Ecofriendly biosynthesis of Ag-NPs by Streptomyces griseus eradicating Candida albicans associated with different types of dysspermatism

doi:10.21203/rs.3.rs-1730237/v1

Silver nanoparticles Ag-NPs have potent biological properties such as antibacterial, antifungal, anti-viral and anti-inflammatory activities. A simple, eco-friendly, inexpensive method was conducted in order to synthesize silver nanoparticles by actinomycetes. A total of 17 actinomycetes isolates were recovered from serially diluted compost sample and subsequently examined for their capabilities to produce Ag-NPs. Alongside, Candida albicans was isolated from human semen that affect male infertility, and subsequently submitted to susceptibility test using the well diffusion method. It was found that the Ag-NPs biosynthesized by Streptomyces griseus Azhar.S2 have high zone of inhibition against Candida albicans which was recorded by measuring the diameter of inhibition zone that was 20.5 ± .05 mm. The biosynthesized Ag-NPs had been characterized using UV-vis spectroscopy, FTIR, EDX and TEM. Conclusively, Candida albicans isolated from different types of dysspermatism human semen was highly eradicated by microbially synthesized Ag-NPs from Streptomyces griseus Azhar.S2.

Silver nanoparticles

green synthesis

Actinomycetes

Candida albicans

human semen

dysspermatism

Nanotechnology is considered a fast-growing discipline of the high-tech economy [1]. Nanotechnology is rapidly finding numerous applications in different areas of medicine, industry, environmental sciences, and food sciences [2]. Nanoscience involves the synthesis and application of nanoscale structures in the range of 1 to 100 nm [3]. In the fast-developing area of nanotechnology, metal nanoparticles are having considerable interest because of its useful applications [4]. Consequently, careful environmental, human and animal health safety assessment should be considered during the commercial use of nanotechnology [5].

The chemical methods lead to particles with strong hydrophobic surface that need to overcome the resulting problems for application [6]. On the other hand, the nanoparticle biosynthesis (green synthesis), due to direct stabilization of the nanoparticles by proteins involved in the synthesis process, yields a more consistent size distribution pattern than the other methods [7]. Metal nanoparticles biogenic synthesis has been proved by many microorganisms including different types of fungi, actinomycetes, bacteria, algae, etc. Although, there are many reports, which have shown that actinomycetes are efficient candidates for the production of different types of metal nanoparticles, both intracellularly and extracellularly, but among the different microorganisms used for the synthesis of metal nanoparticles, still the use of actinomycetes is less known [8]. Different microorganisms were found to convert the soluble toxic selenite or selenate into the insoluble less toxic elemental selenium, forming selenium nanoparticles [9].

Silver nanoparticles Ag-NPs are known to be synthesized by bacteria, yeast, fungi, and actinomycetes [10]. Currently different types of metal inorganic nanoparticles as, titanium, magnesium, copper, ferrous, zinc, gold and silver have been synthesized using various techniques [11]. Biosynthesis of nanoparticles was found to be an alternative method of chemical and physical methods; as many organisms are now used for nanoparticle synthesis because of their effectiveness and flexible biological factors [12]. Ag-NPs most widely used because they have many distinctive properties such as optoelectronic, good electrical conductivity, physicochemical, chemical stability and antibacterial activities [13].

Many types of metal nanoparticles (NPs) have proven to be effective in controlling infection both in vitro and in vivo, including the antibiotic-resistant types [14]. The antimicrobial NPs have several characteristic advantages compared to the commonly used antibiotics, as lower toxicity, lower cost and longer stability. However, there are developing concerns about the safety use of these materials in different areas as hemolytic activity for human red blood cells [15], carcinogenicity, toxicity, and interaction with the immune system [16].

Bacterial infection is well known as a serious safety issue associated with significant mortality and many health problems [17, 18]. Antibiotics are usually used to kill or inhibit the growth of bacteria [19, 20]. But, because of their inappropriate use, bacterial resistance to such antibiotics may occur [21, 22]. Recently, nanoparticles have gained interest as novel antimicrobial agent [23].

