A new mode of swimming in singly flagellated Pseudomonas aeruginosa.

SignificanceThe monotrichous Pseudomonas aeruginosa was usually thought to swim in a pattern of "run and reverse" (possibly with pauses in between), where straight runs alternated with reverses with angular changes of swimming direction near 180°. Here, by simultaneously tracking the cell swimming and the morphology of its flagellum, we discovered a swimming mode in P. aeruginosa-the wrap mode, during which the flagellar filament wrapped around the cell body and induced large fluctuation of the body orientation. The wrap mode randomized swimming direction, resulting in a broad distribution of angular changes over 0 to 180° with a peak near 90°. This allowed the bacterium to explore the environment more efficiently, which we confirmed by stochastic simulations of P. aeruginosa chemotaxis.

Vertical confinement enhances surface exploration in bacterial twitching motility.

Bacteria are often found in environments where space is limited, and they attach themselves to surfaces. One common form of movement on these surfaces is bacterial twitching motility, which is powered by the extension and retraction of type IV pili. Although twitching motility in unrestricted conditions has been extensively studied, the effects of spatial confinement on this behaviour are not well understood. In this study, we explored the diffusive properties of individual twitching Pseudomonas aeruginosa cells in spatially confined conditions. We achieved this by placing the bacteria between layers of agarose and glass, and then tracking the long-term twitching motility of individual cells. Interestingly, we found that while confinement reduced the immediate speed of twitching, it paradoxically increased diffusion. Through a combination of mechanical and geometrical analysis, as well as numerical simulations, we showed that this increase in diffusion could be attributed to mechanical factors. The constraint imposed by the agarose altered the diffusion pattern of the bacteria from normal to superdiffusion. These findings provide valuable insights into the motile behaviour of bacteria in confined environments.

Speed-dependent bacterial surface swimming.

Solid surfaces submerged in liquid in natural environments alter bacterial swimming behavior and serve as platforms for bacteria to form biofilms. In the initial stage of biofilm formation, bacteria detect surfaces and increase the intracellular level of the second messenger c-di-GMP, leading to a reduction in swimming speed. The impact of this speed reduction on bacterial surface swimming remains unclear. In this study, we utilized advanced microscopy techniques to examine the effect of swimming speed on bacterial surface swimming behavior. We found that a decrease in swimming speed reduces the cell-surface distance and prolongs the surface trapping time. Both these effects would enhance bacterial surface sensing and increase the likelihood of cells adhering to the surface, thereby promoting biofilm formation. We also examined the surface-escaping behavior of wild-type Escherichia coli and Pseudomonas aeruginosa, noting distinct surface-escaping mechanisms between the two bacterial species. IMPORTANCE: In the early phase of biofilm formation, bacteria identify surfaces and increase the intracellular level of the second messenger c-di-GMP, resulting in a decrease in swimming speed. Here, we utilized advanced microscopy techniques to investigate the impact of swimming speed on bacterial surface swimming, focusing on Escherichia coli and Pseudomonas aeruginosa. We found that an increase in swimming speed led to an increase in the radius of curvature and a decrease in surface detention time. These effects were explained through hydrodynamic modeling as a result of an increase in the cell-surface distance with increasing swimming speed. We also observed distinct surface-escaping mechanisms between the two bacterial species. Our study suggests that a decrease in swimming speed could enhance the likelihood of cells adhering to the surface, promoting biofilm formation. This sheds light on the role of reduced swimming speed in the transition from motile to sedentary bacterial lifestyles.

Bacterial surface swimming states revealed by TIRF microscopy.

Motility near solid surfaces plays a key role in the life cycle of bacteria and is essential for biofilm formation, biofilm dispersal, and virulence. The alignment of the cell body with the surface during surface swimming impacts bacterial surface sensing. Here, we developed a high-throughput method for characterizing the orientation of the cell body relative to the surface using total internal reflection fluorescence (TIRF) microscopy. The angle between the cell body and the surface was determined by maximizing image cross-correlations between the TIRF image of the cell and a reference library. Utilizing this technique, we surprisingly identified six distinct surface swimming states of Pseudomonas aeruginosa according to the body alignment and the flagellar position. Furthermore, we observed that the near-surface swimming speed is greater in the pull state than in the push state, attributed to hydrodynamic effects near the liquid-solid interface. Hydrodynamic force analysis of the swimming states provided rich insights into the mechanics of bacterial surface swimming. Our technique is readily applicable to the study of surface motility across a wide spectrum of bacterial species.

