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Pseudomonas aeruginosa is a multidrug-resistant Gram-negative pathogen and one of the leading causes of ventilator-associated pneumonia and infections in patients with chronic obstructive pulmonary disease and cystic fibrosis. Murepavadin is a peptidomimetic that specifically targets outer-membrane lipopolysaccharide transport protein LptD of P. aeruginosa. In this study, we find that murepavadin enhances the bactericidal efficacy of ciprofloxacin. We further demonstrate that murepavadin increases intracellular accumulation of ciprofloxacin by suppressing drug efflux. In addition, the murepavadin–ciprofloxacin combination exhibits a synergistic bactericidal effect in an acute murine pneumonia model. In conclusion, our results identify an effective drug combination for the treatment of P. aeruginosa infections. [ABSTRACT FROM AUTHOR]

Drying involves the evaporation of moisture, accompanied by simultaneous heat transfer, mass transfer, and momentum transfer. While the diffusion law is considered an applicable model for explaining the drying phenomenon, the actual drying process cannot be accurately predicted using an analytical solution with a constant diffusion coefficient. Energy efficiency in the drying process is low due to an insufficient understanding of the mechanisms governing moisture migration from solids to air. The development of drying theory has stalled due to an unsolvable discrepancy between experimental results and analytical results. This study analyzes the effect of the binding energy of moisture in paddy rice on the diffusion coefficient. The theoretical relationship between water activity and drying rate in paddy rice was investigated, and the drying process was successfully explained by analyzing free energy transfer and transition theory. The mechanism of rice grain drying was described using a new theoretical solution for the drying process. These results provide new insights into the development of a scientific evaluation standard for assessing the efficiency of actual drying processes. [ABSTRACT FROM AUTHOR]

Ehrlichia chaffeensis infects and proliferates inside monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening tick-borne zoonosis. After internalization, E. chaffeensis resides in specialized membrane-bound inclusions, E. chaffeensis-containing vesicles (ECVs), to evade from host cell innate immune responses and obtain nutrients. However, mechanisms exploited by host cells to inhibit E. chaffeensis growth in ECVs are still largely unknown. Here we demonstrate that host cells recognize E. chaffeensis Ech_1067, a penicillin-binding protein, and then upregulate the expression of PIT1, which is a phosphate transporter and transports phosphate from ECVs to the cytosol to inhibit bacterial growth. We found that host cells upregulate the PIT1 expression upon E. chaffeensis infection using transcriptome sequencing, qRT-PCR and Western blotting, and PIT1 is localized on the ECV membrane in infected THP-1 cells using confocal microscopy. Silence of PIT1 using shRNA enhances E. chaffeensis intracellular growth. Finally, we found that E. chaffeensis Ech_1067 induces the upregulation of PIT1 expression through the MyD88-NF-κB pathway using recombinant protein for stimulation and siRNA for silence. Our findings deepen the understanding of the innate immune responses of host cells to inhibit bacterial intracellular growth and facilitate the development of new therapeutics for HME. [ABSTRACT FROM AUTHOR]

In order to improve the efficiency of fine segmentation and obstacle removal in the road of tea plantation in hilly areas, a lightweight and high-precision DR-YOLO instance segmentation algorithm is proposed to realize environment awareness. Firstly, the road data of tea gardens in hilly areas were collected under different road conditions and light conditions, and data sets were generated. YOLOv8n-seg, which has the highest operating efficiency, was selected as the basic model. The MSDA-CBAM and DR-Neck feature fusion network were added to the YOLOv8-seg model to improve the feature extraction capability of the network and the feature fusion capability and efficiency of the model. Experimental results show that, compared with the YOLOv8-seg model, the DR-YOLO model proposed in this study has 2.0% improvement in AP@0.5 and 1.1% improvement in Precision. In this study, the DR-YOLO model is pruned and quantitatively compressed, which greatly improves the model inference speed with little reduction in AP. After deploying on Jetson, compared with the YOLOv8n-seg model, the Precision of DR-YOLO is increased by 0.6%, the AP@0.5 is increased by 1.6%, and the inference time is reduced by 17.1%, which can effectively improve the level of agricultural intelligent automation and realize the efficient operation of the instance segmentation model at the edge. [ABSTRACT FROM AUTHOR]