Candida albicans is nowadays recognized as one of the most common human pathogens [24]. It has developed several adaptations in order to survive as an opportunistic fungus, as producing hydrolytic enzymes, grouping into biofilms and transit between yeast and hyphal morphology [25]. Several factors play important roles in the pathogenicity of the fungus Candida spp. [26]. It affects the function of the male reproductive system in many different ways as some strains found in semen may act on the sperm cells causing the agglutination of motile sperm, reducing the ability for the acrosome reaction, and alterations in cell morphology [27].

Therefore, the aim of the present study is to prepare a stable-dispersed and pure silver nanoparticles (Ag-NPs) in easy, fast and simple method, approaching the susceptibility of candida albicans isolated from different types of dysspermatism human semen to Ag-NPs biosynthesized by actinomycetes.

2.1. Study participants and semen samples collection

One hundred men patients, attending the fertility clinic at the International Islamic Center for Population Studies and Research (IICPSR), Al-Azhar University, Cairo, Egypt during January 2019 to May 2021. Their ages ranged from 22 to 49 y; their ethical agreements were taken orally. The semen samples were collected from the patients who had 3–7 days of sexual abstinence from intercourse. Fresh ejaculates were obtained by masturbation into sterile plastic containers after 4 days of abstinence [28]. Ejaculate cultures were examined and divided into four groups according to WHO criteria [29]:

Group-1: asthenospermia (the total number of sperm less than39 × 10⁶ /ml).

Group-2: oligospermia (the proportion of progressive motility is less than 32%).

Group-3: azoospermia (the absence of sperm or very low sperm content in semen.

And Group-4: control (fertile men patients).

2.2. Isolation and identification of C. albicans from semen samples

Semen samples were washed in Ham’s F-10 medium (Sigma Chemical Company, St Louis, MO, USA) and prepared by the swim up technique. Briefly, an aliquot of the specimen was mixed with Ham’s F-10 medium (1:2 v/v) using sterile Pasteur pipette after liquefaction, and then centrifuged at 330 g for 10 min. The supernatant was carefully aspirated and the pellet re-suspended in 2 mL of fresh Ham’s F-10 medium. The re-suspended sample was transferred into a 15-mL sterile tube using plastic pipettes and centrifuged at 330 g for 5 min. The tubes were then incubated at an angle of 45 for 1 h in the incubator at 37^°C. After the incubation, the entire supernatant was aspirated and centrifuged at 330 g for 10 min. The supernatant was aspirated and the pellet re-suspended in 1 mL of Ham’s F-10 medium then cultured. The test strain, Candida albicans was isolated from different types of dysspermatism human semen of the patients via standard culture technique using Sabouraud glucose agar medium [30] supplemented with chloramphenicol and inoculated with a cotton swab at 37^°C for 48 h. The identification of isolates of C. albicans was achieved by VITEK® test [31].

2.3. Isolation of actinomycetes from compost samples

Samples of compost were collected from Helwan fertilizer factory- Cairo – Egypt. Sampling was done using sterile polythene bags. Serial dilution method [32, 33] was implemented on yeast extract malt extract medium [34], casein – starch agar medium [35], and starch nitrate agar medium by 0.1 mL of serially diluted samples as an inoculum, followed by incubation at 30^°C for 7 days, and then submitted to further identification based on their morphological characteristics.

2.4. Extracellular silver nanoparticles biosynthesis

All the actinomyces isolates were tested for their ability to synthesize the silver nanoparticles through cultivation on starch nitrate broth media contains (g/l): Soluble starch, 20.0; NaNO₃, 2.0, K₂HPO₄, 2.0; KCl, 0.5; MgSO₄.7H₂O, 0.5; CaCO₃, 2.0; tap water up to 1000 ml, the pH was adjusted at 7-7.4 before sterilization [36]. At the end of the incubation period (7 days under submerged condition), the extracellular supernatant was collected by centrifugation at 10.000 rpm for 10 minutes followed by filtration.1mM of silver nitrate was added to each culture supernatant (19:1 v/v) for the possible synthesis of silver nanoparticles by incubation for 7 days at room temperature in orbital dark shaker, and color shift was detected by UV absorption spectroscopy at 300–700 nm in corresponding to microbial filtrate without addition of silver nitrate [37].

2.5. Susceptibility of candida albicans to biosynthesized Ag-NPs

Agar well diffusion method was conducted to evaluate the fungal activity of the biosynthesized Ag-NPs was carried out against Candida albicans on Sabouraud dextrose agar medium. A well of 6 mm diameter was filled with 10 µl of the Ag-NPs suspension (25 mg/ml) against 10 µl of 1mM AgNO₃ as a control, the plates were incubated for 48 h at 30^°C, and the inhibition zones were recorded.