Rhizophagus Irregularis regulates flavonoids metabolism in paper mulberry roots under cadmium stress.

Broussonetia papyrifera is widely found in cadmium (Cd) contaminated areas, with an inherent enhanced flavonoids metabolism and inhibited lignin biosynthesis, colonized by lots of symbiotic fungi, such as arbuscular mycorrhizal fungi (AMF). However, the physiological and molecular mechanisms by which Rhizophagus irregularis, an AM fungus, regulates flavonoids and lignin in B. papyrifera under Cd stress remain unclear. Here, a pot experiment of B. papyrifera inoculated and non-inoculated with R. irregularis under Cd stress was carried out. We determined flavonoids and lignin concentrations in B. papyrifera roots by LC-MS and GC-MS, respectively, and measured the transcriptional levels of flavonoids- or lignin-related genes in B. papyrifera roots, aiming to ascertain the key components of flavonoids or lignin, and key genes regulated by R. irregularis in response to Cd stress. Without R. irregularis, the concentrations of eriodictyol, quercetin and myricetin were significantly increased under Cd stress. The concentrations of eriodictyol and genistein were significantly increased by R. irregularis, while the concentration of rutin was significantly decreased. Total lignin and lignin monomer had no alteration under Cd stress or with R. irregularis inoculation. As for flavonoids- or lignin-related genes, 26 genes were co-regulated by Cd stress and R. irregularis. Among these genes, BpC4H2, BpCHS8 and BpCHI5 were strongly positively associated with eriodictyol, indicating that these three genes participate in eriodictyol biosynthesis and were involved in R. irregularis assisting B. papyrifera to cope with Cd stress. This lays a foundation for further research revealing molecular mechanisms by which R. irregularis regulates flavonoids synthesis to enhance tolerance of B. papyrifera to Cd stress.

Timescale separation in the coordinated switching of bacterial flagellar motors.

The output of the bacterial chemotaxis signaling pathway, the level of the intracellular regulator CheY-P, modulates the rotation direction of the flagellar motor, thereby regulating bacterial run-and-tumble behavior. The multiple flagellar motors on anE. colicell are controlled by a common cytoplasmic pool of CheY-P. Fluctuation of the CheY-P level was thought to be able to coordinate the switching of multiple motors. Here, we measured the correlation of rotation directions between two motors on a cell, finding that it surprisingly exhibits two well separated timescales. We found that the slow timescale (∼6 s) can be explained by the slow fluctuation of the CheY-P level due to stochastic activity of the chemotactic adaptation enzymes, whereas the fast timescale (∼0.3 s) can be explained by the random pulse-like fluctuation of the CheY-P level, due probably to the activity of the chemoreceptor clusters. We extracted information on the properties of the fast CheY-P pulses based on the correlation measurements. The two well-separated timescales in the fluctuation of CheY-P level help to coordinate multiple motors on a cell and to enhance bacterial chemotactic performance.

Differential Bending Stiffness of the Bacterial Flagellar Hook under Counterclockwise and Clockwise Rotations.

The bacterial hook, as a universal joint coupling rotation of the flagellar motor and the filament, is an important component of the flagellum that propels the bacteria to swim. The mechanical properties of the hook are essential for the flagellum to achieve normal functions. In multiflagellated bacteria such as Escherichia coli, the hook must be compliant so that it can bend for the filaments to form a coherently rotating bundle to generate the thrust when the motor rotates counterclockwise (CCW), yet it also must be rigid so that the bundle can disrupt for the bacteria to tumble to change swimming direction when the motor rotates clockwise (CW). Here, by combining an elastic rod model with high-resolution bead assay to accurately measure the bending stiffness of the hook under CCW or CW rotation in vivo, we elucidate how the hook accomplishes this dual functionality: the hook stiffens under CW rotation, with bending stiffness under CW rotation twice as large as that under CCW rotation. This enables a robust run-and-tumble swimming motility for multiflagellated bacteria.

Uptake of HIV testing and its correlates among sexually experienced college students in Southwestern, China: a Web-Based online cross-sectional study.