Water scarcity poses a significant challenge for people living in arid areas. Despite the effectiveness of many bioinspired surfaces in promoting vapor condensation, their water-harvesting efficiency is insufficient. This is often exacerbated by overheating, which decreases the performance in terms of the micro-droplet concentration and movement on surfaces. In this study, we used a spotted amphiphilic surface to enhance the surfaces' water-harvesting efficiency while maintaining their heat emissivity. Through hydrophilic particle screening and hydrophobic groove modifying, the coalescence and sliding characteristics of droplets on the amphiphilic surfaces were improved. The incorporation of boron nitride (BN) nanoparticles further enhanced the surfaces' ability to harvest energy from condensation. To evaluate the water-harvesting performance of these amphiphilic surfaces, we utilized a real-time recording water-harvesting platform to identify microscopic weight changes on the surfaces. Our findings indicated that the inclusion of glass particles in hydrophobic grooves, combined with 1.0 wt.% BN nanoparticles, enhanced the water-harvesting efficiency of the amphiphilic surfaces by more than 20%. [ABSTRACT FROM AUTHOR]

Ehrlichia chaffeensis infects human monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening zoonosis. After internalization, E. chaffeensis resides in membrane-bound inclusions, E. chaffeensis-containing vesicles (ECVs), which have early endosome-like characteristics and fuse with early autophagosomes but not lysosomes, to evade host innate immune microbicidal mechanisms and obtain nutrients for bacterial intracellular growth. The mechanisms exploited by E. chaffeensis to modulate intracellular vesicle trafficking in host cells have not been comprehensively studied. Here, we demonstrate that E. chaffeensis type IV secretion system (T4SS) effector Etf-3 induces RAB15 upregulation in host cells and that RAB15, which is localized on ECVs, inhibits ECV fusion with lysosomes and induces autophagy. We found that E. chaffeensis infection upregulated RAB15 expression using qRT-PCR, and RAB15 was colocalized with E. chaffeensis using confocal microscopy. Silence of RAB15 using siRNA enhanced ECV maturation to late endosomes and fusion with lysosomes, as well as inhibited host cell autophagy. Overexpression of Etf-3 in host cells specifically induced RAB15 upregulation and autophagy. Our findings deepen the understanding of E. chaffeensis pathogenesis and adaptation in hosts as well as the function of RAB15 and facilitate the development of new therapeutics for HME. [ABSTRACT FROM AUTHOR]

