text
string | predicted_class
string | confidence
float16 |
|---|---|---|
The key novelty of the system presented in this paper is the use of fuzzy-logic and its application in a multi-agent environment. Those agents interact autonomously giving the system greater flexibility and intelligence. We also describe how the open-hardware platform called “Open Garden” is used; it has been designed specifically for the collection of essential agricultural data.
|
other
| 99.94 |
In addition, new interfaces have been implemented. Among these interfaces, the described system is a pioneer in using television as a data representation interface. This is very important because it is one of the devices most commonly used by final users. We should also point out that it is a low cost system, this enables small and medium-sized farmers to access this technology without having to invest a large sum of money.
|
other
| 99.94 |
This article is organized as follows: Section 2 reviews the state of the art and related projects, Section 3 describes the proposed architecture, Section 4 describes the case study conducted to evaluate the system, and finally Section 5 presents the results obtained and our conclusions.
|
other
| 99.94 |
Currently, a variety of automatic crop irrigation systems are available in the literature, and this section provides a detailed analysis of them. Existing solution are limited by various factors: their design does not adapt to the particular requirements of different crop species; the quantity of water supplied by each manufacturer is different, making it difficult to determine the exact amount of water to be supplied to a crop. Moreover, irrigation systems are extremely expensive and are provided by their manufacturers with closed architectures that restrict customization or inter-compatibility with other devices. For example, it is not possible to interconnect sensors from different manufacturers or to integrate data in an application that could be controlled from a smart TV.
|
other
| 99.7 |
The current issue of wasting natural resources has called the European Union to action. The EU is now encouraging the development of solutions that ensure ecological efficiency. Some of the best known projects that have been funded by FP7 include the following:
|
other
| 99.94 |
WATERBEE DA (REF: 283638 Funded FP7-SME): this system allows farmers to save water by watering only at the time and place required . Financed by European Community funds, the project team developed a prototype of a sustainable irrigation system. The tests showed savings of 21%, registering peaks of up to 44%. The impact of irrigation was also reduced to 23%. This system features wireless communication and environmental sensors, providing intelligent, flexible, easy to use, affordable and accurate programming. Moreover, this system can be adapted to the specific requirements of each user, the humidity of the soil and environmental conditions, and to different agricultural management systems . Related works such as include a mobile application that has been used to manage irrigation with pivot in the state of Colorado (USA), while another work in Florida uses evapotranspiration-based irrigation controllers to define schedules. Some works such as operate with a Smart Irrigation Decision Support System; these systems include machine learning techniques such as artificial neural networks, fuzzy decision systems to analyze the water in the soil, or to establish previous irrigation patterns. However, these types of supervised systems require previous expert knowledge to train the algorithms.
|
other
| 99.9 |
OPTIFERT (REF: 2836772 Funded FP7-SME): is based on an innovative automatic irrigation system for medium and large scale agricultural holdings . This system combines fertilization and irrigation, and reflects the increasingly widespread trend among farmers to use computers, making it easier to keep track of the consumption of water and fertilizer. The system is composed of a soil sensor, a data processor, a control and distribution unit that monitors fundamental parameters of soil, and plant requirements in real time. The control software is able to access databases containing information about crop growth and relate it to crop species and soil (type, structure and fertility) data, as well as economic data on costs and prices. It is also possible to get weather forecasts and insert them into the system. In addition, the user can add data, such as reports on crops and planting times. It can obtain data that determine the right amount of water and fertilizer for each stage of crop growth.
|
other
| 99.9 |
ENORASIS (REF: 282949 Funded FP7-SME): by using this system, farmers can install a network of wireless sensors on their farms to gather information on factors affecting the crops’ need for water, including soil moisture, atmospheric temperature, insolation, wind speed and precipitation . The system also has a set of valves to measure any increase in the amount of water. This solution saves water, prevents soil erosion and generates both environmental and economic benefits. This system also uses a weather forecasting model that combines satellite images from the fields and the information from sensors to create a specific meteorological prediction. The model offers such a fine resolution that predictions can focus on areas of up to two square kilometres. Moreover, crop data can be used to prepare a watering plan, allowing the farmers to decide if they need to add more water to the ground .
|
other
| 99.9 |
IRRIMAN LIFE (funded program Life+): granted in 2015, the project is based on an automated system. Using an algorithm , irrigation needs are determined according to the water contained in the soil, the plant, and the atmosphere, all of which are measured on a continual basis using different sensors in the endometrial system. The project ensures the efficient use of water resources, the improvement of the quantitative management of water, and the preservation of the high quality level of water, and avoiding the misuse and deterioration of water resources. This is a very interesting project which has recently started .
|
other
| 99.94 |
This section has provided clear initiatives to implement solutions that combine different sensors for the purpose of using natural resources in a more efficient way e.g., rationing electric power employed by the irrigation equipment or rationing limited resources, such as water. While these systems are composed of different sensors, they use closed platforms and lack the capacity to interact with external agents. Moreover, they lack the intelligence that equips them with learning and adaptation capabilities. Consequently, we need an open and heterogeneous platform that allows us to merge information from all the sensors for subsequent analysis and study.
|
other
| 99.9 |
Having begun as recently as 2014–2015, these projects are still in the development phase. Their use on conventional farms requires a significant investment, making them appropriate only for large areas. Extrapolating these systems for use on smaller areas, such as a small vegetable garden or greenhouse, or using them simply to monitor a crop during a short period of time, would make the cost of acquiring the necessary equipment far too expensive for most farmers.
|
other
| 99.94 |
Nevertheless, a comparative study of commercial solutions has been carried out for small scale farms. The solutions that incorporate sensors do not include systems based on fuzzy logic which allow to establish the watering quantities in a precise way. Aifro WaterEco considers climatology in order to lower or increase irrigation but it is focused on the definition of threshold values and does not include fuzzy logic or sensors, such as soil and land humidity. Blossom , encompasses crop irrigation and generation of calendars, depending on the climate these calendars can be edited manually, it has common functionalities but allows for remote management, it also does not include fuzzy logic in its behavior. BlueSpray includes seasonal information to adjust irrigation as in the previous example, it does not include fuzzy logic based behavior. GreenIQ and IrrigationCaddy are conventional programs that can be managed remotely from mobile applications and include the feature of creating irrigation calendars. Lono incorporates threshold values and seasonal information and reduces crop watering according to the thresholds, as in the previous cases it does not include fuzzy logic and does not have weather sensors.
|
other
| 99.9 |
On the other hand, the Orbit B-Hyve system incorporates a control through smartphones that is able to change some parameters in order to edit the system schedule. The parameters that device takes into account when configuring the irrigation timer are: the slope of the site, the soil type, if it is in the sun or shade, history of rainfall in the area and the current weather. The Rachio Smart Sprinkler Controller system also has a Wi-Fi connection and is able to send the data from the sensor to the user’s smartphone. This device requires an initial configuration which is established by indicating the type of crop and the type of soil. In this way, the system can estimate the irrigation time required by the crop. The fuzzy system is not applied, nor are the flexible rules. Rainmachine is another commercial system which incorporates an automatic irrigation program. It is capable of calculating the percentage of evaporation and transpiration of the soil, according to the weather conditions obtained from the data of the meteorological service. This system, like the others, does not include fuzzy knowledge. The Spruce irrigation system combines the information obtained from all the temperature and humidity sensors and rainfall forecasts. Lastly, we list the Raincommander system for its ease of use and its integration with mobile devices for remote irrigation control. However, this system lacks an intelligent configuration, it has no fuzzy logic rules, and only considers the schedule and the irrigation time that has been configured manually by the user.
|
other
| 99.9 |
After a careful review of the related literature, this work focuses on a novel design of an open architecture composed of virtual agent organizations. The proposed system is economic and can be customized to fit the needs of each farmer making it possible to monitor and automate the irrigation of any crop species. From an analytical point of view, it will be necessary to store the information of each sensor in a remote database, this will allow farmers to examine the effectiveness of the system. Finally, we can deliver these functionalities to the user as services; users will be able to control irrigation from a TV screen, using a remote. In conclusion, the major novelties of this work are: (a) the ability to estimate irrigation time through the use of multi-agent virtual organization technology that executes a fuzzy algorithm, (b) the deployment of agent models in devices with limited capabilities using the PANGEA architecture, (c) the monitoring and control of the irrigation system with a TV remote (thanks to the use of wireless sensors networks).
|
other
| 53.66 |
In the field of computer science and artificial intelligence the use of multi-agent systems deals with the interconnectivity of intelligent agents that collaborate together to solve a complex problem. The use of a combination of agents in wireless sensor networks allows for the design of new platforms with advanced computing capabilities. The design of a multi-agent system based on virtual organizations allows one to monitor and control an irrigation system. The different algorithms that make up the case study should be embedded in embedded devices like sensors or small microcontrollers. To achieve this, we have chosen a multi-task architecture that makes it possible for virtual organizations to have a dialogue between them, this architecture makes up the case study since distributed processing techniques can also be used with it. The proposed architecture must be dynamic, have the ability to merge information from heterogeneous data sources, and contain advanced analysis and prediction capabilities. The dynamism that a multi-agent architecture offers allows us to add new sensors, adapting them to the requirements of the environment. One of the main innovations of this architecture is a design based on organizational theory, which can both imitate and collaborate with human organizations related to crop irrigation. This Section will present the design of an architecture that (1) allows for the creation of an open and self-organizing system, and (2) can handle different types of sensor networks, thus facilitating the dynamic addition of new protocols based on the emergence of new technologies such as Zigbee, RFID, Wi-Fi, and Bluetooth. We will explain the design of the architecture in detail, as well as the agents that make up each virtual organization, as shown in Figure 1.
|
other
| 99.5 |
The architecture is composed of two distinct parts: the bottom is formed by the minimum agents that make up the multi-agent PANGEA system; the top consists of different virtual organizations on which this case study is based, and whose operation is explained below:
|
other
| 99.94 |
Organization Information Fusion: This refers to an organization whose objective is to merge the information provided by the sensor networks (lower layers), which is then integrated with the virtual agent organizations (upper layers). In this organization, agents emulate the human behaviour of adding environmental information, thus making it possible to obtain far more advanced knowledge than what is generally provided by individual data. Also, the information formats controlled by each sensor are transformed to a common and manageable standard for all architecture. The internal message protocol chosen for the communication between agents of the platform is a messaging protocol of plain text that is based on the standard RFC1459 .
|
other
| 99.9 |
Organization Smart Irrigation: This refers to an organization that is in charge of extracting and collecting information from different sensors. Its main function is to transform the physical layer data so that they can be used by other agent organizations. Each agent communicates in a unidirectional way with a central officer who organizes and manages the communication. In this organization, there are two different roles: one held by officers, who obtain the values of the sensors; and another secondary role, in the coordination of tasks and communication with other organizations of the architecture. The different agents that form part of this organization are shown in Table 1. These agents are deployed in the nodes to extract information from the environment, the obtained data are sent to the central node which sends them to the main server.
|
other
| 99.94 |
Organization Control Center: This organization is responsible for monitoring information obtained by agents, and belongs to the Smart Irrigation Organization. The most important task is the intelligent analysis of information and prediction based on the data collected from different sensors. The Crops agent is in charge of coordinating monitoring tasks, analysis and alerts, additionally this agent is responsible for managing the defined rules for each type of crop. In the case of an anomaly, an alerting situation, or a value outside of the usual range, this organization will be responsible for initializing the process of resolving the anomaly, which is then notified to the system administrator.
|
other
| 99.94 |
Organization Application Interface: This organization is in charge of adapting data from the other virtual organizations, and then representing this data in the application layer. As the organization is an interface, the applications inside the client can easily interact with the platform. For example, in the case of an external device that has to request a particular functionality from the system, or any application such as “Web Application or Smart TV”, the data have to be adapted from the raw data to a standard format. This organization will develop an adaptation function, also known as the connector, for later use in any application. The presented case study has several connectors or gateways whose main function is to transform data from the architecture so that the data can then be represented on a smartphone, a Smart TV or a web application.
|
other
| 99.9 |
Pangea MultiAgent System: The decision to use PANGEA was based on its ability to create virtual organizations, which are characterized by their dynamic nature. This is the most singular feature, since other alternatives, such as THOMAS or JADE are not dynamic. PANGEA is a cost-free, multi-agent framework developed by the BISITE research group and anyone can use it. The PANGEA architecture can function with devices with limited computing capabilities, this feature is a big advantage because it enables us to deploy agents embedded in hardware. The fact that sensors are powered by sunlight makes this feature even more essential for the system. Moreover, limited computing capabilities are necessary for the algorithms responsible for data processing, as well as for efficient communication between the sensors in the system. The agents specialized in the management of virtual organizations are defined in , these agents are responsible for managing the agents inside the whole virtual organization. Below, we focus on the basic functions of the agents that manage the virtual organization executed within PANGEA.
|
other
| 99.9 |
DatabaseAgent: This agent plays a storage role in the organization to provide persistence to the information in the organization. It is the only agent with database access privileges. Its objective is to perform backup tasks, as well as to ensure the correct consistency and storage of information. This agent communicates with the rest of the agents in the organization.
|
other
| 99.94 |
Information Agent: This agent manages the services inside the virtual organization. It is also known as the “yellow pages” agent, as it allows other agents to publish the services provided, so that others can access them. When a new device or application uses the architecture for the first time, the corresponding agent should consult the specific services offered in the virtual organization.
|
other
| 99.94 |
Normative Agent: One of the most important aspects in a virtual organization are the norms that govern the organization. This agent is responsible for the security when establishing communication between devices. When an application uses a specific functionality, this agent is in charge of checking whether it is authorized to do so, using a rules engine based on DROOLS .
|
other
| 99.94 |
Service Agent: This agent distributes functionalities as web services. It is also a gateway to communicate external web services outside the system with the agents in the organization. To encourage greater abstraction, functionalities, and different capabilities offered by the architecture, some services are exposed; this mode favors greater integration independent of programming languages.
|
other
| 99.94 |
Organization Manager: This agent plays a very important role in the architecture given that it is responsible for verifying the operation of all the virtual organizations, dealing with security, and balancing and providing encryption of the frames between the most important agents.
|
other
| 99.94 |
In the APP Crop Database different information is included, such as the irrigation rules liked to the type of crop. In these rule we include information on the geographic location, this data base is synchronized with a central server to ease the addition of new crops.
|
other
| 99.94 |
This Section presents a case of study of a small crop environment combining a low cost hardware and multi-agent systems, which allows the fusion of information captured by different sensors. The chosen hardware platform is called the OpenGarden. Due to the wide variety of crops and the source that we can monitor, the architecture can be implemented in three different scenarios: indoors (houses and greenhouses), outdoors (gardens and fields), and with hydroponic agriculture (plants in water-based facilities).
|
other
| 99.9 |
The system must provide the ability to control the state of the plant through the detection of several parameters: moisture in the ground, humidity, brightness sensors for pH, conductivity, temperature, oxygen, and water level. The topology between different sensors is represented in Figure 2. There is a slave node for each type of crop or plant to be monitored, and a single central node that connects the cultivated area. There are two types of nodes: slave nodes, which send the information from the interconnected sensors; and a central or primary node, which acts as a gateway sending data to an agent that resides on a web server, using existing wireless technologies (Wi-Fi, GPRS, 3G).
|
other
| 99.9 |
Below is a detailed explanation of the hardware used in this solution. The Gateway is powered by an Arduino-one controller . The slave nodes send information to the central node via network, with a star topology for the transmission of information, using an Amplitude-Shift Keying (ASK) modulation. The selected band frequency is 433 MHz, due to the autonomy of the devices and the need for efficient communication, where the quantity identity of data shared between the different nodes is not very high. The gateway node is composed of an OpenGarden Shield . The number of central nodes varies depending on the size of the farm, independent subzones can be established with different configurations. The distance between the central zones depends on the visibility of the environment. Using a 433 MHz range we attained interconnectivity between the nodes at a distance of 250 linear meters with total visibility. The functionality offered by each of the controller pins is shown in Figure 3.
|
other
| 99.8 |
The shield of the master node allows us to technologically connect different types of sensors and to gather information from any sensor that is available on the market. In addition, it ensures interconnectivity with external hardware as Arduino or Raspberry by using a serial port. Of note, the shield incorporates a battery for autonomous operation which uses sunlight to power itself during the day. The controller is based on a DS1307 chip to time programming. It has an I2C interface that allows the interconnection of virtually any sensor currently on the market, and an accurate clock that will adjust to time changes. It can detect if there is a fault in the electrical circuit, and consumes less than 500 nA. The central node is capable of expanding its functionalities, providing us with the possibility of adding any type of sensor or functionality that we might need. This expansion port consists of 12 pins (analog and digital) which allow to, for example, adapt the system for activating monitoring systems, monitoring the condition of motors and pumps or if we want to use the system in greenhouses; to control the ventilation system, airflow and motorized doors.
|
other
| 99.9 |
One of the novelties of the system is the use of the light agents that are embedded in the nodes . The light agents are especially designed for implementation in devices and sensors with limited resource constraints. In this case the sensors have limited resources and are therefore embedded in software agents that can communicate with the PANGEA architecture; to reduce computational costs, a simple communication protocol is used. The central node contains an agent that retrieves information for the agents in the Slave Node, this communication is made using the 433 MHz radio frequency. The central node sends the information to the server with REST and the information is made available to the other agent in the virtual organization so that it can be displayed by different devices.
|
other
| 99.94 |
As mentioned in Section 3, the virtual Information Fusion organization aims to adapt and process information from each of the sensors. This organization merges the information collected from each of the individual sensors, and estimates the flow of necessary irrigation at each moment. For the fusion of information from the sensors and the establishing of the volume of water for irrigation, fuzzy logic is used as explained in this section. The reason for using fuzzy logic as opposed to other alternatives, such as Bayes, is because we want to establish a continuous irrigation level and not by categories . A diagram of the flowchart detailing the procedures that take place in the Information Fusion organization in provided in Figure 6. Readers may check for further information.
