KR101765197B1 - Optimized medium composition for lactobacillus sp. autoinducer-2 signal activity assay and the assay method thereof - Google Patents
Optimized medium composition for lactobacillus sp. autoinducer-2 signal activity assay and the assay method thereof Download PDFInfo
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Abstract
Description
The present invention relates to an optimal medium composition for the assay of Autoinducer-2 signal activity of a Lactobacillus sp. Microorganism and an assay method using the same, and more particularly, The present invention relates to a method for the determination of the concentration of a protein by optimizing each component to a specific concentration range of a small amount, such as yeast extract, beef extract, peptone, polysorbate, magnesium sulfate and manganese sulfate, 2 signal activity analysis that can significantly improve the signal activity and the luminescence intensity sensitivity of the autoinducer-2 by stabilizing the expression of the signal or signaling activity of the autoinducer-2 or the strain, and an assay method using the same .
Microbes can increase the probability of survival and adaptation through collective action in a restricted environment. Microbial signaling is one of the mechanisms that enable such collective action. Quorum sensing of microorganisms refers to a phenomenon in which microorganisms secrete interacting factors themselves to adapt to the environment, and other microorganisms recognize these secreted factors. That is, it is one of the signaling features that express promising group behavior when the microorganism reaches a certain level of density. The micro-organism's signaling system is generally divided into intra-species, highly specific signaling and inter-species systems. Inter-species quorum signaling systems of microorganisms are luxS A signal system using Autoinducer-2 (AI-2) as a signaling material by homologue gene is typical (Waters and Bassler, 2005). It is well known that these signaling systems are deeply involved in the formation of biofilms by microorganisms in the natural world (Chen et al., 2011), and many pathogens have been shown to use quorum signaling for efficient virulence expression Active research has been conducted (Xavier and Bassler, 2005).
The quiescent-dependent expression is not directly related to the survival of microorganisms, and the study of antibiotic substitutes that do not cause tolerance through the inhibition of Autoinducer-2 has received the first priority attention. Salmonella (Sallmonella spp.), Escherichia coli (Escherichiacoli), Staphylococcus aureus (Staphylococcusaureus), Listeria monocytogenes (Listreiamonocytogenes), Pseudomonas aeruginosa (Pseudomonasaeruginosa), etc. Most of the major pathogen individual virulence expression control the same type or between heterogeneous quorum signaling pathway And signal inhibitors for neutralizing such signaling systems have been actively studied. In particular, the generation of signal inhibitors by beneficial bacteria such as lactic acid bacteria is considered to be a very useful alternative in this approach. Among them, such as infectious diarrhea by inhibiting bowel disease symptoms signal the activity of Christian Hansen's Lactobacillus also know pillar's (Lactobacillusacidophilus La-5) -2 inducer strains Otto reported that the effect of a series of pathogenic bacteria (Medellin-Peand Griffiths., 2009; Park et al., 2002). Inhibition of the pathogen by inhibition of the autoinducer-2 signaling activity of lactic acid bacteria has also been a subject of interest, based on this finding. 2014).
On the other hand, according to recent studies, the signal activity of the autoinducer-2 itself has a beneficial advantage. On the other hand, the commercially available probiotics LGG ( Lactobacillus rhmanosus GG) or bifidobacteria are using the autoinducer-2 signal to survive and adapt to the intestinal tract of the human body (Lebeer et al., 2008). In E. coli models induced by autoinducer-2 expression, intestinal imbalance (Thompson et al., 2015) have also reported that dysbiosis restores intestinal myelin imbalance in mice induced.
These opposing actions suggest that the role and expected effects of the same lactic acid bacteria are different depending on the signal characteristics, and that further research is needed. In addition, important clues to the development of microbiological treatments such as probiotics and human fecal microbiota transplantation can be obtained through ecological interactions and identification of microbial interactions or microbial and host interactions. "The report said.
To investigate the signaling activity of the autoinducer-2 of these microorganisms and other substances, Vibrio et al. (1998) A harveyi BB170 based autoinducer-2 emission analyzer (bioluminescence assay) is commonly used. BB170 is a strain genetically engineered to produce bioluminescence only when the autoinducer-2 signal is present. Through the generation of its own auto inducer-2 signal, a certain intensity of light emission appears over time. In this case, if an external auto-inducer-2 signal material is added, a relative light unit (in RLU unit) is generated although it is not a light emitting period, Can be analyzed. This method is the most simple and widely used method for analyzing the signal activity of the autoinducer-2, and various studies have been actively carried out to analyze the signal activity of the more sensitive autoinducer-2.