Nerve agents are one of the most dangerous and toxic chemical warfare agents (CWAs). Their rapid detection is very important for specialized persons to take action against these substances. In this work, a salicylaldoximate-based aggregation-induced emission (AIE) probe, 2-hydroxy-5-(1,2,2-triphenylvinyl) benzoxime (TPE-D), was rationally designed for this purpose. The AIE fluorescent sensor has a tetraphenylethylene (TPE) unit combined with a salicylaldoximate moiety as the reaction site for a nerve agent simulant, diethyl chlorophosphate (DCP). TPE-D successively undergoes the formation of nitrile and the phosphorylation of a phenolic hydroxyl group after the salicylaldoximate moiety reacts with DCP, which will give a significant fluorescence "light-up" response due to the AIE effect within minutes. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a ubiquitous pathogen that causes a wide range of acute and chronic infections. Ciprofloxacin, one of the first-line fluoroquinolone class antibiotics, is commonly used for the treatment of P. aeruginosa infections. However, ciprofloxacin-resistant P. aeruginosa is increasingly reported worldwide, making treatment difficult. To determine resistance-related mutations, we conducted an experimental evolution using a previously identified ciprofloxacin-resistant P. aeruginosa clinical isolate, CRP42. The evolved mutants could tolerate a 512-fold higher concentration of ciprofloxacin than CRP42. Genomic DNA reference mapping was performed, which revealed mutations in genes known to be associated with ciprofloxacin resistance as well as in those not previously linked to ciprofloxacin resistance, including the ParER586W substitution and PA0625 frameshift insertion. Simulation of the ParER586W substitution and PA0625 frameshift insertion by gene editing in CRP42 and the model strain PAO1 demonstrated that while the PA0625 mutation does contribute to resistance, mutation in the ParER586W does not contribute to resistance but rather affects tolerance against ciprofloxacin. These findings advance our understanding of ciprofloxacin resistance in P. aeruginosa. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an important nosocomial pathogen which frequently becomes resistant to most antibiotics used in chemotherapy, resulting in treatment failure among infected individuals. Although the evolutionary trajectory and molecular mechanisms for becoming β-lactam resistant have been well established for P. aeruginosa, the molecular basis of reversion from β-lactam resistant to susceptible is largely unexplored. In this study, we investigated the molecular mechanisms by which a ceftazidime-resistant clinical strain is converted to a ceftazidime-susceptible isolate under the clinical setting. RNA sequencing and genomic DNA reference mapping were conducted to compare the transcriptional profiles and chromosomal mutations between these two isolates. Our results demonstrate that a gain-of-function mutation in ampD, via deletion of a 53 bp duplicated nucleotide sequence, is the contributory factor for the conversion. Furthermore, we show for the first time that AmpD is involved in intraspecies competitiveness in P. aeruginosa. We also found that AmpD is not responsible for phenotypic changes between R1 and S2, including growth rate, motilities, pyocyanin, rhamnolipid, and biofilm production. This finding provides novel insights into the alteration of β-lactam sensitivity in P. aeruginosa under the clinical setting. [ABSTRACT FROM AUTHOR]

Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a great challenge to clinical treatment. In this study, we characterized a ST768 MDR P. aeruginosa strain, Pa150, that was isolated from a diabetic foot patient. The minimum inhibitory concentration (MIC) assay showed that Pa150 was resistant to almost all kinds of antibiotics, especially aminoglycosides. Whole genome sequencing revealed multiple antibiotic resistant genes on the chromosome and a 437-Kb plasmid (named pTJPa150) that harbors conjugation-related genes. A conjugation assay verified its self-transmissibility. On the pTJPa150 plasmid, we identified a 16S rRNA methylase gene, rmtB, that is flanked by mobile genetic elements (MGEs). The transfer of the pTJPa150 plasmid or the cloning of the rmtB gene into the reference strain, PAO1, significantly increased the bacterial resistance to aminoglycoside antibiotics. To the best of our knowledge, this is the first report of an rmtB-carrying conjugative plasmid isolated from P. aeruginosa, revealing a novel possible transmission mechanism of the rmtB gene. [ABSTRACT FROM AUTHOR]

Laser powder bed fusion (LPBF) additive manufacturing has been advancing in the fabrication of metallic multi-material structures with intricate structures and refined material layouts. Herein, a comprehensive review of the recent achievements of multi-material structures via LPBF is provided in terms of interface characteristics and strengthening methods, critical technical issues and potential applications. It begins with the introduction of multi-material structures and the scope of the review. The interface characteristics (including representative multi-material types printed by LPBF, interfacial microstructure, defects, etc.) and strengthening methods of multi-material structures are then presented. Thereafter, the critical technical issues in LPBF for multi-material structures are discussed with regard to equipment development, data preparation, thermodynamic calculation and process simulation, and powder cross-contamination and recycling. Moreover, the potential applications (particularly in biomedical, electronic, aerospace) are illustrated and discussed. Finally, the outlook is outlined to provide guidance for future research. [ABSTRACT FROM AUTHOR]