2.6. Morphological identification of the most antagonistic isolate of actinomycetes

The morphological, physical and chemical properties of the most potent isolate were investigated using ISP methods: starch nitrate agar medium and yeast extract-malt extract agar medium [38], inorganic salt-starch agar medium ISP-4, oatmeal agar medium ISP-3, and glycerol–asparagine agar medium ISP-5 [39]. In addition to cover slip culture technique [40], cell wall analysis was carried out using the methods described by [41]. Microscopic examination was performed by high-vacuum mode of a JEOL JSM-5500LV scanning electron microscopy [42].

2.7. Molecular identification of most antagonistic actinomycetes isolate and phylogenetic analysis

Five-day culture pellets were collected by centrifugation, the genomic DNA was extracted by QIAGEN’s DNeasy PowerSoil Pro Kit (Cat. No. 47014), 16S rDNA segment was amplified according to the parameters mentioned in table (1) [43].

Table 1

PCR parameters to amplify 16S rDNA amplicon of the most potent actinomycetes isolate.
Primers sequences	Amplicon size (bp)	General denaturation	Amplification (35 cycles)			Final extension
			Denaturation	Annealing	Extension
AGAGTTTGATCMTGGCTCAG	1485	94˚C 5 min.	94˚C 30 sec.	56˚C 1 min.	72˚C 2 min.	72˚C 10 min.
TACGGYTACCTTGTTACGACTT

PCR products were purified using QIAquick PCR Product extraction kit. (Qiagen, Valencia). Bigdye Terminator V3.1 cycle sequencing kit (Perkin-Elmer) was used for the sequence reaction and then it was purified using Centrisep spin column. DNA sequences were obtained by Applied Biosystems3130 genetic analyzer (HITACHI, Japan), a BLAST® analysis (Basic Local Alignment Search Tool) was initially performed to establish sequence identity to GenBank accessions [44]. The phylogenetic tree was created by the MegAlign module of LasergeneDNAStar version 12.1 and Phylogenetic analyses was done using maximum likelihood, neighbor joining and maximum parsimony in MEGA6 [30].

2.8. Characterization of biosynthesized silver nanoparticles

2.8.1. Particle shape and size characterization

Transmission electron microscopy TEM was used to examine the morphology, size and distribution of synthesized Ag-NPs. For such purpose, an aliquot of aqueous suspension of Ag-NPs was transferred onto a carbon coated copper grid and allowed to dry at room temperature, the grid was then scanned using TEM (JEOL, JEM2100, Electron Microscope, TEMHR, Japan), TEM operated at a voltage of 80 kV. The size of biosynthesized Ag-NPs was analyzed using Image Analysis docu software program [45].

2.8.2. Scanning Electron Microscopy Energy-dispersive X-ray analysis (SEM-EDX)

Energy-dispersive X-ray analysis (EDX) was performed using an X-ray microanalysis system together with a scanning electron microscopy (SEM) [46].

2.8.3. Fourier Transform Infrared (FT-IR) spectrum

Ag-NPs were dried under reduced pressure and the dried sample was packed in KBr-form pellets and A FTIR analysis was performed to determine which compound interferes with the preparation of Ag-NPs. For Fourier transform infrared (FT-IR) measurements, the bio-transformed products found in extracellular supernatants of S. griseus Azhar.S2 were frozen, dried and diluted with potassium bromide (1:100). On a FT-IR instrument attached with diffuse reflectance mode (DRS-800), the spectrum of the sample was monitored spectrum was recorded using FT-IR JASCO 6100, Japan in the range of 400–4000 cm^− 1.at a 4 cm^− 1 resolution. We used the spectral data recorded in the tested sample to identify functional groups based on the reference chart [47].

2.8.4. UV-Visible spectra analysis

The silver nanoparticles were characterized by UV- spectroscopy [48]. The UV absorbance spectra (300 to 700 nm) were taken at various time intervals and different pH values by using spectrophotometer (GBC Equipment Pty Ltd. Australia).