BACKGROUND: The prevalence of human immunodeficiency virus (HIV) is becoming more common among college students in China. However, latest data on the prevalence and correlates of HIV testing among sexually experienced college students is rarely. METHODS: An online survey was conducted among college students aged 18 years or older using multistage stratified cluster sampling from 16 colleges. Data on socio-demographic, HIV testing, HIV-related awareness, attitudes, sexual education and behaviors were collected. Propensity score matching (PSM) and logistic regression model were used to identify factors associated with HIV testing. RESULT: A total of 108,987 students participated the survey, of which 13,201 sexually experienced college students were included in this study. 1,939 (14.69%) college students with sexual experience reported uptake of HIV testing in the preceding year. The uptake of HIV testing increased for college students with a rising HIV knowledge score and sexual health knowledge. Being awareness of HIV-related knowledge (aOR = 1.15, 95%CI: 1.01-1.30), accepting one-night stands (aOR = 1.16, 95%CI:1.03-1.32), obtaining satisfactory sexual interpretation from parent(s) (aOR = 1.24, 95%CI: 1.07-1.43), ever had unintended pregnancy (aOR = 1.78, 95%CI: 1.32-2.38), ever had received HIV-related preventive service(s) (aOR = 1.37, 95%CI: 1.10-1.70), ever had participated HIV-related preventive services (aOR = 3.76, 95%CI: 2.99-4.75) and ever had anal sex (aOR = 2.66, 95%CI: 2.11-3.34) were positively associated with uptake of HIV testing. However, accepting premarital sex (aOR = 0.76, 95%CI: 0.66-0.88), accepting cohabitation (aOR = 0.75, 95%CI: 0.61-0.92), occasionally discussing sex with parent(s) (aOR = 0.68, 95%CI: 0.50-0.91), and being with moderate satisfaction of school sex courses (aOR = 0.74, 95%CI: 0.58-0.95) were negatively associated with uptake of HIV testing. CONCLUSION: The prevalence of HIV testing was relatively low. Participation in HIV-related services and high-risk sexual behaviors were important enablers for testing. Improving sex education for students, increasing HIV preventive services on campus, and improving family sex education are necessary to increase HIV testing among college sexually experienced students.

Co-Immobilization of Lipases with Different Specificities for Efficient and Recyclable Biodiesel Production from Waste Oils: Optimization Using Response Surface Methodology.

Lipase-catalyzed transesterification is a promising and sustainable approach to producing biodiesel. To achieve highly efficient conversion of heterogeneous oils, combining the specificities and advantages of different lipases is an attractive strategy. To this end, highly active Thermomyces lanuginosus lipase (1,3-specific) and stable Burkholderia cepacia lipase (non-specific) were covalently co-immobilized on 3-glycidyloxypropyltrimethoxysilane (3-GPTMS) modified Fe3O4 magnetic nanoparticles (co-BCL-TLL@Fe3O4). The co-immobilization process was optimized using response surface methodology (RSM). The obtained co-BCL-TLL@Fe3O4 exhibited a significant improvement in activity and reaction rate compared with mono and combined-use lipases, achieving 92.9% yield after 6 h under optimal conditions, while individually immobilized TLL, immobilized BCL and their combinations exhibited yields of 63.3%, 74.2% and 70.6%, respectively. Notably, co-BCL-TLL@Fe3O4 achieved 90-98% biodiesel yields after 12 h using six different feedstocks, demonstrating the perfect synergistic effect of BCL and TLL remarkably motivated in co-immobilization. Furthermore, co-BCL-TLL@Fe3O4 could maintain 77% of initial activity after nine cycles by removing methanol and glycerol from catalyst surface, accomplished by washing with t-butanol. The high catalytic efficiency, wide substrate adaptability and favorable reusability of co-BCL-TLL@Fe3O4 suggest that it will be an economical and effective biocatalyst for further applications.

Suppression of cell-cell variation by cooperative interaction of phosphatase and response regulator.