The type III secretion system (T3SS) of Pseudomonas aeruginosa plays a significant role in pathogenesis. We have previously identified type III secretion factor (TSF), which is required for effective secretion of the type III effector molecules, in addition to the low calcium signal. TSF includes many low-affinity high-capacity calcium binding proteins, such as serum albumin and casein. A search for the TSF binding targets on the bacterial outer membrane resulted in identification of PopN, a component of the T3SS that is readily detectable on the bacterial cell surface. PopN specifically interacts with Pcr1, and both popN and pcr1 mutants have a constitutive type III secretion phenotype, suggesting that the two proteins form a complex that functions as a T3SS repressor. Further analysis of the popN operon genes resulted in identification of protein-protein interactions between Pcr1 and Pcr4 and between Pcr4 and Pcr3, as well as between PopN and Pcr2 in the presence of PscB. Unlike popN and pcr1 mutants, pcr3 and pcr4 mutants are totally defective in type III secretion, while apcr2 mutant exhibits reduced type III secretion. Interestingly, PopN, Pcr1, Pcr2, and Pcr4 are all secreted in a type III secretion machinery-dependent manner, while Pcr3 is not. These findings imply that these components have important regulatory roles in controlling type III secretion.

In a search for regulatory genes of the type III secretion system (TTSS) in Pseudomonas aeruginosa, transposon (Tn5) insertional mutants of the prtR gene were found defective in the TTSS. PrtR is an inhibitor of prtN, which encodes a transcriptional activator for pyocin synthesis genes. In P. aeruginosa, pyocin synthesis is activated when PrtR is degraded during the SOS response. Treatment of a wild-type P. aeruginosa strain with mitomycin C, a DNA-damaging agent, resulted in the inhibition of TTSS activation. A prtR/prtN double mutant had the same TTSS defect as the prtR mutant, and complementation by a prtR gene but not by a prtN gene restored the TTSS function. Also, overexpression of the prtN gene in wild-type PAK had no effect on the TTSS; thus, PrtN is not involved in the repression of the TTSS. To identify the PrtR-regulated TTSS repressor, another round of Tn mutagenesis was carried out in the background of a prtR/prtN double mutant. Insertion in a small gene, designated ptrB, restored the normal TTSS activity. Expression of ptrB is specifically repressed by PrtR, and mitomycin C-mediated suppression of the TTSS is also abolished in a ptrB mutant strain. Therefore, PtrB is a new TTSS repressor that coordinates TTSS repression and pyocin synthesis under the stress of DNA damage.

The type III secretion system (T3SS) of Pseudomonas aeruginosa is an important virulence factor. The T3SS of P. aeruginosa can be induced by a low calcium signal or upon direct contact with the host cells. The exact pathway of signal sensing and T3SS activation is not clear. By screening a transposon insertion mutant library of the PAK strain, mutation in the mucA gene was found to cause repression of T3SS expression under both type III-inducing and -noninducing conditions. Mutation in the mucA gene is known to cause alginate overproduction, resulting in a mucoid phenotype. Alginate production responds to various environmental stresses and plays a protective role for P. aeruginosa. Comparison of global gene expression of mucA mutant and wild-type PAK under T3SS-inducing conditions confirmed the down regulation of T3SS genes and up regulation of genes involved in alginate biosynthesis. Further analysis indicated that the repression of T3SS in the mucA mutant was AlgU and AlgR dependent, as double mutants mucA/algU and mucA/algR showed normal type III expression. An algR::Gm mutant showed a higher level of type III expression, while overexpression of the algR gene inhibited type III gene expression; thus, it seems that the AlgR-regulated product inhibits the expression of the T3SS genes. It is likely that P. aeruginosa has evolved tight regulatory networks to turn off the energy-expensive T3SS when striving for survival under environmental stresses.