3.1. Collection of dysspermatism semen samples

The collected samples of men semen were examined and divided into four groups as listed in table (2), as well, semen cultures were prepared for isolating Candida albicans that was later identified by VITEK® system besides the routine lab identification as shown in figure (1). Table (2) showed that the 100 collected dysspermatism semen samples were distributed equally within 4 groups, each group includes 25 patients’ semen that submitted to check Candida albicans growth. Asthenospermia group reported 12 positive Candida albicans culture (48%), oligospermia group reported 10(40%), and the azoospermia group reported 20(80%) for total detected Candida albicans isolates 42 out of 75 (56%) in corresponding to zero detection in the control fertile group as a significant correlation between Candida albicans and the poor fertility of the semen.

Table 2

Categorization of the collected dysspermatism semen samples and the percentage of *Candida albicans* isolated from patients’ semen.
Group	Description	Criteria	Participants	Candida albicans (+)
Group	Description	Criteria	Participants	No.	%
1	Asthenospermia	Sperm count: >39 × 10⁶ /ml	25	12	48
2	Oligospermia	Progressive motility: > 32%	25	10	40
3	Azoospermia	Sperm count: zero or very low	25	20	80
4	Control	Fertile semen	25	0	0

Earlier studies reported that there was a positive correlation (r = .9774) between azoospermia in males and presence of Candida albicans in semen, as well as between the presence of microorganisms and poor semen quality (r = .8563), and the presence of microorganisms and reduced motility (r = .8246) [49]. Additionally, J Rolo, P Caixeirinho, P Gomes-Ruivo, C Gaspar, I Fernandes, R Palmeira‐de‐Oliveira, J Martinez‐de‐Oliveira and A Palmeira‐de‐Oliveira [50] found that semen allowed the growth of C. albicans, and he observed that the increase in viscosity impaired significantly C. albicans growth. It was observed that C. albicans and its filtrates had an inhibitory effect on human sperm motility and impaired the ultrastructure of human spermatozoa, which could be associated with male infertility [51].

3.2. Determination of the most potent isolate of Actinomycetes approaching Ag-NPs biosynthesis

A total of 17 actinomycetes isolates were recovered from the collected compost sample, these isolates were examined for their capability to generate Ag-NPs. During the current study, Ag-NPs were synthesized extracellularly from actinobacteria supernatant. Based on the screening, only one actinomycetes isolate which was designated as Azhar.S2 could do a reduction in Ag⁺ ions when AgNO₃ was added to the supernatant of actinobacteria, and this resulted in a change in color from yellow to dark brown. This result is in harmony with AM Soliman, W Abdel-Latif, IH Shehata, A Fouda, AM Abdo and YM Ahmed [52] who found that metabolites of P. chrysogenum strain F9 were used as a biocatalyst for reducing salt Ag⁺ to nanoscale Ag⁰. Color change due to incubation of biomass filtrate with 1 mM AgNO₃ for 24 h from pale yellow to deep yellowish-brown

3.3. Susceptibility of Candida albicans to biosynthesized silver nanoparticles

Historically, silver has been well known as a disinfectant for long years. The use of silver compounds is reduced due to some limitations. Recently, silver nanoparticles show well antibacterial activity against many microorganisms [53]. The nanoparticles synthesized by the members of actinomycetes showed good polydispersity and stability in addition to significant biocidal activities against various pathogens silver nanoparticles produced by Streptomyces griseus Azhar.S2 have the uppermost zone of inhibition against Candida albicans (20.5 ± .05 mm). Small sized nanoparticles showed more antibacterial activity, indicated by the formation of inhibition zone (mm), than the large sized ones because they affect a large surface area of the bacteria [54].

3.4. Taxonomic characterizations of the actinomycete isolate, Azhar. S2

A scanning electron microscopic examination of actinomycete isolate, Azhar.S2, revealed straight spore chains with smooth surfaces (Fig. 1).

LL-diaminopimelic acid was detected in the entire hydrolysate cell of this strain, but no characteristic sugars were detected. The analysis of the cell wall composition is one of the main methods for identifying the chemotaxonomic characteristics of Streptomyces. Table (3) shows the cultural characteristics of the actinomycete isolate Azhar.S2 on different ISP media. Mycelia of the aerial hyphae of the isolate were yellowish orange with grey aerial hyphae, but no diffused pigment was observed. A study was conducted on carbon and nitrogen utilization, degradation tests, sensitivity tests, tolerance to NaCl, growth pH, and growth temperature (Table 4).