In recent years, new studies showing that the signal activity of the autoinducer-2 of this method can be inhibited by the constituents of the medium present in the culture medium of the strain can inhibit the signal activity of the autoinducer-2, The importance of using selective media is increasing day by day. Specifically, in Bassler's study, early-focused pathogens were generally cultured in LB medium, which did not contain a carbon source such as glucose. In the early Autunducer-2 study, microorganisms capable of culturing in LB medium could not be expected to interfere with the activity of autoinducer-2 in the medium such as glucose.
However, as the research area is expanded to various microorganisms such as lactic acid bacteria, this problem is seriously highlighted and poses a problem of carbon source. The MRS medium (de Man, Rogosa and Sharpe broth), which is generally used as a lactic acid bacteria medium in lactic acid bacteria, is the most efficient and preferentially used nutritional selection medium for culturing lactic acid bacteria. Most of today's studies use this MRS medium as the primary culture medium for testing lactobacilli. Since the measurement of metabolites such as microbial signal substances should be confirmed through the culture medium, the culture medium in the MRS medium is also the basic sample for the assay of the autoinducer-2 signal activity of the lactic acid bacteria.
In particular, glucose, one of the constituents of the MRS medium, has been reported to cause irregular inhibition of bioluminescence by BB170 (Dekeersmaecker and Vanderleyden, 2003). For this reason, researchers in the Lactobacillus genus have used galactose in the MRS medium (Moslehi-Jenabian, 2009), but as a carbon source for galactose in lactobacilli, And the species such as Lactobacillus brevis has a significant decrease in the growth rate and thus has a problem in that the kinds of bacteria that can be applied are limited.
In addition, the remaining components of the MRS medium in addition to glucose have also been found to have serious problems in BB170-based autoinducer-2 signal activity assays. Figure 1 is a graph showing changes in autoinducer-2 signal activity for each component in a conventional MRS medium. Specifically, FIG. 1 shows the result of analyzing the change of the autoinducer-2 signal activity by using the original MRS medium concentration only for each component of the conventional MRS medium. Generally, the spontaneous emission interval of the autoinducer-2 is formed between 3 and 4 hours. If each component does not significantly affect the signal intensity of BB170, it will converge to a relative value of 1. However, Not at all. It can be interpreted that all components affect the signal expression or bioactivity of BB170. Some components such as sodium acetate have some components that exhibit excessive signaling inhibition and some components such as peptone exacerbate excessive signal activity and this tendency is completely different depending on the elapsed time , And it was found that an unspecified signal disturbance could be caused by the MRS medium.
Of course, it is not possible to analyze the signal activity of the autoinducer-2 only if the signal intensity of the sample exceeds the disturbance due to other factors. Therefore, it can be assumed that previous studies have been carried out based on this. However, as described above, the possibility that constituents of this medium may act as a new obstacle to the analysis of signal activity of the autoinducer-2 should not be overlooked. No adequate improvement has been made. Therefore, there is a need for research and development of a new optimized culture composition for more precise and sensitive analysis of the signal activity of Autoinducer-2 expressed in microorganisms.
In order to solve the above problems, the present invention provides a method for assaying the activity of an autoinducer-2 in a conventional MRS medium, which comprises extracting yeast extract, beef extract, peptone, polysorbate, magnesium sulfate, By optimizing the concentrations of manganese sulfate and minimizing each component to a specific concentration range, signal intensity or bioactivity of the strain can be stabilized during luminescence analysis to greatly improve the sensitivity of the autoinducer-2 signal activity and luminescence intensity I realized that it was possible to complete the invention.
Accordingly, it is an object of the present invention to provide an optimum medium composition for analyzing autoinducer-2 signal activity, wherein the sensitivity of the signal activity and the luminescence intensity of the autoinducer-2 is greatly improved.
It is another object of the present invention to provide an optimal medium for the assay of autoinducer-2 signal activity comprising said optimal medium composition.