In bacterial chemotaxis, the output of chemosensing, the concentration of the response regulator CheY-P that was constantly adjusted by the opposing action of the kinase CheA and the phosphatase CheZ, serves as the input of the ultrasensitive flagellar motor that drives bacterial motility. The steady-state kinase activity exhibits large cell-to-cell variation that may result in similar variation in CheY-P concentration. Here, we found that the in vivo phosphatase activity is highly cooperative with respect to CheY-P concentration, and this suppresses the cell-to-cell variation of CheY-P concentration so that it falls within the operational range of the flagellar motor. Therefore, the cooperativity of the CheZ and CheY-P interaction we identified here provided a mechanism of robust coupling between the output of chemosensing and the input of the flagellar motor. Suppression of cell heterogeneity by cooperativity of protein-protein interaction is likely a common feature in many biological signaling systems.

Observation of broken detailed balance in polymorphic transformation of bacterial flagellar filament.

Living systems operate far from thermodynamic equilibrium, which usually manifests as broken detailed balance at the molecular scale. At larger scales with collective function of many molecules, the presence of non-equilibrium thermodynamics may not be evident. In bacterial motility, the switching dynamics of the flagellar rotary motor was recently discovered to be operating in non-equilibrium. However, the resulting motility pattern at the mesoscale, the run-and-tumble behavior, was normally considered to be a Poisson process that can be described by a two-state equilibrium model. Here, we studied the details of the run-and-tumble behavior by following the polymorphic transformation of the flagellar filaments, observing broken detailed balance that reveals its non-equilibrium nature. Evaluation of entropy production provided a direct measure of the lack of detailed balance and a quantification of the rate of energy dissipation for bacterial run-and-tumble regulation.

FliL Differentially Interacts with Two Stator Systems To Regulate Flagellar Motor Output in Pseudomonas aeruginosa.

FliL is present in nearly all flagellated bacterial species and is associated with the flagellar basal body. This protein was found to be important for the function of the flagellar motor, and its absence led to a variety of motility defects in several species. However, the specific function of FliL in Pseudomonas aeruginosa remains elusive. Here, we studied the effects of FliL on motor output in P. aeruginosa using a bead assay, finding that FliL regulates motor output through its differential effects on the two sets of homologous MotAB and MotCD stators. FliL interacts with the MotCD stators to increase the motor torque and the stability of the motor speed, whereas it works with the MotAB stators to maintain a high motor switching rate. These effects of FliL contribute to enhancing P. aeruginosa's motility and chemotaxis. IMPORTANCE FliL emerged as a modulator of flagellar motor function in several bacterial species, but its function in Pseudomonas aeruginosa was unknown. Here, by performing single-motor studies using a bead assay, we elucidated its effects on the flagellar motor in P. aeruginosa. We found that it differentially interacts with two sets of stators (MotAB and MotCD) to regulate different aspects of bacterial motility (motor switching rate and motor rotation speed), thereby enhancing the ability of P. aeruginosa to explore its environment.

The Effect of the Second Messenger c-di-GMP on Bacterial Chemotaxis in Escherichia coli.

c-di-GMP is a ubiquitous bacterial second messenger that plays a central regulatory role in diverse biological processes. c-di-GMP was known to regulate chemotaxis in multiple bacterial species, but its effect on Escherichia coli chemotaxis remained unclear. As an effector of c-di-GMP in E. coli, YcgR when bound with c-di-GMP interacts with the flagellar motor to reduce its speed and its probability of rotating clockwise (CW bias). Here, we found that a significant fraction of the c-di-GMP::YcgR dynamically exchange between the motor and the cytosol. Through fluorescent measurements, we found that there was no competitive binding between the chemotaxis response regulator CheY-P and c-di-GMP::YcgR to the motor. To test the influence of elevated c-di-GMP levels on the chemotaxis pathway, we measured the chemotactic responses of E. coli cells using a FRET assay, finding that elevated c-di-GMP levels had no effect on the upstream part of chemotaxis pathway down to the level of CheY-P concentration. This suggested that the possible effect of elevated c-di-GMP levels on chemotactic motion was through regulation of motor speed and CW bias. Using stochastic simulations of chemotactic swimming, we showed that the effects of reducing motor speed and decreasing CW bias on chemotactic drift velocity are compensating for each other, resulting in minimal effect of elevated c-di-GMP levels on E. coli chemotaxis. Therefore, elevated c-di-GMP levels promote the transition from motile to sedentary forms of bacterial life by reducing the bacterial swimming speed and CW bias, while still maintaining a nearly intact chemotaxis capability in E. coli. IMPORTANCE The ubiquitous bacterial second messenger c-di-GMP was known to regulate chemotaxis in many bacterial species, but its effect on E. coli chemotaxis was unclear. Here we studied the effect of elevated c-di-GMP levels on chemotaxis in E. coli. We found that the binding of c-di-GMP::YcgR (its effector) and the chemotaxis response regulator CheY-P to the flagellar motor are noncompetitive, and elevated c-di-GMP levels do not affect the upstream part of the chemotaxis pathway down to the level of CheY-P concentration. Elevated c-di-GMP levels exert direct effects on the flagellar motor by reducing its speed and CW bias, but the resulting effects on chemotaxis performance are compensating for each other. Our findings here showed that elevated c-di-GMP levels maintain a nearly intact chemotaxis capability when promoting the transition from motile to sedentary forms of bacterial life in E. coli.

