This study was designed to assess the possibility of using bacteriophage-encoded endolysins for controlling planktonic and biofilm cells. The endolysins, LysEP114 and LysEP135, were obtained from plasmid vectors containing the endolysin genes derived from Escherichia coli phages. The high identity (>96%) was observed between LysEP114 and LysEP135. LysEP114 and LysEP135 were characterized by pH, thermal, and lactic acid stability, lytic spectrum, antibacterial activity, and biofilm eradication. The molecular masses of LysEP114 and LysEP135 were 18.2 kDa, identified as muramidases. LysEP114 and LysEP135 showed high lytic activity against the outer membrane-permeabilized E. coli KCCM 40405 at below 37°C, between pH 5 to 11, and below 70 mM of lactic acid. LysEP114 and LysEP135 showed the broad rang of lytic activity against E. coli KACC 10115, S. Typhimurium KCCM 40253, S. Typhimurium CCARM 8009, tetracycline-resistant S. Typhimurium, polymyxin B-resistant S. Typhimurium, chloramphenicol-resistant S. Typhimurium, K. pneumoniae ATCC 23357, K. pneumoniae CCARM 10237, and Shigella boydii KACC 10792. LysEP114 and LysEP135 effectively reduced the numbers of planktonic E. coli KCCM by 1.7 and 2.1 log, respectively, when treated with 50 mM lactic acid. The numbers of biofilm cells were reduced from 7.3 to 4.1 log CFU/ml and 2.2 log CFU/ml, respectively, when treated with LysEP114- and LysEP135 in the presence of 50 mM lactic acid. The results suggest that the endolysins in combination with lactic acid could be potential alternative therapeutic agents for controlling planktonic and biofilm cells.

Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3\'→5\' exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5\'-bond of phosphodiester from 3\' to 5\' end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3\'-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

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Lipases play a vital role in various biological processes, from lipid metabolism to industrial applications. However, the ever-evolving challenges and diverse substrates necessitate the continual exploration of novel high-performance lipases. In this study, we employed an in silico mining approach to search for lipases with potential high sn-1,3 selectivity and catalytic activity. The identified novel lipase, PLL, from Paenibacillus larvae subsp. larvae B-3650 exhibited a specific activity of 111.2 ± 5.5 U/mg towards the substrate p-nitrophenyl palmitate (pNPP) and 6.9 ± 0.8 U/mg towards the substrate olive oil when expressed in Escherichia coli (E. coli). Computational design of cysteine mutations was employed to enhance the catalytic performance of PLL. Superior stability was achieved with the mutant K7C/A386C/H159C/K108C (2M3/2M4), showing an increase in melting temperature (Tm) by 1.9°C, a 2.05-fold prolonged half-life at 45°C, and no decrease in enzyme activity. Another mutant, K7C/A386C/A174C/A243C (2M1/2M3), showed a 4.9-fold enhancement in specific activity without compromising stability. Molecular dynamics simulations were conducted to explore the mechanisms of these two mutants. Mutant 2M3/2M4 forms putative disulfide bonds in the loop region, connecting the N- and C-termini of PLL, thus enhancing overall structural rigidity without impacting catalytic activity. The cysteines introduced in mutant 2M1/2M3 not only form new intramolecular hydrogen bonds but also alter the polarity and volume of the substrate-binding pocket, facilitating the entry of large substrate pNPP. These results highlight an efficient in silico exploration approach for novel lipases, offering a rapid and efficient method for enhancing catalytic performance through rational protein design.