|
other
| 99.9 |
After the initial installation and activation carried out by the farmer, our system begins an auto-evaluation process where it verifies the condition of the installed sensors. If some type of interconnection error occurs, it is reported to the user through an alert. The nodes have an initial connection time of 10 s to connect to the master node. Once the sensors are connected to the WSN and the link with the central node is established via radio, they are ready to collect measurement data.
|
other
| 99.94 |
When the sensors are launched correctly, each one collects data according to the sampling frequency established by the user. Each of the slave nodes is in charge of collecting different measurements from the sensors, converting them into a format that can be read by a human and transmitting them to the central node. Each measure received by the central node is compared with the previous measures and the state of the humidity sensor is analyzed. If the value of the sensors is above 20%, all the information is sent to the central server with the aim of visualizing these data in the developed applications. However, if the humidity sensor displays a value that is below 20%, although all the necessary irrigation conditions are supplied (temperature, radiation, light, humidity) the required irrigation time will also have to be determined apart from sending the data. The sensors’ measures are used as input variables for the fuzzy logic system which measures the exact irrigation time. The empirical rules used by the fuzzy logic system, have been established by a farmer who is an experienced tomato cultivator. These rules can be seen in Figure 7.
|
other
| 99.7 |
As shown in Figure 6, when the crops are being irrigated, sensor readings cannot be taken until 27 min after the irrigation started, this is due to the effects of transpiration. If soil is watered under conditions of extreme heat, the water will evaporate and the ground will not dampen immediately, resulting in an incorrect reading. The 27 min period allows the sensor to retrieve the correct value for subsoil humidity. This time window is fixed and was calculated by performing evaporation tests during the month of July in the town of Salamanca, Spain. It is possible to find literature on how to calculate time dynamically , however it is not the focus of this work.
|
other
| 95.25 |
The goal of the fuzzy logic based algorithm is to determine the volume of water and the duration of irrigation (opening of electric valve) required in each case. Knowledge rules are established for the humidity sensor in three situations: when the sensor is wet, when the sensor is partly wet and finally when the sensor is dry. Table 2 shows the irrigation time for each case.
|
other
| 99.9 |
To determine the reduction of uncertainty levels that comes with the inclusion of these three variables, an analysis of irrigation estimations is carried out through the Bayes application, on the basis of the use of these variables. The accuracy percentages obtained are listed in Table 3. As can be seen, when the three variables are used the accuracy increases. From this we can conclude that the three variables used are important in reducing the uncertainty when estimating the level of irrigation. We should also highlight that when using other classifiers, based on decision trees, such as J48 accuracy rises to 100%, however Bayes has been used given that it is the alternative to fuzzy logic listed in .
|
study
| 97.9 |
The algorithm chosen in this work is based on a Mamdani system , in which the membership functions are trapezoidal. The reason for using this fuzzy system is that the library , which allows one to develop applications with fuzzy logic in microcontrollers based on the ATmega328p chipset, is the only one that possesses the Mamdani fuzzy system. In addition, the Takagi-Sugeno method is less intuitive and more computationally complex. While the defuzzification process can be done using different methods, the centroid technique method was selected in this case. The fuzzy logic system was designed with MATLAB software. Figure 12 shows the general scheme.
|
other
| 99.7 |
As shown in Figure 14, when the temperature is high, irrigation time is completely determined by it, this helps to avoid water evaporation. In addition, Figure 15 shows how irrigation time increases as the humidity sensor approaches dry values and brightness.
|
other
| 99.8 |
Once the defuzzification process has been carried out, the following preliminary conclusions were obtained:The subsoil moisture sensor provides the most important system information; it measures the moisture of the subsoil and indicates when it is necessary to activate the irrigation mechanism. In addition, it estimates the amount of water needed.The outdoor temperature sensor measures the outside temperature. If the temperature is high, this sensor prevents the watering process that, if activated, would simply result in water evaporation and unnecessary water wastage.The solar radiation sensor is as necessary as the outside temperature sensor, since sunlight causes water to evaporate.
|
other
| 99.9 |
All irrigation systems must be controlled and monitored remotely. This section describes the physical connectivity of the system. The developed system can be deployed in any geographical location, provided that there is a data connection (Wi-Fi/3 G GSM) allowing the data from the sensors to be sent to a platform that resides on a central server. All wireless sensors have batteries that are continuously charged by solar energy. The objective of this section is to describe how individuals who are not familiar with technology could use the proposed system to check the state of their plants in real time. The overall architecture, including the screen display, is shown in Figure 16.
|
other
| 99.9 |
The agents embedded in the sensor network send the collected data periodically to organizations of agents located on the central server. This data can be kept and subsequently displayed. The communication between agents is done via RESTful web services, which allows for minimal battery consumption and high speed. Data exchange is done through JSON frames. This format was chosen because the data can be parsed by agents that are embedded in limited computing devices and very little time is required to plot the information. In Figure 17 below we give an example of the information structure.
|
other
| 99.94 |
The most innovative feature presented in this Section is the use of a display agent installed on a Raspberry PI device, which allows us to connect to any type of Smart TV browser that has an HDMI adapter. The goal is to provide all users, particularly elderly farmers, with a visualization agent which will allow them to view the condition of their crops easily and from their own home. To do this, the architectural design was implemented, as shown in the figure below (Figure 18).
|
other
| 99.94 |
The display agent allows the user to interact with the system via the TV remote control. The first time that the farmer opens the application, he has to carry out an initial configuration, in which he chooses the type of crop and its geographic location. In this way, we preload the initial irrigation configuration which the user can modify according to their preferences. Using an infrared sensor, the user will be able to monitor and control the state of the different sensors in a web environment. In addition, the display agent alarms the user if one of the system sensors fails, even if the user is watching TV, a warning will display on the screen. Figure 19 and Figure 20 show a general view of the user interface of the proposed system, which uses a normal television to check the condition of plants.
|
other
| 99.9 |
In Figure 20, we can see the place where the system has been implemented, the farm has a size of 250 m2 and was loaned to us by a farmer for the purpose of this case study. The farm in this case study does not have large dimensions, this is because we wanted to avoid economic loss if the result happened to be negative and growth would be affected.
|
other
| 99.9 |
This work presented the development of an intelligent system based on WSN that monitors and automates crop irrigation in an easy and economical way. The multi-agent architecture chosen to develop the case study is based on PANGEA, due to its ease of use and the ability to deploy agent drivers on computationally limited devices. The low cost of the proposed system (100 €/250 m2) is a key factor, it makes it an accessible tool to the majority of farmers who cannot afford to implement existing solutions.
|
other
| 99.8 |
The location chosen to test and validate the system was a rural garden located in the outskirts of Salamanca, in the town of Roblija de Cojos. The tomato crop in the garden had a WSN composed of various sensors which measured soil moisture, soil temperature, external temperature, light, rain and wind. The nodes were evenly distributed, with one node placed every 5 m2. Since the garden has an area of 250 m2, slave nodes and a central node, which coordinate communication, were also installed. The main characteristics of the field included: clay soil, no crop yield in the last five years, fallow land, no presence of nematodes.
|
other
| 99.9 |
Table 4 provides a comparison between the costs of the commercial systems that have been described in the Background Section 2 and the system proposed in this work. The price of these devices has been calculated on the basis of the configuration that they would require for the case study conducted in this work; a field of 250 m2. The calculated costs do not include additional teleoperator expenses for 3G/GPRS connection. The chosen systems do not have any installation costs since they are self-installing systems and the procedure can be carried out by the user. 250 m2 is established as the baseline size, which is the minimum field range within which the system is useful and its measurements are conclusive. The implementation costs of the other systems are also calculated for a similar area, between 150 and 250 m2. As can be seen, the proposed system has a smaller cost in comparison to the rest of commercial devices, even though the sensitizing areas are broader.
|
study
| 100.0 |
The type of tomato chosen for the testing process was the Pyros tomato. The Pyros tomato is a productive variety, of indeterminate growth, with a similar precocity to the Montfavet variety, resistant to cracking, with an average weight of 130 g, eye-catching green color, and resistant to Verticillium. Surface drip irrigation, 4 L/h of flow drip with a planting framework of 80 cm between rows and 25 cm between plants, with a plant density of 40 plants/100 m2, in a single line of cultivation.
|
other
| 99.9 |
Above we present the diverse results obtained in the case study. Figure 21 shows the different temperature and radiation measurements taken at different times of the day. Figure 22 presents the relationship between temperature and duration of irrigation. We see that at times of extreme temperatures, the irrigation system was not activated in order to avoid water evaporation.
|
study
| 99.9 |
Figure 23 shows how humidity drops to a minimum value at the hottest moments of the day. After irrigation is begun, we can immediately see that humidity increases. Figure 24 displays water consumption levels for an area of tomato crop measuring 50 m2, using conventional programmed irrigation compared with the system proposed in this article.
