The Internet of Things is Applicable in Greenhouses - Juniper Publishers

Robotics & Automation Engineering Journal - Juniper Publishers

Introduction

The industry of growing plants in conditions of artificial microclimate today uses devices that connect to the Internet. The Internet of things in the industry of growing plants create a “smart” greenhouse with improved performance that can bring more profit, to improve the ease of maintenance of technological equipment, and provide efficient flow of the technological processes at all stages. The industry of growing plants and the construction industry of greenhouses are divided sectors, design and construction of greenhouses occur in different geographical regions. Online greenhouse provides a unique opportunity to solve many technical problems at the initial design stage. The system in the presence of sensors in the early stages of construction of the greenhouses will allow you to monitor the progress of construction. Constant monitoring of the process of construction for compliance with the project allows you to cut costs, avoid mistakes and make decisions, checking the results of calculations in real time in the design office at any place of work with the system.

IoT-technology can solve many problems. The energy is distributed rationally between the systems of the microclimate. Monitoring and forecast is made for weather conditions. The control monitors the technical condition and location of equipment in real time. The full provides information about the condition of the plants. The staff analyzes current and historical data, then makes decisions which allows a more efficient use of process equipment, saving time and fuel. The online greenhouse is a from a technological point of view, the consolidation of critical components of the greenhouse as part of a single network to provide them access to each other and exchange information. A network of sensors provides communication. The sensors are part of the technical equipment and interacting with one another. Sensors produce information which is received and accumulated throughout the life cycle of the greenhouse.

IoT- technologies should be used in research work. The problem requires new innovative solutions. Technological method grows plants on the basis of three elements of aeroponic technology with the use of led lighting and instrumentation system for automation of control of technological processes based on IoT technologies. A conceptual model of the industry of growing plants contains two test-object: synthetic adjustable microclimate and plants. Artificial microclimate creates habitat for plants using engineering equipment. Engineering equipment is formed in a separate lighting systems, heating, conditioning, ventilation, irrigation, power, gas composition and so on. The system of microclimate maintained automated control systems using measurement and control equipment to coordinate the operation. Currently ignored three facts. The first fact artificial microclimate shape the technologies that plants subjected to the action of physical fields of different nature: optical, electrical, magnetic and other.

The second fact artificial microclimate depends on the capabilities of biotechnology, where there is a large Arsenal of different tools and techniques to create plants with specific parameters and qualities. The third fact artificial microclimate is not able to establish a direct relationship with the plants. I conducted an analytical review of existing scenarios, organization of a scientific experiment. The object of research is led lighting in the artificial microclimate and the response of the plants. Problem associated with LEDs and plants showed that despite some progress, the technology remains poorly applicable in practice. The results of many studies pay attention to individual approach to each plant. The choice of factors and the criterion is done empirically. According to the analysis made preliminary conclusions that the problem for the industry of growing plants is:

1) Construction of an artificial microclimate require large capital investments. Evaluation of the efficiency of greenhouse production gives a low degree of forecast process

2) Development of technologies for growing plants in conditions of artificial microclimate associated with a long preparatory phase material and time costs

3) Requires large number of laboratory data obtained in experimental models

4) The results obtained in the study correlated poorly with the scale of production

5) Scatter the recommended parameters of the artificial climate for different crops is quite wide, and therefore requires an individual approach to each plant according to varietal characteristics

6) Empirical search of certain combinations of characteristics of temperature and humidity environment, light environment, a gas environment and a nutritious basal medium

7) Search for plants with the appropriate internal genetic potential for adaptive plasticity

Our study is intended to prove that the solution of problems related to the methodology of scientific research system of artificial climate, requires a comprehensive interdisciplinary approach. The main goal of our study is to develop a structural organization of a scientific experiment to study the response of plants to an led system using IoT-technology.

Research is important to overcome the difficulties associated with the study of the peculiarities of the process of growing plants under LEDs in conditions of artificial microclimate. Difficulties arise in the complexity of the organization of the technological process, which is a combination of many interrelated subsystems, as well as imperfect methods and the mathematical apparatus, which does not allow to adequately describe such a set.