It is still another object of the present invention to provide a method for assaying autoinducer-2 signal activity using the optimal medium.
The present invention relates to a method for the treatment and / or prevention of manganese sulfate, which comprises extracting 0.1 to 10 mg / L of yeast extract, 0.5 to 15 mg / L of beef extract, 0.5 to 15 mg / L of peptone, 0.05 to 5 mg / L of polysorbate, 0.05 to 1 mg / An autoinducer-2 microorganism autoinducer comprising an optimal medium composition for assaying Autoinducer-2 signal activity with the addition of 0.01 to 0.1 mg / L, wherein glucose and galactose are removed. 2 (Autoinducer-2) signal activity assays.
delete
The present invention also relates to a method for producing a microorganism, which comprises primary culturing a microorganism belonging to the genus Lactobacillus in a primary culture medium; Washing the primary cultured microorganism with a first anti-optimal medium; Culturing the washed microorganism in the optimal medium of claim 1; And analyzing the autoinducer-2 signal activity of the secondary cultured microorganism using a luminescence analyzer; (Autoinducer-2) signal activity of a microorganism belonging to the genus Lactobacillus.
In addition, the primary culturing step is performed at a temperature of 18 ° C to 45 ° C for 12 hours to 16 hours, and the secondary culturing step is performed at a temperature of 18 ° C to 45 ° C for 30 minutes to 2 hours. It is possible to provide a method for analyzing autoinducer-2 signal activity of microorganisms of the genus Bacillus.
The optimal culture composition according to the present invention is characterized by comprising yeast extract, beef extract, peptone, polysorbate, magnesium sulfate and manganese sulfate as essential components, and optimizing each component to a specific concentration range in a small amount, Or stabilize the physiological activity of the strain, thereby greatly improving the sensitivity of the signal intensity and emission intensity of Autoinducer-2.
It can also be used for analytical instruments, metabolites and functional studies using microorganisms.
Figure 1 is a graph showing changes in autoinducer-2 signal activity for each component in a conventional MRS medium.
FIG. 2 is a graph showing changes in autoinducer-2 signal activity by concentration for each component of the optimum medium (AL medium) composition prepared in the production example of the present invention.
FIG. 3 is a graph showing changes in signal activity of the autoinducer-2 using the optimum medium prepared in the production example of the present invention, the normal MRS medium, and the mMRS medium.
4 is a graph showing the intensity of signal emission of the autoinducer-2 using the strains cultured in Examples 1 and 2 and Comparative Examples 1 and 2 of the present invention.
FIG. 5 is a graph showing changes in the number of viable cells of the strains used in Examples 1 and 2 according to the optimum medium of the present invention. FIG.
Hereinafter, the present invention will be described in more detail with reference to one embodiment.
The present invention relates to a pharmaceutical composition containing yeast extract, beef extract, peptone,
The optimal media composition of the present invention excludes all components such as ammonium citrate, sodium acetate, and Dipotassium Phosphate (DPP), which are factors that inhibit the signal activity of Autoinducer-2 in the constituents of conventional MRS media Which is characterized by comprising yeast extract, beef extract, peptone, polysorbate, magnesium sulfate and manganese sulfate as essential constituents, with their respective constituents being minimized to a small specific concentration range, And an optimal culture medium composition optimized for analysis.
According to a preferred embodiment of the present invention, the composition comprises 0.1 to 10 mg / L yeast extract, 0.5 to 15 mg / L beef extract, 0.5 to 15 mg / L peptone, 0.05 to 5 mg / L polysorbate, 0.05 to 1 mg / L of sulfate and 0.01 to 0.1 mg / L of manganese sulfate.
Specifically, each component of the optimal culture medium composition can be greatly reduced to a minimum specific concentration ratio compared to the concentration of the conventional MRS medium components, and particularly optimized within a range that does not significantly affect the bioluminescence of BB170. In addition, when minimizing one, such as the original composition ratio of the medium, it may be possible to uniformly minimize other components depending on the magnification.
The optimum medium composition can be mixed with the minimum concentration of the four components (yeast extract, beef extract, peptone, and polysorbate) used as the main nutrients of the lactic acid bacteria, among the essential constituents. The yeast extract may contain 0.1 to 10 mg / L of the total optimum medium composition. If the concentration of the yeast extract is less than 0.1 mg / L, the microorganism can maintain its physiological activity and metabolite production for a certain period of time There is no problem, and if it is more than 10 mg / L, the autoinducer-2 signal intensity can be excessively increased.