|
other
| 99.9 |
The results of the case study have been compared with the traditional automatically programmed irrigation system. Concretely, an irrigation programming device has been used, it is called Orbit B-Hyve with a cost of 130 $. A description and an image are included below (Figure 25).
|
other
| 99.3 |
The crop was always irrigated at dawn, using 1 L of water, and in the evening, using 0.5 L of water. As shown in the image, water consumption in a traditional system is linear, and does not consider any external factors, meaning that the amount of water used for irrigation is always the same and occurs at the same hours of the day. However, the use of the proposed architecture guarantees that the precise amount of water is used, depending on the sensors values and the weather. Both systems were evaluated during 30 days, in comparison to the traditional system, 37% were achieved with the traditional system.
|
other
| 96.8 |
As mentioned before, the conventional system was applied to an area of 10 m2 while the new system was used on an area of 50 m2. The location was contiguous and there was no difference between the crops. Although less water was used with the proposed system, crop production per square meter is very similar; the proposed system 4.73 kg/m2 tomatoes were collected as opposed to the 4.65 kg/m2 of the commercial one. Production is not very high, given that it follows a normal cycle; crops are planted in January and harvested in summer. In other regions, where crops are grown in greenhouses, up to 10 kg/m2 can be obtained.
|
other
| 99.9 |
Clonorchis sinensis, a human liver fluke, is a major food-borne parasite in China . Long-term infections can lead to liver fibrosis, cirrhosis and even carcinogenesis [2, 3]. Some epidemiological studies and clinical researchers have shown that the incidence rate of primary hepatocellular carcinoma (HCC) is much higher in patients infected with C. sinensis than in non-infected patients [4–6].
|
study
| 88.9 |
A homologue of granulin from C. sinensis (CsGRN) was identified in our previous studies, which was predicted to be a component of excretory/secretory products (ESPs) [7, 8]. Granulins are a family of secreted, glycosylated peptides that are cleaved from a single precursor protein with 7.5 repeats of a highly conserved 12-cysteine granulin/epithelin motif . As independent growth factors, granulin family members are excessively expressed in various tumour tissues and are important in normal development, wound healing, and tumorigenesis . Overexpression of progranulin (PGRN), which is a 60 to 90 kDa glycoprotein containing seven tandemly repeated granulin motifs in mammals, is linked to tumorigenesis in numerous human tissues, including liver. It is also associated with an aggressive and invasive tumour phenotype . The ov-grn-1, a granulin among the ESPs of Opisthorchis viverrini, may establish a tumorigenic environment that ultimately manifests as cholangiocarcinoma (CCA) [12, 13]. FhGLM, a granulin-like molecule in Fasciola hepatica that is likely to be secreted through a nonclassical pathway, might exert a proliferative action on host cells during fascioliasis .
|
study
| 100.0 |
ESPs of C. sinensis (CsESPs) were reported as one of the most important factors for pathogenesis . Our previously studies showed that some molecules from CsESPs could cause obvious apoptotic inhibition, and promote proliferation and migration of human HCC cells, which might exacerbate the process of HCC patients combined with C. sinensis infection [16, 17].
|
study
| 100.0 |
Given the close phylogenetic relationship with liver flukes and topologically similarity to both ov-grn-1 and PGRN, we proposed that CsGRN may have a similar biological function of other granulin superfamily members as a growth factor. In the present study, the molecular characteristics of CsGRN and its potential roles in the pathogenesis of clonorchiasis were investigated.
|
study
| 100.0 |
Adult worms of C. sinensis were isolated from the bile ducts of infective cats. After washing procedures, the adult worms were used for tissue sections preparation or total RNA extraction. Adult worms were also cultivated to collect CsESPs and eggs, according to the method described previously .
|
study
| 99.94 |
The mRNA sequence annotated with granulin (CsGRN) was selected from our C. sinensis cDNA plasmid library and was identified by DNA sequencing. The domains, physicochemical properties and some structures of the translated amino acid sequence were predicted with proteomics tools at NCBI (http://www.ncbi.nlm.nih.gov) and ExPaSy website (http://www.expasy.org/), and the disulphide bonds were analysed through the website (http://scratch.proteomics.ics.uci.edu/). The multiple alignments of sequences with homologues from human and helminths were carried out by Vector NTI software, and the phylogenetic tree was constructed with corresponding sequences from 14 other species using the software MEGA version 6.0 .
|
study
| 100.0 |
The ORF of CsGRN (GenBank KY855531) was 714 bp and the specific primers were as follows: forward 5′-CGC GGA TCC TGT AAA TAT AAC CAG ACT TG-3′ (BamH I) (Thermo Scientific, Waltham, USA), Reverse: 5′-TTA CTC GAG CGG AGC ACA GGT GTA GTG AT-3′ (Xhol I) (Thermo Scientific). The underlined bases indicated restrictions sites. cDNA was synthesised from total RNA, which was isolated from frozen C. sinensis adult tissues. The amplification conditions were 94 °C for 1 min, 55 °C for 1 min and 72 °C for 1 min for 30 cycles, plus 72 °C for 10 min. The purified PCR products were ligated into the pGEM-T-Easy vector (Promega, Madison, USA), followed by transformation into E. coli DH5α (Promega). The resulting plasmid DNA was digested with the appropriate restriction enzymes, ligated into the pET-28a (+) expression vector (Novagen, Darmstadt, Germany), and then transformed into E. coli BL21 (DE3) (Promega). Selected clones were grown and induced with 1 mM isopropyl-β-d-thiogalactoside (IPTG, Sigma, Guangzhou, China) at 20 °C for 12–18 h. The bacterial cells were collected by centrifugation and were sonicated on ice. The supernatant was collected, and recombinant protein was purified using the His-Bind Purification Kit (Novagen). The lysates of purified protein were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of purified recombinant protein was determined by using the BCA protein assay kit (Novagen).
|
study
| 100.0 |
The recombinant CsGRN (rCsGRN) and CsESPs were emulsified with complete Freund’ s adjuvant (Sigma-Aldrich, Guangzhou, China) and were subcutaneously immunised with 200 μg of protein for each rat initially. Subsequently, each rat was given 100 μg of protein (emulsified with equivalent incomplete Freund’s adjuvant (Sigma-Aldrich) for three booster injections at 2-week intervals. Anti-CsGRN serum was collected every 2 weeks. Two weeks after the final boosting, the rats were sacrificed, and the sera were collected. Sera from naïve rats were also collected as a control. The antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. For the production of mouse anti-CsGRN serum, Balb/c mice were initially immunised with 100 μg of purified rCsGRN followed by 50 μg the next three times, as described in the above method. The mice were sacrificed, and the liver tissues and serum were collected every 2 weeks from the final boosting for 8 weeks. The liver tissues and serum from normal mice during the same periods were also collected as a control.
|
study
| 100.0 |
Freshly prepared C. sinensis adult worms were washed with phosphate buffer solution (PBS, 20 mM, pH 7.4) and then were fixed with 4% paraformaldehyde. The worms were dehydrated by a graded ethanol series, embedded in paraffin blocks and stored in a desiccator until use. Sections (4 μm thickness) were mounted on slide glasses, deparaffinized, rehydrated and rinsed with PBS. The slides were incubated with rat anti-CsGRN or rat normal sera (1:50 dilutions) in PBS at room temperature for 2 h and then were washed with PBS three times. After incubation with Cy™3-conjugated anti-rat IgG (1:400 dilutions; Proteintech Group, Chicago, USA) for 2 h, the slides were washed with PBS three times and were observed under a light/fluorescence microscope (Olympus BX63, Hatagaya, Japan).
|
study
| 99.9 |
Female Balb/c mice (6–8 weeks of age) in the C. sinensis group were intragastrically infected with metacercariae (30 metacercariae per mouse). We determined the success of infection by stool examination. Five mice from each group were randomly selected and were sacrificed at 7, 30, 60, 90, 120 and 180 days postinfection (uninfected mice in same terms were used as controls). The liver tissues were extracted, and tissue sections were prepared. These samples were fixed and cut by a microtome of 4 μm sections. The sections were incubated with mouse anti-CsGRN serum or mouse naïve serum (1:50 dilutions) overnight at 4 °C after being dewaxed in xylene, dehydrated in ethanol, and blocked with normal goat serum. The sections were washed with PBST (0.1% Tween-20 in PBS) and were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:400 dilutions; Proteintech Group) for 1 h. The sections were rinsed with PBS for 15 min, after which the slides were developed with diaminobenzidine (DAB). Next, the sections were counterstained with Mayer’s haematoxylin, dehydrated, cleared in xylene and mounted in PermountH. Images were captured with a microscope (Olympus BX51, Hatagaya, Japan).