Plant as an object of study should be characterized with more General methodological positions. As one of them can be a system approach, is the methodology of scientific research and practical development of complex object. In this case, in the first place is not an analysis of the constituent parts of the object, and the characteristics of the system as a whole, disclosure of the mechanisms and linkages that maintain the integrity of the object. Structural-parametric modeling and optimization of led lighting, underlying the proposed information technology are the rational change components of the light environment, depending on the condition of the plants in the dynamics of the vegetation development. Thus, the concept of biofeedback through the accumulation of a complete image as metamerism design of set logic the system should be more efficient than the classical concept of traditional Cybernetics and systems analysis. Attempts to mathematically postulate what should be a parametric climate, how it should react the plant, the phenomena outside their holistic entirety, will remain in the framework of private applied research. The proposed concept will now, at the level of modern technical equipment and data management systems to guarantee the proper functioning of the systems of artificial microclimate, create intelligent bioengineering systems such as virtual greenhouses.

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Effect of Lupeol, A Triperpenoid Compound on the Drug Resistant Gene, MecA of Methicillin Resistant Staphylococcus Aureus - Juniper Publishers

 Biotechnology & Microbiology - Juniper Publishers

Abstract

mec A gene is found to be responsible for drug resistance in Methicillin Resistant Staphylococcus aureus. Lupeol obtained from aqueous extract of bark of Alstonia scholaris, a traditional medicinal plant inhibited the growth of MRSA. The aqueous extract obtained from the bark of the tree analyzed by LC-MS and found to contain lupeol. On treating with MRSA cultures, it is found to have an inhibitory action at concentration of 50μg/ml. On RT-PCR analysis and mec A expression protein analysis, it is revealed that lupeol inhibits cell wall synthesis at sub inhibitory concentration of 20μg/ml when added in the growth medium. It inhibits mec A gene.

Keywords: MRSA; Lupeol; Alstonia scholaris; RT-PCR, MRSA; MecA

Introduction

Emergence of drug resistance is an alarming threat to the medical field. Microbes develop resistance due the regular use antibiotics. Methicillin Resistant Staphylococcus aureus (MRSA) is one such organisms. There are different mechanisms which causes the development of drug resistance. mecA is the gene associated with drug resistance in MRSA [1,2]. Plant antimicrobials are a cheap alternative against MDR. The gene responsible for different drug resistance have been reported in S. aureus [3-5]. Lupeol is a triterpenoid compound found in vegetables like cabbage, tomato etc. It is an anti-inflammatory agent and can affect the pathways having nuclear factor kappa B (NFκB), cFLIP, Fas, Kras, phosphatidylinositol-3-kinase (PI3K)/Akt and Wnt/β-catenin in a variety of cells. The action of Lupeol obtained from the extract bark of Alstonia scholaris was tested against the MRSA in the present study [6].

Material and Methods

Plant material was selected locally and identified and deposited in the herbarium, Department of Botany, University of Calicut (Voucher no.6234). Extract was prepared as described earlier with some modifications. In brief the bark collected was air dried and extracted with sterile water [7].10 gm of the sample was mixed with 50 of sterile water and kept on the shaker at 200rpm for 10 hrs. The supernatant was collected and filtered and dried. The extract was subjected to bioassay guided fractionation with increasing order of polarity, starting from solvent with least polarity. Solvents such as hexane, chloroform, methanol and water were used for extraction. The final extract with water has subjected to dryness. The dry mass was then dissolved to get a extract of concentration 1mg/ml and stored under refrigeration as stock till further use. MRSA (ATTC 1109) strain was procured from the Jubilee hospital, Thrissur used for the study. The culture was screened with oxacillin at 5μg/ml concentration and screened for resistance using nutrient agar plates using Bauer Kirby method and as per the NCCLS, 2000.