In addition, the beef extract and the peptone may contain a concentration of 0.5 to 15 mg / L, respectively, as a complementary nutrient component to the yeast extract, for the total optimum medium composition. If the concentration is less than 0.5 mg / L Similarly, there is a problem that the physiological activity of the microorganism itself and the production of metabolites can not be maintained for a certain period of time, and when it is more than 15 mg / L, the autoinducer-2 signal intensity can be excessively increased.
The polysorbate may contain a concentration of 0.05 to 5 mg / L with respect to the total optimum medium composition. If the concentration is less than 0.05 mg / L, the physiological activity of the microorganism itself and the problem of lowering metabolite production If it is more than 5 mg / L, the autoinducer-2 signal intensity can be excessively increased.
On the other hand, the present invention provides an optimal medium for the assay of autoinducer-2 signal activity comprising the optimal medium composition.
The present invention also relates to a method for producing a microorganism, which comprises: primary culturing a microorganism on a primary culture medium; Washing the primary cultured microorganism with the optimal medium; Secondary culturing the washed microorganism in the optimal medium; And analyzing the secondary incubated microorganism with an autoinducer-2 signal activity using a luminescence analyzer.
According to a preferred embodiment of the present invention, the first primary culture medium may be an MRS medium used as a conventional culture medium for lactic acid bacteria, or an mMRS medium prepared by replacing glucose in MRS medium with galactose have.
According to a preferred embodiment of the present invention, the microorganism may be a strain of the genus Lactobacillus. Specifically, the strain of the genus Lactobacillus is selected from the group consisting of Lactobacillus rhamnosus , lactobacillus plantarum , Lactobacillus < RTI ID = 0.0 > acidophilus , Lactobacillus < RTI ID = 0.0 > sakei , Lactobacillus casei ), Lactobacillus ( Lactobacillus gasseri , Lactobacillus < RTI ID = 0.0 > paracasei , Lactobacillus < RTI ID = 0.0 > ferementum , Lactobacillus reuteri , Lactobacillus < RTI ID = 0.0 > delbrueckii , Lactobacillus < RTI ID = 0.0 > johnsonii , Lactobacillus < RTI ID = 0.0 > helveticus , and Lactobacillus bulgaricus may be used. Preferably, Lactobacillus < RTI ID = 0.0 > rhamnosus GG), lactobacillus ( lactobacillus < RTI ID = 0.0 > plantarum ) and Lactobacillus acidophilus ( Lactobacillus acidophilus , and more preferably lactobacillus splantarum may be used.
According to a preferred embodiment of the present invention, the primary culturing step is performed at a temperature of 18 to 45 ° C for 12 to 16 hours, and the secondary culturing step is performed at a temperature of 18 to 45 ° C for 30 minutes to 2 hours .
Specifically, in the primary culturing step, the culture can be performed at a temperature of 18 to 45 ° C for 12 to 16 hours. If the culture time is less than 12 hours, microbial culture may not be properly performed, The microorganism enters the death chamber and may not exhibit optimum signal activity.
In the secondary culturing step, the culture can be performed at a temperature of 18 to 45 ° C. for 30 minutes to 2 hours. If the incubation time is less than 30 minutes, the time required for generating and discharging the signal substance and metabolites is not sufficient And when the time exceeds 2 hours, the restriction of the nutrients supplied at the minimum may cause the microorganisms to stop the intended reaction and to manifest physiological activation mechanisms depending on the extreme environment.
According to a preferred embodiment of the present invention, in the step of analyzing the signal activity of the autoinducer- 2, the emission analyzer can be analyzed using vibrioharveyi BB170.
Thus, the optimal culture composition of the present invention is a yeast extract, a beef extract, a peptone, a polysorbate, a magnesium sulfate, and a manganese sulfate other than some constituents that interfere with the signal activity analysis of the autoinducer-2 in the conventional MRS medium By optimizing the effective components, minimizing each component to a specific concentration range can stabilize the signal expression or the physiological activity of the strain during luminescence analysis, thereby greatly improving the sensitivity of the autoinducer-2 to signal activity and luminescence intensity.
Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by the following Examples.
Preparation Example: Preparation of Optimal Medium Composition
After adding 5 mg of yeast extract, 10 mg of peptone, 10 mg of peptone, 1 mg of
Example 1
1% (v / v) of lactobacillus rhamnosus GG (LGV), a commercial probiotic strain, was inoculated on MRS medium and then primary cultured at 37 ° C for 12 to 16 hours. Then, the cultured LGG was washed three times at a concentration of 0.1% of the optimum medium prepared in the above production example to completely remove the constituents of the MRS medium used for the initial culture. Then, the washed LGG was inoculated into the optimal medium (concentration of 0.1% of the MRS medium) and then incubated at 37 ° C for 1 hour.
Example 2
In place of LGG used in Example 1, lactobacillus , a commercially available probiotic strain,
Comparative Example 1
The procedure of Example 1 was repeated except that the LGG used in Example 1 was not cultured in the optimal medium.
Comparative Example 2
The same procedure as in Example 2 was carried out except that the 299v used in Example 2 was not cultured to the optimal medium.
Experimental Example 1: Analysis of Autoinducer-2 signal activity by concentration for each component of the optimum medium (AL medium) composition prepared in the above Preparation Example
The effect of each component of the optimum medium prepared in the above preparation example on the autoinducer-2 signal activity by concentration of peptone, beef extract, yeast extract and polysorbate, which are the main nutrients of lactic acid bacteria growth, was analyzed. Specifically, the above four components were diluted by concentration (1/10, 1/100, 1/1000), respectively. The analytical method was measured in a Reluminant Light Unit (RLU) unit in a luminometer using an autoinducer-2 emission assay, the results of which are shown in FIG.
FIG. 2 is a graph showing changes in the activity of the autoinducer-2 signal for each component of the optimum medium (AL medium) composition prepared in the above Preparation Example. As shown in FIG. 2, when the concentration was 1/10 (10%), the peptone, beef extract, and yeast extract showed significant signal activities. It was also confirmed that the concentration of 1/100 (1%) and 1/1000 (0.1%) exhibited a signal activity inhibition lower than that of 1/10. However, in the case of
The higher the dilution factor when the signal stability is comparable, the more advantageous is the limited amount of lactic acid produced by the lactic acid bacteria during the 1 hour incubation time to obtain the supernatant. The production of the lactic acid lowers the pH of the supernatant, which has a problem that greatly affects the bioactivity of the BB170 strain. In consideration of this point, in the above example, the optimum culture composition was selected as 1/1000 (0.1%), and the same concentrations were applied to the remaining manganese and magnesium sulfate.
Experimental Example 2: Analysis of Autoinducer-2 Signal Activity by Concentration of Optimal Medium Composition
The degree of autoinducer-2 signal activity (signal disturbance) was analyzed using the optimal medium prepared in the above Preparation Example, and the normal MRS medium and mMRS medium. Specifically, the existing MRS medium and the mMRS medium prepared by replacing the glucose of the MRS medium with galactose were used as
(MRS medium)
(mMRS)
(10% concentration)
(1% concentration)
(0.1% concentration)
(Polysorbate 80)
(Magnesium Sulfate)
(Manganese Sulfate)
(Ammonium Citrate)
(Sodium Acetate)
(Dipotassium Phosphate, DPP)
FIG. 3 is a graph showing changes in the signal activity of the autoinducer-2 using the optimal medium prepared in the above-mentioned production example, the normal MRS medium and the mMRS medium. As can be seen from FIG. 3, in the case of the conventional MRS medium and mMRS medium, the initial autoinducer-2 signal activity was not converged to the relative value 1, and the signal activity was irregular as the incubation time was increased. It was found that disturbance or signal disturbance occurred due to unstable physiological activity.
In contrast, in the
Experimental Example 3: Autoinducer-2 signal emission analysis using strains cultured in Examples 1 and 2 and Comparative Examples 1 and 2
In order to analyze the degree of luminescence of the autoinducer-2 signal activity using the strains cultured in Examples 1 and 2 and Comparative Examples 1 and 2 as follows, Vibrio harveyi BB170-based autoinducer-2 luminescence analysis Autoinducer-2 bioluminescence assay). The results are shown in FIG.