Ehrlichia chaffeensis , a small Gram-negative obligatory intracellular bacterium, infects human monocytes or macrophages, and causes human monocytic ehrlichiosis, one of the most prevalent, life-threatening emerging zoonoses. Reactive oxygen species are produced by the host immune cells in response to bacterial infections. The mechanisms exploited by E. chaffeensis to resist oxidative stress have not been comprehensively demonstrated. Here, we found that E. chaffeensis encodes two functional enzymes, GshA and GshB, to synthesize glutathione that confers E. chaffeensis the oxidative stress resistance, and that the expression of gshA and gshB is upregulated by CtrA, a global transcriptional regulator, upon oxidative stress. We found that in E. chaffeensis , the expression of gshA and gshB was upregulated upon oxidative stress using quantitative RT-PCR. Ehrlichia chaffeensis GshA or GshB restored the ability of Pseudomonas aeruginosa GshA or GshB mutant to cope with oxidative stress, respectively. Recombinant E. chaffeensis CtrA directly bound to the promoters of gshA and gshB , determined with electrophoretic mobility shift assay, and activated the expression of gshA and gshB determined with reporter assay. Peptide nucleic acid transfection of E. chaffeensis , which reduced the CtrA protein level, inhibited the oxidative stress-induced upregulation of gshA and gshB. Our findings provide insights into the function and regulation of the two enzymes critical for E. chaffeensis resistance to oxidative stress and may deepen our understanding of E. chaffeensis pathogenesis and adaptation in hosts. [ABSTRACT FROM AUTHOR]

Carbon metabolism plays an important role in bacterial physiology and pathogenesis. The type III secretion system (T3SS) of Pseudomonas aeruginosa is a virulence factor that contributes to acute infections. It has been demonstrated that bacterial metabolism affects the T3SS. Meanwhile, expression of T3SS genes is negatively regulated by the small RNAs RsmY and RsmZ. In this study, we studied the relationship between the dihydrolipoamide acetyltransferase gene aceF and the T3SS. Our results reveal an upregulation of RsmY and RsmZ in the aceF mutant, which represses the expression of the T3SS genes. Meanwhile, the aceF mutant is more tolerant to hydrogen peroxide. We demonstrate that the expression levels of the catalase KatB and the alkyl hydroperoxide reductase AhpB are increased in the aceF mutant. The simultaneous deletion of rsmY and rsmZ in the aceF mutant restored the expression levels of katB and ahpB, as well as bacterial susceptibility to hydrogen peroxide. Thus, we identify a novel role of AceF in the virulence and oxidative response of P. aeruginosa. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an important opportunistic pathogen capable of causing variety of infections in humans. The type III secretion system (T3SS) is a critical virulence determinant of P. aeruginosa in the host infections. Expression of the T3SS is regulated by ExsA, a master regulator that activates the expression of all known T3SS genes. Expression of the exsA gene is controlled at both transcriptional and posttranscriptional levels. Here, we screened a P. aeruginosa transposon (Tn5) insertional mutant library and found rplI, a gene coding for the ribosomal large subunit protein L9, to be a repressor for the T3SS gene expression. Combining real-time quantitative PCR (qPCR), western blotting and lacZ fusion assays, we show that RplI controls the expression of exsA at the posttranscriptional level. Further genetic experiments demonstrated that RplI mediated control of the exsA translation involves 5' untranslated region (5' UTR). A ribosome immunoprecipitation assay and qPCR revealed higher amounts of a 24 nt fragment from exsA mRNA being associated with ribosomes in the ΔrplI mutant. An interaction between RplI and exsA mRNA harboring its 24 nt, but not 12 nt, 5' UTR was confirmed by RNA Gel Mobility Shift and Microscale Thermophoresis assays. Overall, this study identifies the ribosomal large subunit protein L9 as a novel T3SS repressor that inhibits ExsA translation in P. aeruginosa. Author summary: Ribosomes provide all living organisms the capacity to synthesize proteins. The production of many ribosomal proteins is often controlled by an autoregulatory feedback mechanism. P. aeruginosa is an opportunistic human pathogen and its type III secretion system (T3SS) is a critical virulence determinant in host infections. In this study, by screening a Tn5 mutant library, we identified rplI, encoding ribosomal large subunit protein L9, as a novel repressor for the T3SS. Further exploring the regulatory mechanism, we found that the RplI protein interacts with the 5' UTR (5' untranslated region) of exsA, a gene coding for transcriptional activator of the T3SS. Such an interaction likely blocks ribosome loading on the exsA 5' UTR, inhibiting the initiation of exsA translation. The significance of this work is in the identification of a novel repressor for the T3SS and elucidation of its molecular mechanism. Furthermore, this work provides evidence for individual ribosomal protein regulating mRNA translation beyond its autogenous feedback control. [ABSTRACT FROM AUTHOR]