Table 3

Cultural characteristics of the actinomycete isolate, Azhar.S2 on different nutrient media.
Types of media	Growth	Substrate mycelium color	Aerial mycelium color	Diffusible pigments
Starch-nitrate agar	Good	Deep orange (51)	Gray (265)	None
Inorganic-trace salt- starch agar (ISP 4)	Good	Deep orange (51)	Gray (265)	None
Glycerol asparagine agar (ISP 5)	Moderate	Vivid orange yellow (66)	Light gray (264)	None
Yeast extract- malt extract agar (ISP 2)	Moderate	Vivid orange yellow (66)	Light gray (264)	None
Oatmeal agar (ISP 3)	Moderate	Yellowish orange (67)	Yellowish gray	None
Melanin pigment media: 1- Tryptone yeast extract broth (ISP 1) 2- Peptone yeast extract iron agar (ISP 6) 3- Tyrosine agar (ISP 7)	Weak Weak Moderate	Vivid yellow (82) Vivid yellow (82) Vivid yellow (82)	Light gray (264) Gray (265) Gray (265)	None None None

Table 4

Chemotaxonomic, morphological and biochemical characteristics of the actinomycete isolate, Azhar.S2.
Character	Results
Chemotaxonomic analyses: Cell wall hydrolysis for: Detection of diaminopimelic acid (DAP) isomers Sugar pattern	LL-DAP ND
Morphological characteristics: Spore chains Spore surface Spore mass color Substrate mycelial color Diffusible pigment produced	Straight Smooth Gray Yellowish orange -
Range of pH for growth: (3.0:7.5) Optimum temperature for growth (°C) :( 25.0:35.0) Optimum Salinity for growth (%): (1.0: 4.0)	+ + +
Sensitivity assay: - Penicillin G (10 IU) Growth in the presence of sodium azide (0·01 %, w/v)	+ -
Biochemical characteristics: - Utilization of citrate Urease production Hydrogen sulfide production Starch hydrolysis Gelatin liquefaction Methyl red test Vogues-proskauer test Indole production Lipase	+ + - - + + - - +
Utilization of carbon source: - Raffinose Xylose Glucose Arabinose Sucrose	- - + + -
Utilization of nitrogen source: - L-Arginine L-Asparagine L-Cysteine L-Methionine L- Tryptophan L-Serine Valine L-Leucine L- Lysine	+ + + + - + + + +
(+) = positive, (-) = negative, (ND) = Not Detected.

3.5. Molecular identification and phylogenetic analysis of Streptomyces sp.

C. albicans infection may decrease the functional competence of spermatozoa by reducing motility and membrane mitochondrial potential and by promoting molecular apoptosis mechanisms [55]. The presence of C. albicans affects seminal parameters. The effect is related to incubation time and yeast concentration, it can be supposed that the yeast sperm interaction is mediated through the mannose sperm receptor [27]. It was reported that an infertile patient with male accessory gland infection due to C. albicans was unable to achieve fertilization even after assisted reproductive techniques. The was also confirmed by different authors [56].

Actinomycetes are aerobic, Gram-positive bacteria, generally exhibiting branched filamentous growth and contain high guanine plus cytosine content in their DNA Using multiple sequence alignment, the 16S rRNA sequences of the selected Azhar.S2 isolate, and Streptomyces sp. helped identify the actinomycete isolate. An agarose gel electrophoresis was used to study the electrophoresis of PCR amplification. The multiple sequence alignment in Figure (3) show that this isolate is close to Streptomyces griseus ATCC10137 by 98.5%. The sequence was submitted to the GenBank1 Nucleotide Sequence Database (GenBank: SUB10781491 Streptomyces OL773497).

3.6. Characterization of Ag-NPs

3.6.1. Particle size and distribution by TEM of biosynthesized Ag-NPs

Transmission Electron Microscopy (TEM) analysis of silver nanoparticles (Ag-NPs) are shown in figure (4) to measure the size and shape of Ag-NPs. Figures (4) shows variable shaped nanoparticles, most of which are spherical in shape. a good quality results have been shown with particles mean size of 14.54 nm with Dev(rms) equal 3.121 nm. This result is in accordance with M Shu, F He, Z Li, X Zhu, Y Ma, Z Zhou, Z Yang, F Gao and M Zeng [57] who found that the morphology and size of the Ag NPs characterized by high-resolution TEM ranged in diameter from 10.3 to 18.9 nm, with an average size of 13.8 nm, also, the typical SEM image showed that the synthesized Ag NPs have a spherical shape and fine size.