Specifically, in the autoinducer-2 luminescence analysis, Vibrio harveyi BB170 strain was cultured in LB medium for 16 hours at 30 ° C and 120 rpm as Surrett and Bassler suggested. Next, strain BB170 was diluted in AB medium at a ratio of 1: 2000, and 90 μl of the diluted BB170 strain was added to a 96-well microtiter white plate (Whatman 7701-3350) and the above-mentioned Examples 1 and 2 and Comparative Examples 1 and 2 Were added to each well and 10 μl of each strain was added thereto. The relative light unit (RLU) value was measured with a luminometer (GloMax Microplate Luminometer, Promega) at intervals of 30 minutes for 2-5 hours.
FIG. 4 is a graph showing signal intensity of the autoinducer-2 using the strains cultured in Examples 1 and 2 and Comparative Examples 1 and 2. As shown in FIG. 4, in the case of Comparative Examples 1 and 2, it was confirmed that the intensity of autoinducer-2 signal emission was as low as about 200 S / N regardless of the type of strain.
On the other hand, in the case of Examples 1 and 2, the intensity of autoinducer-2 signal emission was higher than that of Comparative Examples 1 and 2 cultured in a normal mMRS medium or significantly improved to about 2 times or more And no loss of sensitivity due to luminescence was observed at all. It was confirmed that when the commercial probiotic strains were cultured in the novel optimal medium of the present invention, signal disturbance of the autoinducer-2 was minimized and the intensity of actually measured signal light emission could be greatly improved compared to the conventional method.
Experimental Example 4: Analysis of the number of viable cells in the strains used in Examples 1 and 2
In order to confirm the change in the viable cell counts of the strains used in Examples 1 and 2, the change in the viable cell counts of the strains was analyzed in the following manner, and the results are shown in FIG. Specifically, the strains used in Examples 1 and 2 were cultured for 90 minutes at 37 ° C in the optimal medium prepared in the above Preparation Examples, respectively, and then the number of viable cells was observed using a flat plate culture method .
FIG. 5 is a graph showing changes in viable cell counts of the strains used in Examples 1 and 2 according to the optimum medium. As can be seen from FIG. 5, the 299v and LGG strains were further cultured for 90 minutes in the optimum medium prepared in the above-mentioned preparation example, but it was confirmed that no significant change in the number of bacteria was observed. From these results, it can be seen that the experimental result of Experimental Example 3 is not caused by the change of the viable cell count of the strains due to the use of the novel optimum medium different from the conventional MRS medium.
Therefore, the optimum culture composition prepared in the above Preparation Example contains Yeast Extract, Beef Extract, Peptone, Polysorbate, Magnesium Sulfate and Manganese Sulfate as essential components, and optimizes each component to a specific concentration range of a small amount It was confirmed that signal induction or stabilization of the bacterial physiological activity during the luminescence analysis significantly improved the signal activity and sensitivity of the luminescence intensity of the autoinducer-2.
Claims (7)
Washing the primary cultured microorganism with a first anti-optimal medium;
Culturing the washed microorganism in the optimal medium of claim 1; And
Analyzing the autoinducer-2 signal activity of the secondary cultured microorganism using a luminescence analyzer;
(Autoinducer-2) signal activity of a microorganism of the genus Lactobacillus.
Wherein said primary culturing is carried out at a temperature of 18 ° C to 45 ° C for 12 to 16 hours and said secondary culturing is carried out at a temperature of 18 ° C to 45 ° C for 30 minutes to 2 hours, Autoinducer-2 signaling activity assay of microorganisms.
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| CN114540230A (en) * | 2022-02-24 | 2022-05-27 | 浙江工商大学 | Preparation method and application of culture medium suitable for producing exopolysaccharides by lactobacillus rhamnosus |
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| Moslehi-Jenabian 등. International Journal of Food Microbiology. Vol. 135, No. 3, 페이지 295-302(2009.08.14.)* |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540230A (en) * | 2022-02-24 | 2022-05-27 | 浙江工商大学 | Preparation method and application of culture medium suitable for producing exopolysaccharides by lactobacillus rhamnosus |
| CN114540230B (en) * | 2022-02-24 | 2024-04-19 | 浙江工商大学 | Preparation method and application of extracellular polysaccharide production culture medium suitable for lactobacillus rhamnosus |
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