The emergence and rapid spread of multi-drug resistant (MDR) bacteria pose a serious threat to global healthcare. There is an urgent need for new antibacterial substances or new treatment strategies to deal with the infections by MDR bacterial pathogens, especially the Gram-negative pathogens. In this study, we show that a number of synthetic cationic peptides display strong synergistic antimicrobial effects with multiple antibiotics against the Gram-negative pathogen Pseudomonas aeruginosa. We found that an all-D amino acid containing peptide called D-11 increases membrane permeability by attaching to LPS and membrane phospholipids, thereby facilitating the uptake of antibiotics. Subsequently, the peptide can dissipate the proton motive force (PMF) (reduce ATP production and inhibit the activity of efflux pumps), impairs the respiration chain, promote the production of reactive oxygen species (ROS) in bacterial cells and induce intracellular antibiotics accumulation, ultimately resulting in cell death. By using a P. aeruginosa abscess infection model, we demonstrate enhanced therapeutic efficacies of the combination of D-11 with various antibiotics. In addition, we found that the combination of D-11 and azithromycin enhanced the inhibition of biofilm formation and elimination of established biofilms. Our study provides a realistic treatment option for combining close-to-nature synthetic peptide adjuvants with existing antibiotics to combat infections caused by P. aeruginosa. Author summary: Antimicrobial resistance is a global public health problem that is limiting our antibiotic capacity to control bacterial infections. Despite the enormous efforts in drug development, very few clinically relevant drugs have been approved in the last decades. This is especially critical for Gram-negative bacteria, which remain the most problematic group, because, among other reasons, the outer membrane acts as a permeability barrier preventing antibiotics to reach their targets. Here, we describe the action mechanism through which a human close-to-nature synthetic "helper" peptide is able to sensitize Pseudomonas aeruginosa to a broad range of antibiotics for which it is normally resistant. We demonstrate that this peptide is active at different levels (membrane, proton motive force, respiration, ATP synthesis, ROS accumulation and efflux pump activity), facilitating antibiotic accumulation inside the bacteria and promoting cell death. Moreover, we have determined the efficacy of the synergistic combinations in complex relevant environments, such as Pseudomonas biofilms, human blood and in vivo in a mice infection model. This work demonstrates the effectiveness of synergistic combinations for fighting an important lung pathogen, providing a new therapeutical option for the treatment of P. aeruginosa infection and extending the use of the current antibiotics. [ABSTRACT FROM AUTHOR]

Therapy for Pseudomonas aeruginosa infections is hard due to its high natural and acquirable antibiotic resistance. After colonization in the hosts, P. aeruginosa commonly accumulates genomic mutations which confer them antibiotic resistance and better adaptations to the host environment. Deciphering the mechanisms of antibiotic resistance development in the clinical setting may provide critical insights into the design of effective combinatory antibiotic therapies to treat P. aeruginosa infections. In this work, we demonstrate a resistance mechanism to aztreonam of a clinical isolate (ARP36) in comparison with a sensitive one (CSP18). RNAseq and genomic DNA resequencing were carried out to compare the global transcriptional profiles and in the clinical setting genomic profiles between these two isolates. The results demonstrated that hyperexpression of an efflux pump MexAB-OprM caused by a R70Q substitution in MexR, contributed to the increased resistance to aztreonam in the isolate ARP36. Simulation of mexR of ARP36 by gene editing in CSP18 conferred CSP18 an ARP36-like susceptibility to the aztreonam. The R70Q substitution prevented MexR from binding to the intergenic region between mexR and mexAB-oprM operon, with no impact on its dimerization. The presented experimental results explain for the first time why the clinically relevant R70Q substitution in the MexR derepresses the expression of mexAB-oprM in P. aeruginosa. [ABSTRACT FROM AUTHOR]