|
study
| 100.0 |
RBE (human cholangiocarcinoma cell line, ATCC), and PLC (human hepatocarcinoma cell line, ATCC) were maintained as specified by ATCC protocols. RBE cells were cultured in RPMI 1640 (Gibco, Carlsbad, USA) while PLC was routinely maintained in DMEM medium (Gibco). These cell lines were supplemented with 10% fetal bovine serum (FBS, Gibco) and penicillin-streptomycin (100 units/ml) in 5% CO2 at 37 °C.
|
study
| 99.94 |
Standard molecular biology techniques were used for the construction of the pEGFP-C1-CsGRN recombinant plasmid. The PCR product of the CsGRN fragment was cut and inserted between the Xhol I (Thermo Scientific) and EcoR I (Thermo Scientific) restriction sites in the pEGFP-C1 vector (Promega). The forward and reverse primers (restriction sites are underlined) used to amplify this fragment included forward 5′-GCG CCT CGA GTG TAA ATA TAA CCA GAC-3′ (Xhol I) and reserve 5′-ATA AGG ATC CCG GAG CAC AGG TGT AG-3′ (EcoR I) respectively, based on the following conditions: 30 s denaturation at 94 °C, 30 s annealing at 60 °C, and 1 min extension at 72 °C for 30 cycles, plus 72 °C for 10 min. Cells (1 × 106) plated in a 6-well cell culture cluster were transfected with either 0.8 μg of pGFP-C1 or pGFP-C1-CsGRN using lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. Two days after transfection, the stable cell line selection was started using the optimal concentration of G418. The medium was changed every 2–3 days, and the cells were split when necessary. After 2–4 weeks, all of the non-transfected cells disappeared, and isolated colonies began to appear. The selective overexpression of GFP or CsGRN cells was designated as PLC-GFP/RBE-GFP cells and PLC-GRN/RBE-GRN cells, respectively. CsGRN overexpression in these cells was checked by a fluorescence microscope, fluorescence activated cell sorting (FACS) analysis, qRT-PCR analysis and Western blotting first incubated with mouse anti-CsGRN sera (1:100 dilution).
|
study
| 100.0 |
To further confirm the role of CsGRN in human cancer progression, wound-healing assays were performed to evaluate the effect of CsGRN on cell migration as described previously . The transfected PLC and RBE cells seeded in 6-well plates were grown to 80% confluence and were wounded by scratching with p200 pipette tips. Wounds were observed and photographed under a light microscope (Leica DMI3000B, Wetzlar, Germany) every 24 h for 72 h. The distances between the parallel cell edges were measured at each time point using Image J software. For each well, three different fields along the scratch were analysed in triplicate. Cell motility was measured as the percentage of the cell migration distance, which was regarded as the initial scratch distance. To evaluate the effect of CsGRN on cell invasion, we performed transwell assays as described previously . The PLC-GFP/RBE-GFP cells and PLC-GRN/RBE-GRN cells were suspended at 5.0 × 104 per insert with serum-free media and then were transferred to wells filled with a culture medium containing 10% FBS as a chemoattractant. After 24 h of incubation, non-invading cells on the top of the membrane were removed with a cotton swab. The migrated cells on the underside of the filter membrane were fixed and stained with 0.1% crystal violet. The number of migrated cells on the membrane was counted in five randomly selected microscopic fields, and the cells were photographed. The protocol used for the invasion assay was the same as that used for the migration assay, except that the transwell insert was coated with Matrigel (BD Biosciences, Heidelberg, Germany).
|
study
| 100.0 |
Total RNA of transfected cells was isolated using the Trizol reagent and was reverse transcribed to cDNA using ABM’s 5× All-In-One RT Master Mix (Transgen, Beijing, China). For qRT-PCR, SYBR Premix Ex Taq (Takara, Dalian, China) was used according to the manufacturer’s instructions. The primers were as follows: for CsGRN, forward 5′-CGC GGA TCC TGT AAA TAT AAC CAG ACT TG-3′ and reverse 5′-TTA CTC GAG CGG AGC ACA GGT GTA GTG AT-3′; for MMP2, forward 5′-TAC AGG ATC ATT GGC TAC ACA CC-3′ and reverse 5′-GGT CAC ATC GCT CCA GAC T-3′;for MMP9, forward 5′-TGT ACC GCT ATG GTT ACA CTC G-3′ and reverse 5′-GGC AGG GAC AGT TGC TTC T-3′; for human actin, forward 5′-GGC ACT CTT AGC CTT CCT TCC T-3′ and reverse 5′-GCC AGA CAG CAC TGT GTT GGC GT-3′. Reactions were conducted under the following conditions: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 20 s. The melting curves were analysed automatically by a collection of the fluorescence signals, and the expression of mRNA was calculated and normalised using the 2-ΔΔCt method relative to actin with CFX96 software (Bio-Rad, Hercules, USA). Independent experiments were performed in triplicate.
|
study
| 99.94 |
CsGRN (5 μg) and CsESPs (30 μg) were subjected to 12% SDS-PAGE and then were electro-transferred onto polyvinylidene difluoride (PVDF) membranes (Whatman, Maidstone, UK) at 100 V for 1 h in a Trans-Blot transfer (Bio-Rad). The membranes were blocked with 5% (w/v) skimmed milk in PBS for 2 h at room temperature and then were probed with mouse anti-His tag monoclonal antibody (1:2000, Novagen), rat anti-CsGRN serum (1:200), rat anti-CsESPs serum (1:200) and rat naïve serum (1:200) at 4 °C overnight, respectively. After washing with PBS, the membranes were successively incubated with HRP-conjugated rabbit anti-rat or rabbit anti-mouse IgG at 1:2000 dilution (Proteintech Group) at room temperature for 1 h. The blots were visualised by enhanced chemiluminescence (ECL, Millipore, Billerica, USA). We also used Western blotting to detect the mechanism of cancer progression induced by CsGRN. In detail, the total proteins of transfection cells were extracted using radioimmunoprecipitation assay lysis buffer (RIPA, Beyotime, Shanghai, China) and then were electrotransferred onto PVDF membranes. The membranes were incubated at 4 °C overnight with E-cadherin (1:2,000), vimentin (1:2,000), N-cadherin (1:2,000), ZO-1 (1:2,000), β-catenin (1:2,000), or GAPDH (1:2,000) diluted in blocking solution. In addition, the membranes with total proteins from Balb/c mice liver tissues were incubated at 4 °C overnight with E-cadherin (1:2,000), vimentin (1:2,000), N-cadherin (1:2,000), ZO-1 (1:2,000), β-catenin (1:2,000), p-ERK (1:2,000), ERK (1:2,000), p-AKT (1:2,000) or GAPDH (1:2,000) diluted in blocking solution, all antibody were products from cell signaling technology (CST, Boston, USA).
|
study
| 100.0 |
Experimental data were obtained from three independent experiments with a similar pattern; data are expressed as the means ± standard deviation. The Student’s t-test and ANOVA were used to determine the statistical significance of the data obtained, and the means were compared between the groups using SPSS21.0 statistical software. A P < 0.05 represented a statistically significant difference.
