Suppression of Pseudomonas aeruginosa type III secretion system by a novel calcium-responsive signaling pathway.

Expression of the type III secretion system (T3SS) in Pseudomonas aeruginosa is exquisitely controlled by diverse environmental or host-related signals such as calcium (Ca2+), however, the signal transduction pathways remain largely elusive. In this study, we reported that FleR, the response regulator of the two-component system FleS/FleR, inhibits T3SS gene expression and virulence of P. aeruginosa uncoupled from its cognate histidine kinase FleS. Interestingly, FleR was found to repress T3SS gene expression under Ca2+-rich conditions independently of its DNA-binding domain. FleR activates the elevation of intracellular c-di-GMP contents and FleQ serves as the c-di-GMP effector to repress T3SS gene expression through the Gac/Rsm pathway. Remarkably, we found that AmrZ, a member of the FleR regulon, inhibits T3SS gene expression by directly targeting the promoter of exsCEBA in an expression level-dependent manner. This study revealed an intricate regulatory network that connects P. aeruginosa T3SS gene expression to the Ca2+ signal.

A type I-F CRISPRi system unveils the novel role of CzcR in modulating multidrug resistance of Pseudomonas aeruginosa.

Pseudomonas aeruginosa has abundant signaling systems that exquisitely control its antibiotic resistance in response to different environmental cues. Understanding the regulation of antibiotic resistance will provide important implications for precise antimicrobial interventions. However, efficient genetic tools for functional gene characterizations are sometimes not available, particularly, in clinically isolated strains. Here, we established a type I-F CRISPRi (CSYi) system for programmable gene silencing. By incorporating anti-CRISPR proteins, this system was even applicable to bacterial hosts encoding a native type I-F CRISPR-Cas system. With the newly developed gene-silencing system, we revealed that the response regulator CzcR from the zinc (Zn2+)-responsive two-component system CzcS/CzcR is a repressor of efflux pumps MexAB-OprM and MexGHI-OpmD, which inhibits the expression of both operons by directly interacting with their promoters. Repression of MexAB-OprM consequently increases the susceptibility of P. aeruginosa to multiple antibiotics such as levofloxacin and amikacin. Together, this study provided a simple approach to study gene functions, which enabled us to unveil the novel role of CzcR in modulating efflux pump genes and multidrug resistance in P. aeruginosa. IMPORTANCE P. aeruginosa is a ubiquitous opportunistic pathogen frequently causing chronic infections. In addition to being an important model organism for antibiotic-resistant research, this species is also important for understanding and exploiting CRISPR-Cas systems. In this study, we established a gene-silencing system based on the most abundant type I-F CRISPR-Cas system in this species, which can be readily employed to achieve targeted gene repression in multiple bacterial species. Using this gene-silencing system, the physiological role of Zn2+ and its responsive regulator CzcR in modulating multidrug resistance was unveiled with great convenience. This study not only displayed a new framework to expand the abundant CRISPR-Cas and anti-CRISPR systems for functional gene characterizations but also provided new insights into the regulation of multidrug resistance in P. aeruginosa and important clues for precise anti-pseudomonal therapies.

Online and Robust Intermittent Motion Planning in Dynamic and Changing Environments.

In this article, we propose RRT-Q X∞ , an online and intermittent kinodynamic motion planning framework for dynamic environments with unknown robot dynamics and unknown disturbances. We leverage RRT X for global path planning and rapid replanning to produce waypoints as a sequence of boundary-value problems (BVPs). For each BVP, we formulate a finite-horizon, continuous-time zero-sum game, where the control input is the minimizer, and the worst case disturbance is the maximizer. We propose a robust intermittent Q-learning controller for waypoint navigation with completely unknown system dynamics, external disturbances, and intermittent control updates. We execute a relaxed persistence of excitation technique to guarantee that the Q-learning controller converges to the optimal controller. We provide rigorous Lyapunov-based proofs to guarantee the closed-loop stability of the equilibrium point. The effectiveness of the proposed RRT-Q X∞ is illustrated with Monte Carlo numerical experiments in numerous dynamic and changing environments.

Enhanced ethanol oxidation reaction of CoSeO3 by Ni-doping for water electrolysis and an innovative zinc-ethanol-air battery.

By doping Ni into m-CoSeO3, the structure of the catalyst was modified to improve the Ethanol Oxidation Reaction (EOR) catalytic performance. The catalyst exhibited excellent EOR catalytic activity (j10 = 1.35 V) and high stability. Therefore, this catalyst is used in an innovative zinc-ethanol-air battery, which is more efficient and stable than the traditional zinc-air battery.

CzcR Is Essential for Swimming Motility in Pseudomonas aeruginosa during Zinc Stress.