Philadelphia chromosome-positive (Ph+) leukemia is a fatal hematological malignancy. Although standard treatments with tyrosine kinase inhibitors (TKIs) have achieved remarkable success in prolonging patient survival, intolerance, relapse, and TKI resistance remain serious issues for patients with Ph+ leukemia. Here, we report a new leukemogenic process in which RAPSYN and BCR-ABL co-occur in Ph+ leukemia, and RAPSYN mediates the neddylation of BCR-ABL. Consequently, neddylated BCR-ABL enhances the stability by competing its c-CBL-mediated degradation. Furthermore, SRC phosphorylates RAPSYN to activate its NEDD8 E3 ligase activity, promoting BCR-ABL stabilization and disease progression. Moreover, in contrast to in vivo ineffectiveness of PROTAC-based degraders, depletion of RAPSYN expression, or its ligase activity decreased BCR-ABL stability and, in turn, inhibited tumor formation and growth. Collectively, these findings represent an alternative to tyrosine kinase activity for the oncoprotein and leukemogenic cells and generate a rationale of targeting RAPSYN-mediated BCR-ABL neddylation for the treatment of Ph+ leukemia.
Chronic myeloid leukemia (CML for short) accounts for about 15% of all blood cancers diagnosed in adults in the United States. The condition is characterized by the overproduction of immature immune cells that interfere with proper blood function. It is linked to a gene recombination (a type of mutation) that leads to white blood cells producing an abnormal ‘BCR-ABL’ enzyme which is always switched on. In turn, this overactive protein causes the cells to live longer and divide uncontrollably. Some of the most effective drugs available to control the disease today work by blocking the activity of BCR-ABL. Yet certain patients can become resistant to these treatments over time, causing them to relapse. Other approaches are therefore needed to manage this disease; in particular, a promising avenue of research consists in exploring whether it is possible to reduce the amount of the enzyme present in diseased cells. As part of this effort, Zhao, Dai, Li, Zhang et al. focused on RAPSYN, a scaffolding protein previously unknown in CML cells. In other tissues, it has recently been shown to participate in neddylation – a process by which proteins receive certain chemical ‘tags’ that change the way they behave. The experiments revealed that, compared to healthy volunteers, RAPSYN was present at much higher levels in the white blood cells of CML patients. Experimentally lowering the amount of RAPSYN in CML cells led these to divide less quickly – both in a dish and when injected in mice, while also being linked to decreased levels of BCR-ABL. Additional biochemical experiments indicated that RAPSYN sticks with BCR-ABL to add chemical ‘tags’ that protect the abnormal protein against degradation, therefore increasing its overall levels. Finally, the team showed that SRC, an enzyme often dysregulated in emerging cancers, can activate RAPSYN’s ability to conduct neddylation; such mechanism could promote BCR-ABL stabilization and, in turn, disease progression. Taken together, these experiments indicate a new way by which BCR-ABL levels are controlled. Future studies should investigate whether RAPSYN also stabilizes BCR-ABL in patients whose leukemias have become resistant to existing drugs. Eventually, RAPSYN may offer a new target for overcoming drug-resistance in CML patients.

This study aims to present a comprehensive study on the risks associated with the residual presence and transport of Escherichia coli (E. coli) in soil following the application of livestock manure in Chinese farmlands by integrating machine learning algorithms with mechanism-based models (Phydrus). We initially review 28 published papers to gather data on E. coli\'s die-off and attachment characteristics in soil. Machine learning models, including deep learning and gradient boosting machine, are employed to predict key parameters such as the die-off rate of E. coli and first-order attachment coefficient in soil. Then, Phydrus was used to simulate E. coli transport and survival in 23692 subregions in China. The model considered regional differences in E. coli residual risk and transport, influenced by soil properties, soil depths, precipitation, seasonal variations, and regional disparities. The findings indicate higher residual risks in regions such as the Northeast China, Eastern Qinghai-Tibet Plateau, and pronounced transport risks in the fringe of the Sichuan Basin fringe, the Loess Plateau, the North China Plain, the Northeast Plain, the Shigatse Basin, and the Shangri-La region. The study also demonstrates a significant reduction in both residual and transport risks one month after manure application, highlighting the importance of timing manure application and implementing region-specific standards. This research contributes to the broader understanding of pathogen behavior in agricultural soils and offers practical guidelines for managing the risks associated with manure use. This study\'s comprehensive method offers a potentially valuable tool for evaluating microbial contaminants in agricultural soils across the globe.