For RT-PCR study MRSA broth were prepared in Muller-Hinton medium with the extract of concentration 25μg/ml. The experiment was done with PCR primers as reported by Mariana et al, [8] with some modifications [8]. Total RNA was extracted from cells in the logarithmic phase of growth and purified using Guanidium Isothiocyanate. Unless otherwise specified, cells were grown to mid logarithmic phase in NB (pH 6.6) at 36°C, conditions optimum for expression of the toxic proteins. 750μl culture was centrifuged and resuspended in 600μl Lysis buffer (freshly supplemented with 0.7% β-ME) and mixed well by vortex. 60μl of 2M Na-acetate (pH-4.0) was added and mixed with vortex. An equal volume of hot phenol (68°C) saturated with DEPC water (pH 4.0) was added and vortex vigorously for 5 minutes. The mixture was incubated at 68°C for 10 minutes, cooled, 120μl of chloroform was added and vortex vigorously for 15 minutes with intermittent incubation on ice.

The mixture was centrifuged, 150μl of the aqueous phase was transferred to a fresh micro-centrifuge tube and equal volume of isopropanol was added. The solutions were mixed well and incubated at -20C for 1-2 hours. RNA was precipitated by centrifugation at 13,000 rpm for 20 minutes and the pellet was dissolved in 500μl of Lysis solution. RNA was re-precipitated by adding an equal volume of isopropanol, kept at -20°C for 1-2 hrs. After centrifugation, the pellet was washed in 80% ethanol, dried at room temperature and dissolved in 10μl of DEPC treated water. RNA was quantified by measuring the absorbance at 260nm (A)*. For all experiments with RNA, extensive precautions against RNase contamination were taken.

Semi-quantitative RT-PCR

Reverse transcription of the isolated RNA was performed to synthesize the first strand of cDNA with reverse primer and then amplification of cDNA was done using specific Primer sets.

Procedure

Isolated RNA samples were subjected to DNase treatment to make them free from any contaminating DNA.

a. For cDNA synthesis a total of 200ng of RNA was taken, incubated at 70°C with specific anti sense primer for 10 minutes annealing in a thermal cycler.
b. Add 5X buffer, and then Superscript RT at 42°C, allow the reaction to carry in thermal cycler for successful reverse transcription.
c. Following cDNA synthesis, amplification of specific genes responsible for cell wall synthesis (mecA genes) was done using specific primer sets.
d. Amplification was for 35 cycles (each cycle consisted of 94°C for 30 seconds, 50°C for 30 seconds and 72°C for 30 seconds, followed by a seven-minute extension at 72°C)
e. Genomic DNA served as a positive control, and DNase treated RNA that had not been reverse transcribed was used as a negative control.
f. Aliquots removed at 25, 30 and 35cycles for each PCR product was electrophoresed, and the gels were analyzed with a Gel Doc System.
g. PCR products were normalized according to the amount of 16S rRNA detected in the same cDNA sample.16S rRNA is a housekeeping gene and is constitutively expressed.
h. Each set of experiments was repeated at least thrice.

DNase Experiment Set Up

DNase treatment of isolated RNA samples were done before cDNA preparation to remove any DNA contamination with RNA, so that Reverse Transcriptase can only reverse transcribes the mRNA to prepare the complementary DNA.

a. The procedure generally follows the given set up:

b. Autoclaved water -- As required to make the total volume 10μl.

c. RNase Inhibitor -- 0.5μl

d. RNA -- Desired volume in microliter to have total 1micogram

e. DNase Enzyme -- 1μl

f. (Total 10μl of reaction set up)

g. This reaction mixture was kept for 30 minutes at 37°C water bath. The reaction was stopped by adding 1micoliter EDTA to each microfuge tube, to chelate Mg2+ ions. Finally, the heat inactivation of DNase enzyme was done at 70°C water bath for 10 minutes.

LC-MS analysis of the extract
5μl of the sample was injected to the HPLC. The system was with dual pump, rhedyne injector SPD photodiode array detector and 6.12 sp5 integration software. The compound was identified as lupeol by LC MS analysis [9].

Discussion

LC-MS analysis showed the presence of lupeol in the extract (Figures 1-3). PCR results clearly indicate that lupeol inhibits the mecA synthesis in MRSA at mRNA level (Figure 4 & 5). Lupeol can be used to develop a new drug against MRSA infections. Use of antimicrobials against drug resistance was described earlier by different workers [10-12]. Earlier research showed that lupeol is a strong anticancer agent. The action mechanism against amino acid synthesis has to be studied for further verification of the result.

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