Two-component system (TCS) plays a vital role in modulating target gene expression in response to the changing environments. Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can survive under diverse stress conditions. The great adaptability of P. aeruginosa relies heavily on the abundant TCSs encoded by its genome. However, most TCSs in P. aeruginosa have not been well-characterized. CzcS/CzcR is a metal responsive TCS which displays multiple regulatory functions associated with metal hemostasis, quorum sensing activity and antibiotic resistance. In this study, we found that swimming motility of P. aeruginosa was completely abolished during zinc (Zn2+) stress when the czcR gene from the TCS CzcS/CzcR was deleted. Noticeably, CzcR was dispensable for swimming without the stress of Zn2+ excess. CzcR was shown to be activated by Zn2+ stress possibly through inducing its expression level and triggering its phosphorylation to positively regulate swimming which was abolished by Zn2+ stress in a CzcR-independent manner. Further TEM analyses and promoter activity examinations revealed that CzcR was required for the expression of genes involved in flagellar biosynthesis during Zn2+ stress. In vitro protein-DNA interaction assay showed that CzcR was capable of specifically recognizing and binding to the promoters of operons flgBCDE, flgFGHIJK, and PA1442/FliMNOPQR/flhB. Together, this study demonstrated a novel function of CzcR in regulating flagellar gene expression and motility in P. aeruginosa when the pathogen encounters Zn2+ stress conditions. IMPORTANCE The fitness of bacterial cells depends largely on their ability to sense and respond quickly to the changing environments. P. aeruginosa expresses a great number of signal sensing and transduction systems that enable the pathogen to grow and survive under diverse stress conditions and cause serious infections at different sites in many hosts. In addition to the previously characterized functions to regulate metal homeostasis, quorum sensing activity, and antibiotic resistance, here we report that CzcR is a novel regulator essential for flagellar gene expression and swimming motility in P. aeruginosa during Zn2+ stress. Since swimming motility is important for the virulence of P. aeruginosa, findings in this study might provide a new target for the treatment of P. aeruginosa infections with Zn2+-based antimicrobial agents in the future.

Fiber-Reinforcing Effect in the Mechanical and Road Performance of Cement-Emulsified Asphalt Mixtures.

Cement-emulsified asphalt mixture (CEAM), a kind of cold mix asphalt mixture, has the advantages of energy conservation and emission reduction as well as easy construction. However, the performance of CEAM is not as good as hot mixed asphalt mixtures. Hence, in this study, two different fibers were adopted as the reinforcing phase to improve the comprehensive properties of CEAM. The results indicated that the addition proportion and curing time were crucial to fiber-reinforced cement-emulsified asphalt mixture (FRCEAM). The compressive strengths, water stability, and raveling resistances of FRCEAM preparations with polyester or brucite fibers (FRCEAM-PF and -BF, respectively) were enhanced significantly. FRCEAM-PF had the maximum flexural tensile strength and strain, which meant that its low-temperature performance was the best compared to FRCEAM-PF and CEAM. However, the contribution of PF to CEAM high-temperature stability was greater than that of BF. Fiber addition to CEAM not only enhanced the cycles of fatigue loading but also reduced sensitivity to changes in stress level. Furthermore, FRCEAM-BF durability was slightly better than that of FRCEAM-PF. SEM analysis indicated that fibers provided bridging and meshing effects. Although PF and BF showed different enhancement effects, both mixtures met the requirements for hot mixed asphalt mixtures.

First Report of Leaf Spot Caused by Corynespora cassiicola on Acanthus ilicifolius in China.