BACKGROUND: Medical dressings are designed to promote wound healing and reduce infection. The aim of project is to investigate the effect of natural brown colored cotton dressings on the healing of infected wounds in E.coli animals.
METHODS: In this study, degreased white cotton gauze was used as the control group, with degreased brown cotton gauze and degreased bleached brown cotton gauze as the experimental group 1 and experimental group 2, to investigate the effect on the repair of post-infectious wound damage in animals by establishing an infected wound model in rats with E.coli as the infecting organism.
RESULTS: The ability to promote healing of infected wounds was investigated by analyzing the wound healing status, macroscopic wound healing rate, hematoxylin-eosin staining, Masson staining, secretion of inflammatory factors by Elisa assay. The result showed that at day 14 of wound healing, the macroscopic wound healing rate was greater than 98% for all three groups of dressings; the collagen content reached 49.85 ± 5.84% in the experimental group 1 and 53.48 ± 5.32% in the experimental group 2, which was higher than the control group; brown cotton gauze promotes skin wound healing by shortening the inflammatory period in both groups. The expression of three inflammatory factors THF-α, IL-2, and IL-8 and three cytokines MMP-3, MMP-8, and MMP-9 were lower than that of the control group.
CONCLUSIONS: It was found that natural brown cotton gauze has better repairing and promoting healing effect on infected wounds. It opens up the application of natural brown cotton gauze in the treatment of infected wounds.

The metabolic environment is responsible for antibiotic resistance, which highlights the way in which the antibiotic resistance mechanism works. Here, GC-MS-based metabolomics with iTRAQ-based proteomics was used to characterize a metabolic state in tetracycline-resistant Escherichia coli K12 (E. coli-RTET) compared with tetracycline-sensitive E. coli K12. The repressed pyruvate cycle against the elevation of the proton motive force (PMF) and ATP constructed the most characteristic feature as a consequence of tetracycline resistance. To understand the role of the elevated PMF in tetracycline resistance, PMF inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the pH gradient were used to investigate how the elevation influences bacterial viability and intracellular antibiotic concentration. A strong synergy was detected between CCCP and tetracycline to the viability, which was consistent with increasing intracellular drug and decreasing external pH. Furthermore, E. coli-RTET and E. coli-RGEN with high and low PMF concentrations were susceptible to gentamicin and tetracycline, respectively. The elevated PMF in E. coli-RTET was attributed to the activation of other metabolic pathways, except for the pyruvate cycle, including a malate-oxaloacetate-phosphoenolpyruvate-pyruvate-malate cycle. These results not only revealed a PMF-dependent mechanism for tetracycline resistance but also provided a solution to tetracycline-resistant pathogens by aminoglycosides and aminoglycoside-resistant bacteria by tetracyclines.

Bacteria in biofilms secrete potassium ions to attract free swimming cells. However, the basis of chemotaxis to potassium remains poorly understood. Here, using a microfluidic device, we found that Escherichia coli can rapidly accumulate in regions of high potassium concentration on the order of millimoles. Using a bead assay, we measured the dynamic response of individual flagellar motors to stepwise changes in potassium concentration, finding that the response resulted from the chemotaxis signaling pathway. To characterize the chemotactic response to potassium, we measured the dose-response curve and adaptation kinetics via an Förster resonance energy transfer (FRET) assay, finding that the chemotaxis pathway exhibited a sensitive response and fast adaptation to potassium. We further found that the two major chemoreceptors Tar and Tsr respond differently to potassium. Tar receptors exhibit a biphasic response, whereas Tsr receptors respond to potassium as an attractant. These different responses were consistent with the responses of the two receptors to intracellular pH changes. The sensitive response and fast adaptation allow bacteria to sense and localize small changes in potassium concentration. The differential responses of Tar and Tsr receptors to potassium suggest that cells at different growth stages respond differently to potassium and may have different requirements for potassium.