Metachronal patterns by magnetically-programmable artificial cilia surfaces for low Reynolds number fluid transport and mixing.

Motile cilia can produce net fluid flows at low Reynolds number because of their asymmetric motion and metachrony of collective beating. Mimicking this with artificial cilia can find application in microfluidic devices for fluid transport and mixing. Here, we study the metachronal beating of nonidentical, magnetically-programmed artificial cilia whose individual non-reciprocal motion and collective metachronal beating pattern can be independently controlled. We use a finite element method that accounts for magnetic forces, cilia deformation and fluid flow in a fully coupled manner. Mimicking biological cilia, we study magnetic cilia subject to a full range of metachronal driving patterns, including antiplectic, symplectic, laeoplectic and diaplectic waves. We analyse the induced primary flow, secondary flow and mixing rate as a function of the phase lag between cilia and explore the underlying physical mechanism. Our results show that shielding effects between neighboring cilia lead to a primary flow that is larger for antiplectic than for symplectic metachronal waves. The secondary flow can be fully explained by the propagation direction of the metachronal wave. Finally, we show that the mixing rate can be strongly enhanced by laeoplectic and diaplectic metachrony resulting in large velocity gradients and vortex-like flow patterns.

Collective motion enhances chemotaxis in a two-dimensional bacterial swarm.

In a dilute liquid environment in which cell-cell interaction is negligible, flagellated bacteria, such as Escherichia coli, perform chemotaxis by biased random walks alternating between run-and-tumble. In a two-dimensional crowded environment, such as a bacterial swarm, the typical behavior of run-and-tumble is absent, and this raises the question whether and how bacteria can perform chemotaxis in a swarm. Here, by examining the chemotactic behavior as a function of the cell density, we showed that chemotaxis is surprisingly enhanced because of cell crowding in a bacterial swarm, and this enhancement is correlated with increase in the degree of cell body alignment. Cells tend to form clusters that move collectively in a swarm with increased effective run length, and we showed analytically that this resulted in increased drift velocity toward attractants. We also explained the enhancement by stochastically simulating bacterial chemotaxis in a swarm. We found that cell crowding in a swarm enhances chemotaxis if the cell-cell interactions used in the simulation induce cell-cell alignment, but it impedes chemotaxis if the interactions are collisions that randomize cell moving direction. Therefore, collective motion in a bacterial swarm enhances chemotaxis.

Upcoming flow promotes the bundle formation of bacterial flagella.

Flagellated bacteria swim by rotating a bundle of helical flagella and commonly explore the surrounding environment in a "run-and-tumble" motility mode. Here, we show that the upcoming flow could impact the bacterial run-and-tumble behavior by affecting the formation and dispersal of the flagellar bundle. Using a dual optical tweezers setup to trap individual bacteria, we characterized the effects of the imposed fluid flow and cell body rotation on the run-and-tumble behavior. We found that the two factors affect the behavior differently, with the imposed fluid flow increasing the running time and decreasing the tumbling time and the cell body rotation decreasing the tumbling time only. Using numerical simulations, we computed the flagellar bundling time as a function of flow velocity, which agrees well with our experimental observations. The mechanical effects we characterized here provide novel, to our knowledge, ingredients for further studies of bacterial chemotaxis in complex environments such as dynamic fluid environments.

Dynamics of the Two Stator Systems in the Flagellar Motor of Pseudomonas aeruginosa Studied by a Bead Assay.