3.6.2. Energy-Dispersive Analysis of X-ray of biosynthesized Ag-NPs

The EDX provides both qualitative and quantitative information regarding elements potentially involved in the formation of Ag-NPs (Fig. 5). EDX micro-analysis is performed by measuring the energy and intensity distribution of X-ray signals with 28.790 ± 5.85% silver content. Figures (5) represents the EDX spectrum of biosynthesized Ag-NPs using Streptomyces isolate Azhar S2. These planes confirmed the face centered crystalline structure of nano silver. Other studies recommend using of XRD and Energy Dispersive X-Ray spectroscopy (EDX) to examine the purity of the biosynthesized CuO-NPs produced by actinomycetes [58].

3.6.3. Fourier transform infrared (FTIR) spectroscopic analysis of the produced Ag-NPs

FTIR spectroscopic analysis of the produced Ag-NPs as shown in Figure (6) indicates the presence of amino acid molecules which are the building blocks of the protein structure. Bonds represented by peak 3447.09 may correspond to the primary amine or amide owing to bond N-H. While bonds represented by peak at 1719.42 and peaks at 2924.05 and 2851.06 may correspond to the C = O and O-H of the carboxylic acid, respectively. These bonds confirm the presence of amide or amine and carboxylic acid groups of the amino acids. Most likely, this protein is the enzyme(s) catalyzing the reduction of silver nitrate into elemental silver. FTIR analysis could qualitatively identify some materials, and it could do quantitative analysis [59]. Several studies aimed to examine silver nanoparticles by FTIR which could interpret the functional groups included in binding mechanisms [60].

3.6.4. Ultraviolet (UV) spectroscopic analysis of the biosynthesized Ag-NPs

The biosynthesis of Ag-NPs was characterized by UV–visible spectrum (Fig. 7) that showed one major broad band at 207 nm with absorbance 2.54 au, another minor band at 433 with absorbance 0.44 au which is a typical band for Ag-NPs as mentioned in previous studies [61]. Moreover, the broadness of the band may be related to the method [45].

The aim of this study is to prepare stable-dispersed and pure silver nano-particles (Ag-NPs) in an easy, fast and simple method. The susceptibility of candida albicans isolated from different types of dysspermatism human semen (which affects motility of human spermatozoa and may cause infertility) to silver nanoparticles biosynthesized by actinomycetes was studied. It was found that the biosynthesized silver nanoparticles produced by Streptomyces griseus Azhar.S2 have high zone of inhibition against Candida albicans.

Acknowledgement

The authors appreciate sincerely efforts of secretariat assistance of the International Islamic Center for Population Studies and Research (IICPSR), Al-Azhar University, Cairo, Egypt. As well as, the authors would like to thank botany and microbiology department, faculty of science, Al-Azhar University, Cairo, Egypt. For providing lab facilities. Also, the authors thank the regional center for mycology and biotechnology (RCMB), Al-Azhar University, in which the characterization of Ag-NPs was performed.

Authors’ contributions

All authors contribute in equal proportions to produce this article, E.E.H was responsible for semen collection and oral ethical approval, W.B.S and E.S.A. wrote the manuscript text, I.M.A. was responsible for spectroscopic characterization, all authors performed all experimental work and interpretation, reviewed the manuscript as well.

Funding

The authors declare that they did not receive any financial support from any funding agency.

Availability of data and materials

That all data and materials are available and included in the article

Ethics approval and consent to participate

Approval for this study was obtained from the Ethics and Research Committee of Islamic Center for Population Studies and Research (IICPSR), Al-Azhar University, Cairo, Egypt. This study was carefully explained to the participants and their informed consent obtained before being recruited into the study. Participants were assured of anonymity and confidentiality, the rights of patients to participate or not were respected.

Competing interests

The authors declare that there are no conflicts of interest.

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No competing interests reported.

Ecofriendly biosynthesis of Ag-NPs by Streptomyces griseus eradicating Candida albicans associated with different types of dysspermatism

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Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods