chinese VAI (CVAI) have been considered to be associated with the risk of non-alcoholic fatty liver disease (NAFLD), but the association between abdominal obesity indices and the pathological features of NAFLD is uncertain. This study aims to explore the associations between these indices and the pathological features of NAFLD. METHODS: A total of 147 patients with biopsy-confirmed NAFLD were enrolled in the final analysis. General information, biochemical tests, and pathological information of patients were collected. VAI, LAP, and CVAI were calculated. Spearman's correlation analysis and logistics regression analysis were applied to assess the relationship between abdominal obesity indices and the pathological features of NAFLD. Receiver operating characteristic curve analyses were used to assess the value of abdominal obesity indices in predicting liver fibrosis and non-alcoholic steatohepatitis. RESULTS: Non-alcoholic fatty liver disease activity score (NAS) ≥ 5 significantly correlated with WC, LAP, VAI, and CVAI both in univariate and multivariate analyses (P < 0.05). Fibrosis was significantly and positively correlated with WC, LAP, and CVAI (P < 0.05). After adjustment for potential confounders, fibrosis remained associated with CVAI (P < 0.05). CONCLUSION: CVAI is significantly associated with the pathological features of NAFLD, and CVAI shows the most superior efficacy in diagnosing fibrosis among these indices.

Rational design, synthesis, antifungal evaluation and docking studies of antifungal peptide CGA-N12 analogues based on the target CtKRE9.

Candida tropicalis is a major non-albicans species that causes invasive candidiasis. CGA-N12, an anti-Candida peptide found by our group, disrupted cell wall architecture by inhibiting the activity of the protein killer-resistant 9 (KRE9), a ß-1,6-glucan synthase specific to Candida spp. and plants. Herein, a set of CGA-N12 analogues were rationally designed based on the interaction networks between CGA-N12 and C. tropicalis KRE9 (CtKRE9). Seven CGA-N12 analogues with significantly improved antifungal activity against C. tropicalis were screened by reducing the docking energy of CGA-N12 and CtKRE9 and increasing the number of positive charges on CGA-N12 based on a stable three-dimensional model of CtKRE9. CGA-N12 and its analogues exhibited antifungal activity against C. tropicalis and its persist cells; they also inhibited biofilm formation and eradicated preformed biofilms. Compared with fluconazole, they displayed higher activities against the growth of persister cells and more effective preformed biofilm eradication. Among them, CGA-N12-0801, CGA-N12-0902 and CGA-N12-1002 displayed much higher activity and anti-proteinase digestion stability than CGA-N12. Specifically, CGA-N12-0801 was the optimal analogue, with a minimum inhibitory concentration of 3.46 µg/mL and a therapeutic index of 158.07. The results of electronic microscopy observations and KRE9 activity inhibition assays showed that CGA-N12 and its analogues killed C. tropicalis by disrupting the architecture of the cell wall and the integrity of the cell membrane. In conclusion, for the first time, we provide a simple and reliable method for the rational design of antimicrobial peptides and ideal candidates for treating Candida infections that not effectively eliminated by azole drugs.

Metal-organic framework-modulated Fe3O4 composite au nanoparticles for antibacterial wound healing via synergistic peroxidase-like nanozymatic catalysis.

Bacterial wound infections are a serious threat due to the emergence of antibiotic resistance. Herein, we report an innovative hybrid nanozyme independent of antibiotics for antimicrobial wound healing. The hybrid nanozymes are fabricated from ultra-small Au NPs via in-situ growth on metal-organic framework (MOF)-stabilised Fe3O4 NPs (Fe3O4@MOF@Au NPs, FMA NPs). The fabricated hybrid nanozymes displayed synergistic peroxidase (POD)-like activities. It showed a remarkable level of hydroxyl radicals (·OH) in the presence of a low dose of H2O2 (0.97 mM). Further, the hybrid FMA nanozymes exhibited excellent biocompatibility and favourable antibacterial effects against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The animal experiments indicated that the hybrid nanozymes promoted wound repair with adequate biosafety. Thus, the well-designed hybrid nanozymes represent a potential strategy for healing bacterial wound infections, without any toxic side effects, suggesting possible applications in antimicrobial therapy.

Single-cell analysis reveals immune cellular components in odontogenic keratocysts.

OBJECTIVES: Various types of cells comprising a complex and diverse cell population are required for the biological activities of odontogenic keratocyst (OKC). Immune and non-immune cells collaborate via cytokine- or chemokine-mediated communication and direct cell-cell interactions. This study aimed to characterize the immune ecosystem and understand the potential chemotactic role of OKC fibroblasts in immune cell migration. MATERIALS AND METHODS: Mass cytometry of 41 markers was employed for the classification of OKC cells from six OKC samples. Immunofluorescence staining and single-cell RNA sequencing (GSE176351) were used for the detection of fibroblast subpopulations. Enzyme-linked immunosorbent assay and immunofluorescence staining were employed for chemokine detection in hypoxia- and/or HIF-1α inhibitor-treated OKC fibroblasts and tissues. Chemotaxis assay was employed to determine the chemotactic effect of fibroblasts via co-culture with peripheral blood mononuclear cells. A cell communication network was constructed based on the single-cell RNA sequencing data. RESULTS: The characterization of the immune cell types of OKC evidenced the enrichment of macrophages, neutrophils and B cells. The majority (41.5%) of fibroblast subsets consisted of chemokine ligand-enriched myofibroblasts. The activation of the HIF-1α signaling pathway in fibroblasts was associated with chemokine release. The chemokines released by OKC fibroblasts remarkably promoted the migration of peripheral blood mononuclear cells in the co-culture system. Close interactions between myofibroblasts and immune cells were validated by cell-cell interaction analysis. Increased RANKL expression was detected in OKC fibroblasts in the co-culture system with peripheral blood mononuclear cells. CONCLUSIONS: Our results provided deep insights into the immune ecosystem and highlighted the potential chemotactic effects of chemokine-enriched myofibroblasts within OKCs. The close interaction between immune cells and fibroblasts demonstrated in this study may be responsible for the osteoclastogenic effects of OKC fibroblasts.

Salivary non-apoptotic tumoral microvesicles: A potential progressive marker in oral cancer patients.

Tumour cell-secreted microvesicles (MVs) contribute immensely to tumour progression. However, the role of tumoral salivary MVs in oral squamous cell carcinoma (OSCC) remains unclear. Herein, we elucidated the role of non-apoptotic salivary tumoral MVs in OSCC development, especially relating to the migration ability. We purified and compared non-apoptotic salivary tumoral MVs from 63 OSCC patients and orthotopic OSCC mice model. Next, we compared the protein difference between apoptotic and non-apoptotic MVs by Western blot, proteomics and flow cytometry from saliva and CAL27 cells. Finally, we collected the non-apoptotic MVs and co-cultured with normal oral epithelial cells, the migration ability was examined by wound healing assay and Western blot assay. Our results indicated that the levels of non-apoptotic tumoral S-MVs were significantly higher in OSCC patients with T3 to T4 stages than in patients with T1 to T2 stages or healthy donors. In OSCC mice model, we found elevations of non-apoptotic tumoral MVs associated with tumoral volume. EGFR overexpression increased the generation of non-apoptotic tumoral MVs which could significantly promote normal epithelial cell migration. In conclusion, elevated levels of non-apoptotic tumoral S-MVs are associated with clinicopathologic features of OSCC patients, implying that non-apoptotic tumoral S-MVs are a potential progressive marker of OSCC.

Early high-dose continuous veno-venous hemofiltration alleviates the alterations of CD4+ T lymphocyte subsets in septic patients combined with acute kidney injury.

BACKGROUND: This study aims to determine whether early high-dose continuous venous-venous hemofiltration (CVVH) alleviates the alterations in CD4+ T lymphocyte subsets in septic patients combined with acute kidney injury. METHODS: Enrolled septic patients combined with acute kidney injury were randomized into CVVH (n = 50) and conventional treatment (non-CVVH, n = 53) groups. Healthy volunteers (n = 21) were enrolled. CVVH was initiated within 12 h of intensive care unit (ICU) admission with doses of 35~60 ml/kg/h and maintained for at least 72 h. Th1, Th2, Th17, and Treg were measured by flow cytometry on days 1, 3, and 7 of ICU admission. Sequential organ failure assessment (SOFA) scores were calculated. RESULTS: Th1 percentages and Th1/Th2 ratios were lower, and Th2, Th17, and Treg percentages and Th17/Treg ratios were higher in septic patients compared to healthy volunteers. CVVH significantly increased Th1 percentages and Th1/Th2 ratios, and significantly decreased Th2, Th17, and Treg percentages and Th17/Treg ratios compared to non-CVVH. Th1 percentages and Th1/Th2 ratios were negatively correlated with SOFA scores, while Th2, Th17, and Treg percentages and Th17/Treg ratios were positively correlated with SOFA scores. Patients with CVVH had significantly lower SOFA scores on day 7 of ICU admission and a shorter ICU stay compared to those with non-CVVH. CONCLUSIONS: Septic patients combined with acute kidney injury exhibit different alterations of CD4+ T lymphocyte subsets. Early high-dose CVVH alleviates the alterations, which may be one of the factors associated with improved sepsis severity.

Internalization and membrane activity of the antimicrobial peptide CGA-N12.

Antimicrobial peptides (AMPs) are conventional antibiotic alternatives due to their broad-spectrum antimicrobial activities and special mechanisms of action against pathogens. The antifungal peptide CGA-N12 was originally derived from human chromogranin A (CGA) and consists of the 65th to 76th amino acids of the CGA N-terminal region. In the present study, we found that CGA-N12 had fungicidal activity and exhibited time-dependent inhibition activity against Candida tropicalis. CGA-N12 entered the cells to exert its antagonist activity. The internalization of CGA-N12 was energy-dependent and accompanied by actin cytoskeleton-, clathrin-, sulfate proteoglycan-, endosome-, and lipid-depleting agent-mediated endocytosis. Moreover, the CGA-N12 internalization pathway was related to the peptide concentration. The effects of CGA-N12 on the cell membrane were investigated. CGA-N12 at a low concentration less than 4 × MIC100 did not destroy the cell membrane. While with increasing concentration, the damage to the cell membrane caused by CGA-N12 became more serious. At concentrations greater than 4 × MIC100, CGA-N12 destroyed the cell membrane integrity. Therefore, the membrane activity of CGA-N12 is concentration dependant.

The phytopathogen Xanthomonas campestris utilizes the divergently transcribed pobA/pobR locus for 4-hydroxybenzoic acid recognition and degradation to promote virulence.

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot in crucifers. Our previous findings revealed that Xcc can degrade 4-hydroxybenzoic acid (4-HBA) via the ß-ketoadipate pathway. This present study expands on this knowledge in several ways. First, we show that infective Xcc cells induce in situ biosynthesis of 4-HBA in host plants, and Xcc can efficiently degrade 4-HBA via the pobA/pobR locus, which encodes a 4-hydroxybenzoate hydroxylase and an AraC-family transcription factor respectively. Next, the transcription of pobA is specifically induced by 4-HBA and is positively regulated by PobR, which is constitutively expressed in Xcc. 4-HBA directly binds to PobR dimers, resulting in activation of pobA expression. Point mutation and subsequent isothermal titration calorimetry and size exclusion chromatography analysis identified nine key conserved residues required for 4-HBA binding and/or dimerization of PobR. Furthermore, overlapping promoters harboring fully overlapping -35 elements were identified between the divergently transcribed pobA and pobR. The 4-HBA/PobR dimer complex specifically binds to a 25-bp site, which encompasses the -35 elements shared by the overlapping promoters. Finally, GUS histochemical staining and subsequent quantitative assay showed that both pobA and pobR genes are transcribed during Xcc infection of Chinese radish, and the strain ΔpobR exhibited compromised virulence in Chinese radish. These findings suggest that the ability of Xcc to survive the 4-HBA stress might be important for its successful colonization of host plants.

Effects of N-terminal modifications on the stability of antimicrobial peptide SAMP-A4 analogues against protease degradation.

Infections with multidrug-resistant (MDR) pathogens are increasingly concerning for public health. Synthesized antimicrobial peptide A4 (SAMP-A4), a peptide computationally designed by our research team, is a potential drug candidate. However, the antimicrobial peptide SAMP-A4 is easily degraded in serum. To obtain SAMP-A4 analogues with high biostability, chemical modifications at its N-terminus, including fatty acid conjugation, glycosylation and PEGylation, were carried out. The results showed that the introduction of hydrophobic fatty acids at the N-terminus of SAMP-A4 is better than hydrophilic glycosylation and PEGylation. With increasing fatty acid chain length, the stability of SAMP-A4 analogues in serum and trypsin solutions is significantly improved, and the activities against MDR bacteria and Candida are significantly enhanced. There is no obvious change in haemolysis even when hexanoic acid is coupled with SAMP-A4, so the resulting analogue SAMP-A4-C6, SAMP-A4 conjugated with hexanoic acid, is the most likely of the analogues to become a drug.