We developed a robust bead assay for studying flagellar motor behavior of Pseudomonas aeruginosa. Using this assay, we studied the dynamics of the two stator systems in the flagellar motor. We found that the two sets of stators function differently, with MotAB stators providing higher total torque and MotCD stators ensuring more stable motor speed. The motors in wild-type cells adjust the stator compositions according to the environment, resulting in an optimal performance in environmental exploration compared to that of mutants with one set of stators. The bead assay we developed in this investigation can be further used to study P. aeruginosa chemotaxis at the level of a single cell using the motor behavior as the chemotaxis output. IMPORTANCE Cells of Pseudomonas aeruginosa possess a single polar flagellum, driven by a rotatory motor powered by two sets of torque-generating units (stators). We developed a robust bead assay for studying the behavior of the flagellar motor in P. aeruginosa, by attaching a microsphere to shortened flagellar filament and using it as an indicator of motor rotation. Using this assay, we revealed the dynamics of the two stator systems in the flagellar motor and found that the motors in wild-type cells adjust the stator compositions according to the environment, resulting in an optimal performance in environmental exploration compared to that of mutants with one set of stators.

Swimming Escherichia coli Cells Explore the Environment by Lévy Walk.

Escherichia coli cells swim in aqueous environment in a random walk of alternating runs and tumbles. The diffusion characteristics of this random walk remains unclear. In this study, by tracking the swimming of wild-type cells in a three-dimensional (3D) homogeneous environment, we found that their trajectories are superdiffusive, consistent with Lévy walk behavior. For comparison, we tracked the swimming of mutant cells that lack the chemotaxis signaling noise (the steady-state fluctuation of the concentration of the chemotaxis response regulator CheY-P) and found that their trajectories are normal diffusive. Therefore, wild-type E. coli cells explore the environment by Lévy walk, which originates from the chemotaxis signaling noise. This Lévy walk pattern enhances their efficiency in environmental exploration.IMPORTANCEE. coli cells explore the environment in a random walk of alternating runs and tumbles. By tracking the 3D trajectories of E. coli cells in an aqueous environment, we found that their trajectories are superdiffusive, with a power-law shape for the distribution of run lengths, which is characteristics of Lévy walk. We further show that this Lévy walk behavior is due to the random fluctuation of the output level of the bacterial chemotaxis pathway, and it enhances the efficiency of the bacteria in exploring the environment.

Dynamics of Switching at Stall Reveals Nonequilibrium Mechanism in the Allosteric Regulation of the Bacterial Flagellar Switch.

Behavior of the bacterial flagellar motor depends sensitively on the external loads it drives. Motor switching, which provides the basis for the run-and-tumble behavior of flagellated bacteria, has been studied for motors under zero to high loads, revealing a nonequilibrium effect that is proportional to the motor torque. However, behavior of the motor switching at stall (with maximum torque) remains unclear. An extrapolation from previous studies would suggest the maximum nonequilibrium effect for motor switching at stall. Here, we stalled the motor using optical tweezers and studied the motor switching with a high time resolution of about 2 ms. Surprisingly, our results showed exponentially distributed counterclockwise (CCW) and clockwise (CW) intervals, indicating that motor switching at stall is probably an equilibrium process. Combined with previous experiments at other loads, our result suggested that the nonequilibrium effect in motor switching arises from the asymmetry of the torque generation in the CCW and CW directions. By including this nonequilibrium effect in the general Ising-type conformation spread model of the flagellar switch, we consistently explained the motor switching over the whole range of load conditions. We expect to see a similar mechanism of nonequilibrium regulation in other molecular machines.

Direct Mapping from Intracellular Chemotaxis Signaling to Single-Cell Swimming Behavior.

Bacterial chemotaxis allows bacteria to sense the chemical environment and modulate their swimming behavior accordingly. Although the intracellular chemotaxis signaling pathway has been studied extensively, experimental studies are still lacking that could provide direct link from the pathway output (the intracellular concentration of the phosphorylated form of the response regulator phosphorylated CheY (CheY-P)) to single-cell swimming behavior. Here, we measured the swimming behavior of individual Escherichia coli cells while simultaneously detecting the intracellular CheY-P concentration, thereby providing a direct relationship between the intracellular CheY-P concentration and the single-cell run-and-tumble behavior. The measured relationship is consistent with the ultrasensitivity of the motor switch and a "veto model" that describes the interaction among individual flagella, although contribution from the voting mechanism could not be ruled out.