|
other
| 99.6 |
The sequence of CsGRN encoding 238 amino acids was predicted with molecular weight 25.8 kDa, theoretical point isoelectric (PI) of 8.5. It contains two granulin domains from sequence released in the GenBank (GAA54205.1), which is presumed without N-terminal signal peptide. Multiple alignments of the sequences among GRN and nine members from other organisms (Fig. 1a) indicated that it belongs to the granulin family with a conservative domain with rich cysteine peptides. They share low sequence identity to each other except for O. viverrini. In addition, the sequence is abundant with cysteines being presumed to contain thirteen disulphide bonds. Moreover, the phylogenetic analysis (Fig. 1b) suggested that the granulin protein from C. sinensis has a very close relationship to O. viverrini. Furthermore, the evolution of the members from host and trematode or nematode may vary greatly.Fig. 1 a Multiple alignment of sequences with one core granulin domain from various species are performed with Vector NTI software. Identical sequences are in black and conservative sequences are in grey; theoretical disulphide bonds are numbered one to six above each cysteine residue. b Phylogenetic tree for the granulins from a range of phyla constructed by MEGA 6.0 using the neighbor-joining method, Poisson correction is required and bootstrap values are 1,000. The abbreviations and accession numbers of the sequences are as follows: Clonorchis sinensis (C. sinensis, GAA54205.1); Opisthorchis viverrini (O. viverrini, XP_009174632.1); Caenorhabditis elegans (C. elegans, NP_492982.1); Dicentrarchus labrax (D. labrax, CBN81737.1); Fasciola hepatica (F. hepatica, ID BN1106_s891B000441 in the WormBase ParaSite); Schistosoma japonicum (S. japonicum, CAX73857.1); Schistosoma haematobium (S. haematobium, XP_012796138.1); Homo sapiens (H. sapiens, NP_002078.1); Mus musculus (M. musculus, NP_032201.2); Rattus norvegicus (R. norvegicus, AAH72469.1); Ixodes scapularis (I. scapularis, XP_002415868.1); Trichinella spiralis (T. spiralis, XP_003371171.1); Anolis carolinensis (A. carolinensis, XP_008111426.1); Haemonchus contortus (H. contortus, CDJ86608.1), Schistosoma mansoni (S. mansoni, CCD75903.1)
|
study
| 100.0 |
a Multiple alignment of sequences with one core granulin domain from various species are performed with Vector NTI software. Identical sequences are in black and conservative sequences are in grey; theoretical disulphide bonds are numbered one to six above each cysteine residue. b Phylogenetic tree for the granulins from a range of phyla constructed by MEGA 6.0 using the neighbor-joining method, Poisson correction is required and bootstrap values are 1,000. The abbreviations and accession numbers of the sequences are as follows: Clonorchis sinensis (C. sinensis, GAA54205.1); Opisthorchis viverrini (O. viverrini, XP_009174632.1); Caenorhabditis elegans (C. elegans, NP_492982.1); Dicentrarchus labrax (D. labrax, CBN81737.1); Fasciola hepatica (F. hepatica, ID BN1106_s891B000441 in the WormBase ParaSite); Schistosoma japonicum (S. japonicum, CAX73857.1); Schistosoma haematobium (S. haematobium, XP_012796138.1); Homo sapiens (H. sapiens, NP_002078.1); Mus musculus (M. musculus, NP_032201.2); Rattus norvegicus (R. norvegicus, AAH72469.1); Ixodes scapularis (I. scapularis, XP_002415868.1); Trichinella spiralis (T. spiralis, XP_003371171.1); Anolis carolinensis (A. carolinensis, XP_008111426.1); Haemonchus contortus (H. contortus, CDJ86608.1), Schistosoma mansoni (S. mansoni, CCD75903.1)
|
study
| 100.0 |
The ORF of CsGRN was cloned into the pET-28a (+) expression vector, and the recombinant plasmids were confirmed by restriction enzyme identification and DNA sequencing (not shown). The one mM IPTG-induced rCsGRN was purified and analysed by SDS-PAGE, and the purified protein was obtained from the supernatant using the His-Bind Purification Kit (Fig. 2a).Fig. 2Expression and identification of rCsGRN. a rCsGRN was identified by 12% SDS-PAGE. Protein molecular weight markers (Lane M), lysate of E. coli containing pET-28a (+) without induction (Lane 1) and with induction by IPTG (Lane 2), lysate of E. coli containing pET-28a (+)-CsGRN without induction (Lane 3) and with induction by IPTG (Lane 4), supernatant (Lane 5) and precipitate (Lane 6) of the lysate of E. coli containing the recombinant plasmid after induction, the purified recombinant CsGRN protein (Lane 7). b rCsGRN protein was identified as a component of CsESPs. rCsGRN protein was probed by mouse anti-His-tag serum, rat anti-CsGRN serum, rat anti-CsESPs serum and naïve serum (Lanes 1–4, respectively). In addition, CsESPs were probed with rat anti-CsGRN serum and naïve serum (Lanes 5, 6). Protein molecular weight markers (Lane M)
|
study
| 100.0 |
Expression and identification of rCsGRN. a rCsGRN was identified by 12% SDS-PAGE. Protein molecular weight markers (Lane M), lysate of E. coli containing pET-28a (+) without induction (Lane 1) and with induction by IPTG (Lane 2), lysate of E. coli containing pET-28a (+)-CsGRN without induction (Lane 3) and with induction by IPTG (Lane 4), supernatant (Lane 5) and precipitate (Lane 6) of the lysate of E. coli containing the recombinant plasmid after induction, the purified recombinant CsGRN protein (Lane 7). b rCsGRN protein was identified as a component of CsESPs. rCsGRN protein was probed by mouse anti-His-tag serum, rat anti-CsGRN serum, rat anti-CsESPs serum and naïve serum (Lanes 1–4, respectively). In addition, CsESPs were probed with rat anti-CsGRN serum and naïve serum (Lanes 5, 6). Protein molecular weight markers (Lane M)
|
study
| 100.0 |
rCsGRN was probed with mouse anti-His tag serum, rat anti-CsGRN serum, rat anti-CsESPs serum, and rat naïve serum as a control (Fig. 2b, Lanes 1–4). In addition, CsESPs was probed with rat anti-CsGRN serum (Lane 5) and rat naïve serum as a control (Lane 6). Rat anti-CsGRN serum could specifically recognise CsESPs, and rat anti-CsESPs serum could react with rCsGRN (Fig. 2b, Lanes 2, 5), confirming that CsGRN was an excretory-secretory product of C. sinensis.
|
study
| 100.0 |
Immunofluorescence localisation analysis showed that CsGRN was mainly localised in the tegument and testes of C. sinensis adult worm (Fig. 3).Fig. 3Immunolocalization of CsGRN in C. sinensis adult worm. Sections of C. sinensis adult worm were probed with rat anti-CsGRN serum (a, b, e, f) or normal rat sera (c, d, g, h) followed by my3-conjugated anti-rat IgG. Sections were imaged under fluorescence light (b, d, f, h), or white light (a, c, e, g). CsGRN was distributed in the tegument and testes of the adult worm (b, f). Blue arrow: tegument; Yellow arrow: testes. The images were magnified at 100× (scale-bar: 200 μm) for adult worm
|
study
| 100.0 |
Immunolocalization of CsGRN in C. sinensis adult worm. Sections of C. sinensis adult worm were probed with rat anti-CsGRN serum (a, b, e, f) or normal rat sera (c, d, g, h) followed by my3-conjugated anti-rat IgG. Sections were imaged under fluorescence light (b, d, f, h), or white light (a, c, e, g). CsGRN was distributed in the tegument and testes of the adult worm (b, f). Blue arrow: tegument; Yellow arrow: testes. The images were magnified at 100× (scale-bar: 200 μm) for adult worm
|
study
| 100.0 |
The liver tissues from Balb/c mice infected with C.sinensis were analysed by immunohistochemistry using mouse anti-CsGRN sera (Fig. 4). Liver sections from infected and uninfected mice probed with naive serum were not shown here. Positive staining was indicated with brown. Compared with sections from normal mice, strong staining was detected in the bile duct and the liver tissue after 1 month of parasite infection. Additionally, the level of CsGRN was higher with extended time. Most importantly, brown granules were found around the cell nuclei, explaining that CsGRN entered the biliary epithelium cells and hepatic cells across the cell membrane (Fig. 4). CsGRN was also seen in the bile ducts that were distant from the liver flukes, while normal mouse biliary epithelium and liver tissues were unstained (Fig. 4a, c, e, g, i, k).Fig. 4Immunohistochemical localisation of CsGRN in infected Balb/c mouse livers. Sections of Balb/c mouse livers infected with C. sinensis treated with mouse anti-CsGRN serum (b, d, f, h, j and l) or mouse naïve serum (a, c, e, g, i and k). During early infection (1 month), CsGRN was observed mainly surrounding the bile ducts where parasites reside, and brown granules were found around cell nuclei, which explained how CsGRN entered the biliary epithelium cells across the cell membrane (d). From 2 months post-infection, CsGRN was also seen within the hepatocytes (f, h, j and l). With prolonged infection, more CsGRN gathered in bile duct epithelial cells and hepatocytes. Positive hepatocytes and biliary epithelium cells were even found in the smaller bile duct where adult worm could not reach. a, b normal, c, d 1 month, e, f 2 months, g, h 3 months, i, j 4 months and (k, l) 6 months post-infection. Arrows indicate positive cells. The images were magnified at 1,000× (scale-bar: 20 μm)
|
study
| 100.0 |
Immunohistochemical localisation of CsGRN in infected Balb/c mouse livers. Sections of Balb/c mouse livers infected with C. sinensis treated with mouse anti-CsGRN serum (b, d, f, h, j and l) or mouse naïve serum (a, c, e, g, i and k). During early infection (1 month), CsGRN was observed mainly surrounding the bile ducts where parasites reside, and brown granules were found around cell nuclei, which explained how CsGRN entered the biliary epithelium cells across the cell membrane (d). From 2 months post-infection, CsGRN was also seen within the hepatocytes (f, h, j and l). With prolonged infection, more CsGRN gathered in bile duct epithelial cells and hepatocytes. Positive hepatocytes and biliary epithelium cells were even found in the smaller bile duct where adult worm could not reach. a, b normal, c, d 1 month, e, f 2 months, g, h 3 months, i, j 4 months and (k, l) 6 months post-infection. Arrows indicate positive cells. The images were magnified at 1,000× (scale-bar: 20 μm)
|
study
| 100.0 |
The ORF of CsGRN was cloned into the pEGFP-C1 eukaryotic expression vector, and the recombinant plasmids were confirmed by sequencing (Additional file 1: Figure S1). Viable cell images of most cells showed green fluorescence and grew well after the recombinant plasmid pEGFP-C1-CsGRN transduction (Fig. 5a). To exclude the possibility that transfection plasmids could affect cell survival, the cellular viability of the transfected cells was detected by Annexin V-APC/7AAD staining with FACS analysis. Neither the pEGFP-C1 plasmids nor the pEGFP-C1-CsGRN plasmids had any effect on cell viability (Fig. 5b). The qRT-PCR and Western blotting results showed the relative mRNA level and the protein expression of CsGRN were significantly raised in the PLC-GRN and RBE-GRN cells compared with that in PLC-GFP/RBE-GFP cells (Fig. 5c, d), indicating that overexpressed-CsGRN PLC/RBE cell line was successfully constructed.Fig. 5Successful construction of stable CsGRN-overexpressed hepatoma cells and cholangiocarcinoma cells. a Green fluorescence was observed under an inverted microscope and was emitted by PLC and RBE cells transformed with pGFP-C1 and pGFP-C1-CsGRN, respectively. The images were magnified at 100× (scale-bar: 200 μm).b Cellular viability of transfected PLC and RBE cells (24 h, 48 h and 72 h after transfection) was detected by Annexin V-APC/7AAD double staining with FACS analysis. c qRT-PCR was used to detect the expression of CsGRN mRNA between the experimental group and control group. d Western blot analysis was used to determine the CsGRN protein expression between the experimental group and control group. *P < 0.05, ***P < 0.001 compared with a matched group
|
study
| 100.0 |
Successful construction of stable CsGRN-overexpressed hepatoma cells and cholangiocarcinoma cells. a Green fluorescence was observed under an inverted microscope and was emitted by PLC and RBE cells transformed with pGFP-C1 and pGFP-C1-CsGRN, respectively. The images were magnified at 100× (scale-bar: 200 μm).b Cellular viability of transfected PLC and RBE cells (24 h, 48 h and 72 h after transfection) was detected by Annexin V-APC/7AAD double staining with FACS analysis. c qRT-PCR was used to detect the expression of CsGRN mRNA between the experimental group and control group. d Western blot analysis was used to determine the CsGRN protein expression between the experimental group and control group. *P < 0.05, ***P < 0.001 compared with a matched group
|
study
| 100.0 |
To determine whether CsGRN could play any role in cancer cell migration, we carried out wound-healing assays. The results showed that PLC/RBE-GRN cells could induce significant cell migration when compared with PLC/RBE-GFP cells, respectively (Fig. 6). Similarly, transwell cell migration/invasion assay results indicated that the upregulation of CsGRN in PLC and RBE cells significantly increased migration/invasive activity compared with that in the control group (Fig. 7). Therefore, CsGRN could significantly improve the migration/invasion ability of CCA and HCC in vitro.Fig. 6 CsGRN promotes increased cell migration. Cell migration of PLC and RBE cells shown by wound-healing assay (a and c). Cells were observed using light microscope under 5× objective (scale-bar: 100 μm). Assays were performed in triplicate. Relative cell migration level was calculated by normalising to cell migration level at 0 h. ***P < 0.001, compared with the control group. Data are presented graphically in panels b and d Fig. 7Cell migration and invasion triggered by CsGRN in the transwell assay. PLC-GFP/GRN cells (a) or RBE-GFP/GRN cells (c) were suspended in serum-free media for 24 h. PLC-GFP cells and RBE-GFP cells were the negative control. Invasion assays were performed using Matrigel-coated membranes. The migration assay was similar to the invasion assay, except that the upper side of the membranes was not coated with the matrigel. Cells attached to the lower surface of the membranes at 24 h were counted under a light microscope. Cells were observed using a light microscope under a 10× objective (scale-bar: 100 μm). Ten random visual fields were selected to quantify the migration and invasion (b and d) using Image J software. **P < 0.01, ***P < 0.001, compared with control group
|
study
| 100.0 |
CsGRN promotes increased cell migration. Cell migration of PLC and RBE cells shown by wound-healing assay (a and c). Cells were observed using light microscope under 5× objective (scale-bar: 100 μm). Assays were performed in triplicate. Relative cell migration level was calculated by normalising to cell migration level at 0 h. ***P < 0.001, compared with the control group. Data are presented graphically in panels b and d
|
study
| 100.0 |
Cell migration and invasion triggered by CsGRN in the transwell assay. PLC-GFP/GRN cells (a) or RBE-GFP/GRN cells (c) were suspended in serum-free media for 24 h. PLC-GFP cells and RBE-GFP cells were the negative control. Invasion assays were performed using Matrigel-coated membranes. The migration assay was similar to the invasion assay, except that the upper side of the membranes was not coated with the matrigel. Cells attached to the lower surface of the membranes at 24 h were counted under a light microscope. Cells were observed using a light microscope under a 10× objective (scale-bar: 100 μm). Ten random visual fields were selected to quantify the migration and invasion (b and d) using Image J software. **P < 0.01, ***P < 0.001, compared with control group
|
study
| 100.0 |
To investigate the underlying mechanisms by which CsGRN promotes CCA and HCC cell migration and invasion, Western blotting was performed to show the expression levels of epithelial-mesenchymal transition (EMT) relevant markers. The results indicated that overexpressed-CsGRN PLC and RBE cell significantly increased vimentin, N-cadherin and β-catenin, and decreased ZO-1 compared with the control group, indicating that CsGRN is involved in the EMT process in CCA and HCC cells (Fig. 8a, b). The relative mRNA expression of matrix metalloproteinases (MMPs) in transfected PLC and RBE cells showed that the MMP2 level was higher in PLC-GRN cells and that the MMP2 and MMP9 levels were higher in RBE-GRN cells than in PLC-GFP cells or RBE-GFP cells (Fig. 8c). To further validate the effects of CsGRN on promoting cancer metastasis, we conducted animal experiments. By detecting the EMT-related indicators and relevant signalling pathway markers in Balb/c mouse liver immunised with rCsGRN protein (Fig. 8d), we found that the level of E-cadherin increased, but vimentin decreased with the immune time extended. The signal indictors p-ERK and p-AKT were used to determine the mechanisms of migration. The expression levels of p-ERK and p-AKT were dynamically changed. In detail, p-ERK reached a peak in 4 weeks after immunisation while p-AKT did in 2 weeks. The statistical data of Fig. 8a, b, d are shown in Additional file 2: Figure S2.Fig. 8 CsGRN promotes mesenchymal characteristics. a, b Western blotting was used to detecting the expression of EMT-relevant markers in transfected PLC and RBE cells. PLC-GRN/RBE-GRN cells showed the upregulation of vimentin, N-cadherin and β-catenin, and downregulation of ZO-1; however, the expression level of E-cadherin had not changed. c Relative mRNA expression of MMPs in transfected PLC and RBE cells. MMP2 expression was higher in PLC-GRN cells, and MMP2 and MMP9 were higher in RBE-GRN cells than in PLC-GFP cells or RBE-GFP cells. **P < 0.01, compared with control group. d The expression of EMT-relevant markers and relevant signalling pathways indicators was detected in the liver from Balb/c mice injected with rCsGRN protein. The level of E-cadherin increased, and the level of vimentin decreased in rCsGRN protein-treated mice with an extended immune time extended (no change in N-cadherin). The expression levels of p-ERK and p-AKT were dynamically changed. p-ERK reached a peak in 4 weeks after immunisation, while p-AKT did at 2 weeks
|
study
| 100.0 |
CsGRN promotes mesenchymal characteristics. a, b Western blotting was used to detecting the expression of EMT-relevant markers in transfected PLC and RBE cells. PLC-GRN/RBE-GRN cells showed the upregulation of vimentin, N-cadherin and β-catenin, and downregulation of ZO-1; however, the expression level of E-cadherin had not changed. c Relative mRNA expression of MMPs in transfected PLC and RBE cells. MMP2 expression was higher in PLC-GRN cells, and MMP2 and MMP9 were higher in RBE-GRN cells than in PLC-GFP cells or RBE-GFP cells. **P < 0.01, compared with control group. d The expression of EMT-relevant markers and relevant signalling pathways indicators was detected in the liver from Balb/c mice injected with rCsGRN protein. The level of E-cadherin increased, and the level of vimentin decreased in rCsGRN protein-treated mice with an extended immune time extended (no change in N-cadherin). The expression levels of p-ERK and p-AKT were dynamically changed. p-ERK reached a peak in 4 weeks after immunisation, while p-AKT did at 2 weeks
|
study
| 100.0 |
Long-standing infections with C. sinensis eventually lead to clonorchiasis, which results in cholangiectasis, cholecystitis, cholelithiasis, hepatic fibrosis, and even CCA and HCC . Additionally, researchers have proposed that mechanical damage caused by the activities of the parasites and CsESPs excreted and secreted from the liver fluke is the primary pathogenic mechanism . In our previous studies , CsGRN, one of an ingredient of CsESPs, was identified and the prokaryotic expression of the recombinant CsGRN protein was carried out.
|
study
| 100.0 |
Sequence analysis showed that CsGRN was a granulin member that has 28% overall identity with human PGRN. It shared the characteristic granulin motif that was reported to be a potent proliferative agent. Additionally, CsGRN may promote carcinoma progression by prompting angiogenesis, insensitivity to apoptosis, tumour invasion and anchorage independence, all of which support tumour expansion in the unfavourable interstitial environment [25, 26]. Although the deduced protein was predicted without signal peptides, the localisation of CsGRN in the tegument and testes of C. sinensis adult worm indicated that the protein could be secreted through other pathways such as that used by F. hepatica .
|
study
| 100.0 |
The protein was also found in the hepatocytes and cholangiocytes of infected Balb/c mice, possibly implying CsGRN could be internalised by various mammalian cells via an endocytotic mechanism [27–29]. Moreover, the surprising finding provided a clue about the protein pertaining to its function on interaction with host cells as an ingredient of CsESPs. According to some reports, granulin plays a central role in the carcinogenesis of a range of malignancies . For instance, PGRN is highly expressed in ovarian tumours , breast cancer , glioblastomas , and gastric cancer . Moreover, as an important mediator of tumorigenesis and wound repair, it is associated with aggressive and invasive tumour phenotypes by stimulating invasion, protecting against anoikis, and supporting tumour expansion in the unfavourable interstitial environment [34, 35]. Based on these aspects, the overexpressed-CsGRN stable cell lines named as PLC-GRN and RBE-GRN cells were successfully constructed, and the wound-healing assay and transwell assay were performed to observe the ability of the migration and invasion of CCA and HCC in vitro. The results displayed that they could stimulate cancer cell migration, which was similar to the action exerted by other recombinant proteins [36, 37]. Compared with cells co-cultured with protein, PLC-GRN and RBE-GRN cells were more approximate to the real situation that the internalised protein of this parasite might interfere with signalling and displayed the carcinogenic nature of biliary epithelial cells and hepatocytes. Likewise, Helicobacter pylori CagA protein targets PAR1/MARK kinase after delivery into gastric epithelial cells and disrupts cell polarity, resulting in disorganisation of the gastric epithelial architecture, inflammation and carcinogenesis . Furthermore, similar to lipoprotein, internalisation induced by activating the fuel-sensing enzyme adenosine monophosphate-activated protein kinase (AMPK) is crucial to maintaining glioblastoma cell growth .
|
study
| 99.94 |
However, the mechanisms by which CsGRN enhances tumour metastasis are unclear. And we hypothesised that they could be pertaining to EMT process, which endows cancer cells with a more aggressive phenotype, finally leading to tumour progression, including invasion and metastasis [40, 41]. Various biomarkers have been screened to demonstrate the EMT process, including the upregulation of mesenchymal markers such as N-cadherin, β-catenin and vimentin, as well as the loss of epithelial markers such as E-cadherin and ZO-1 .
|
study
| 100.0 |
Cadherins are transmembrane glycoproteins of cellular junctions that mediate calcium-dependent cell-cell adhesion and alterations in cadherin function have been implicated in tumorigenesis . Although the loss of E-cadherin has been seen as a hallmark of EMT, recent evidence have indicated that a gain of expression of N-cadherin but without E-cadherin change in tumour cells contributes directly to an increased invasive potential and is independently associated with an early stage of metastasis [44, 45]. The catenins, serving to link the cadherin to the cytoskeleton, can regulate alterations in cadherin function . Disruption of the connection between the cadherins and the cytoskeleton by mutations in β-catenin inactivates the adhesive function of E-cadherin in tumour cells and results in a non-adhesive phenotype. Thus, the increased expression of β-catenin is intimately related to the decline of E-cadherin . Our experiments confirm these findings. Similarly, in PLC-GRN/RBE-GRN cells, the up-modulation of N-cadherin, β-catenin and vimentin, as well as the downregulation of ZO-1while the decrease of E-cadherin were not detected in vitro. Therefore, we can also draw the conclusion that CsGRN induced the mesenchymal characteristics of PLC and RBE cells in responsible for cell invasion and metastasis. In contrast, in the liver of Balb/c mice immunised with rCsGRN protein, the upregulation of E-cadherin and downregulation of vimentin indicated the block of EMT progress. In other words, anti-CsGRN, a specific antibody produced by immunisation with rCsGRN protein, probably prevents the tumour cells from invasion and metastasis.
|
study
| 100.0 |
As many studies have reported, MMPs play a predominant role in the process of tumour cell intravasation, and the dynamic interplay between N-cadherin and epidermal growth factor receptor (EGFR) leads to MMPs gene transcription . As shown in our data, the expression of N-cadherin and MMPs was increased in PLC-GRN/RBE-GRN cells; therefore, we speculated that the level of MMPs was increased under the action of the N-cadherin and EGFR through stimulation by CsGRN, eventually leading to the degradation of basement membranes and metastasis.
|
study
| 100.0 |
Numerous studies have shown that β-catenin, a key downstream effector in the Wnt signalling pathway, is considered to be a cell-cell adhesion protein, and most likely promotes tumour progression once activated . We investigated the overexpression of β-catenin in vitro but without a change in vivo depending on our study, which prompted the speculation that β-catenin only acted as a biomarker of EMT but didn’t activate the β-catenin/wnt pathway.
|
study
| 100.0 |
To further study the potential downstream effectors modulated by CsGRN promotion progression of HCC and CCA, we also tested the signalling molecules AKT and ERK using Western blotting. Our results showed that the expression of p-ERK and p-AKT were dynamically changed when p-ERK reached a peak in 4 weeks after immunisation while p-AKT did at 2 weeks. These data implied that the PI3K/AKT and ERK pathways participated in the promotion of EMT induced by CsGRN, and they might not be activated at the same time.
|
study
| 100.0 |
As we know, the disease is often caused by complicated factors. This study observed that CsGRN, as a CsESP like CsCBs and Csseverin, has also been shown to promote cell migration and invasion. In consideration of the complex constitution of CsESPs, including proteases, antioxidant enzymes and metabolic enzymes [24, 50], interactions among these components are required to determine the authentic factors for pathogenesis in the future. We will explore the actions of granulin members with one or more typical domains in C. sinensis to clarify the difference between these molecules in our further studies.
|
study
| 100.0 |
Overall, we identified CsGRN was a growth factor of C. sinensis and a vital constituent of CsESPs. To date, we have observed the potential promotion cell migration and invasion of CsGRN. We also showed that the EMT process can be triggered by CsGRN and involved in HCC and CCA metastasis via the activation of the PI3K/AKT and ERK pathways.
|
study
| 100.0 |
In summary, we identified CsGRN as belonging to the granulin family through bioinformatics and phylogenetic analyses. We also expressed and purified soluble rCsGRN in E.coli and discovered it was an important component of CsESPs. In adult worm, CsGRN is mainly located in the tegument and testes, which might be involved in parasite growth and development or even in the pathopoiesis of the parasite. CsGRN could be detected at a high expression level in clonorchiasis-induced Balb/c mouse liver tissues and was even observed in hepatocytes and cholangiocytes. In addition, the secretory eukaryotic expression vector pEGFP-C1-CsGRN was generated and employed to intervene in the expression and secretion of CsGRN in PLC and RBE cells. An enhancement of cell migration and invasion was observed, and the current results suggested that CsGRN is likely to promote cell migration and invasion by inducing liver EMT via the ERK and PI3K/AKT signalling pathways. The present study supports the involvement of CsGRN in the pathogenesis of CCA and HCC.
|
study
| 100.0 |
Additional file 1: Figure S1.Successful construction of the eukaryotic expression plasmid pEGFP-C1-CsGRN. a Restriction enzyme identification of the recombinant plasmid pEGFP-C1-CsGRN. DNA ladder 5000 (Lane M), double enzyme digestion of pEGFP-C1-CsGRN (Lane 1), recombinant plasmid pEGFP-C1-CsGRN (Lane 2), empty vector pEGFP-C1 (Lane 3). b Sequencing data from recombinant plasmid pEGFP-C1-CsGRN and CsGRN gene were completely matched. (TIF 206 kb) Additional file 2: Figure S2.Densitometric analysis of genes from Fig. 8a, b, d. Densitometric results were analysed with Image J software. Statistical comparisons between more than two experimental groups were made with one-way ANOVA tests followed by Tukey’s multiple comparisons test. Results are reported as the mean ± standard error of the mean (SEM), and P was set to 0.05. For all analyses, Prism 5.0 software (Graph Pad Software, San Diego, USA) was used. a *P < 0.05, **P < 0.01, compared with the control group. b *P < 0.05, **P < 0.01 and ***P < 0.001, indicate difference from experimental treatment. ## P < 0.01 and ### P < 0.001, compared with the matched pair. (TIF 910 kb)
|
study
| 100.0 |
Successful construction of the eukaryotic expression plasmid pEGFP-C1-CsGRN. a Restriction enzyme identification of the recombinant plasmid pEGFP-C1-CsGRN. DNA ladder 5000 (Lane M), double enzyme digestion of pEGFP-C1-CsGRN (Lane 1), recombinant plasmid pEGFP-C1-CsGRN (Lane 2), empty vector pEGFP-C1 (Lane 3). b Sequencing data from recombinant plasmid pEGFP-C1-CsGRN and CsGRN gene were completely matched. (TIF 206 kb)
|
study
| 99.75 |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.