Acanthus ilicifolius, also referred to as holly mangrove, usually grows in the coastal areas in southern China. It is not only a pioneer species of mangroves but also an important medicinal mangrove plant. In June 2019, leaf spots on A. ilicifolius with disease incidence of about 30 to 40% were observed in about 150 plants in a field located in Zhanjiang City, Guangdong Province, China. The disease mainly occurred on the leaf margins or tips. Initial symptoms of the disease were dark brown spots (5 to 9 mm) surrounded by a yellow halo, that expanded irregularly or semicircularly, until finally the leaves turned chlorotic and defoliated. Six samples of symptomatic leaves were excised, surface sterilized with 75% ethanol 30 s, 0.1% HgCl2 30 s, and rinsed three times with sterile water, and cut into small pieces (5 mm × 5 mm), placed on potato dextrose agar (PDA) at 25°C and darkness, and two isolates with different morphological characteristics were obtained after 3 days. For pathogenicity tests of the isolates, wounded and non-wounded leaves were inoculated. Fresh wounds were made with a sterile needle. Mycelial plugs from 8-day-old cultures of two isolates, ALY1 and ALY2, on PDA were inoculated on adaxial surface of wounded and non-wounded healthy young leaves of the A. ilicifolius seedlings (2 years old, 4 leaves per plant, 2 replicate plants per isolate), covered with wet cotton and kept in moist chambers at 25 ± 1°C, 80% relative humidity. Leaves inoculated with sterile PDA plugs were used as the control. After 3-5 days, the wounded and non-wounded leaves inoculated with ALY1 showed symptoms of leaf spots, similar to those observed in the field, while leaves inoculated with ALY2 and the control leaves remained symptomless. The pathogenicity tests were repeated three times under the same conditions and similar results were observed. ALY1 was confirmed as the pathogen causing the leaf spot. On PDA, ALY1 produced a gray to grayish brown colony. The conidia were obclavate to cylindrical, straight to slightly curved, pale brown, with 6 to 18 pseudosepta, 62.5 to 225.0×3.8 to 14.8 µm (n=17). According to the morphological characteristics, ALY1 was preliminarily identified as Corynespora cassiicola (Zhang et al.2018). For molecular identification, the internal transcribed spacer (ITS), the actin gene (ACT), the ß-tubulin gene (TUB2) and translation elongation factor 1-alpha gene (TEF1-α) of the three single-spore isolates from ALY1 (ALY1-1 to ALY1-3) were amplified and sequenced with primers ITS4/ITS5 (White et al.1990), ACT512F/ACT783R(Carbone and Kohn 1999), T1/T2 (Glass and Donaldson 1995), and EF1-728F/EF1-986R (O'Donnell et al.1998) respectively. The ITS region of ALY1 (-1, -2, -3) (GenBank accession number MN860006, -07, -08) was 99-100% identical (579/585, 586/586, 581/584 base pairs) to a corresponding ITS (MH255527) of C. cassiicola. Similarly, the ACT (MN887504, -05, -06), TUB2 (MN887507, -08, -09) and TEF1-α (MN887501, -02, -03) were 100% (342/342, 342/342, 342/342 base pairs), 100% (664/664, 662/662, 664/664 base pairs) and 100% (290/290, 290/290, 290/290 base pairs) to corresponding genes FJ853033, MH763700, and MK589886 sequences of C. cassiicola in GenBank, respectively. Phylogenetic tree generated with MEGA7 by the neighbor-joining analysis revealed that the isolate's ITS, ACT, TUB2 and TEF1-α sequences were grouped in the same clade to C. cassiicola. Based on both the morphological characteristics and sequences analyses, the pathogen causing leaf spot of A. ilicifolius was identified as C. cassiicola. This species had been reported on the A. ilicifolius in Guam America and Hong Kong China (Lu et al. 2000; Zhuang 2001; Dixon et al. 2009; Sumabat et al. 2018). The best of our knowledge, it is the first report of C. cassiicola causing leaf spot of A. ilicifolius in China. This disease may a potential threat to A. ilicifolius along the coastal regions of southern China.

Pathways connecting two opposed bilayers with a fusion pore: a molecularly-informed phase field approach.

A phase field model with two phase fields, representing the concentration and the head-tail separation of amphiphilic molecules, respectively, has been constructed using an extension of the Ohta-Kawasaki model (Macromolecules, 1986, 19, 2621-2632). It is shown that this molecularly-informed phase field model is capable of producing various self-assembled amphiphilic aggregates, such as bilayers, vesicles and micelles. Furthermore, pathways connecting two opposed bilayers with a fusion pore are obtained by using a combination of the phase field model and the string method. Multiple fusion pathways, including a classical pathway and a leaky pathway, have been obtained depending on the initial separation of the two bilayers. The study shed light on the understanding of the membrane fusion pathways and, more importantly, laid a foundation for further investigation of more complex membrane morphologies and transitions.

Vehicle autonomous localization in local area of coal mine tunnel based on vision sensors and ultrasonic sensors.

This paper presents a vehicle autonomous localization method in local area of coal mine tunnel based on vision sensors and ultrasonic sensors. Barcode tags are deployed in pairs on both sides of the tunnel walls at certain intervals as artificial landmarks. The barcode coding is designed based on UPC-A code. The global coordinates of the upper left inner corner point of the feature frame of each barcode tag deployed in the tunnel are uniquely represented by the barcode. Two on-board vision sensors are used to recognize each pair of barcode tags on both sides of the tunnel walls. The distance between the upper left inner corner point of the feature frame of each barcode tag and the vehicle center point can be determined by using a visual distance projection model. The on-board ultrasonic sensors are used to measure the distance from the vehicle center point to the left side of the tunnel walls. Once the spatial geometric relationship between the barcode tags and the vehicle center point is established, the 3D coordinates of the vehicle center point in the tunnel's global coordinate system can be calculated. Experiments on a straight corridor and an underground tunnel have shown that the proposed vehicle autonomous localization method is not only able to quickly recognize the barcode tags affixed to the tunnel walls, but also has relatively small average localization errors in the vehicle center point's plane and vertical coordinates to meet autonomous unmanned vehicle positioning requirements in local area of coal mine tunnel.