Dioscorea bulbifera Mediated Synthesis of Novel AucoreAgshell Nanoparticles with Potent Antibiofilm and Antileishmanial Activity

1 Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, India 2Nanomechanics Laboratory, Department of Physics, Indian Institute of Science Education and Research, Pune 411008, India 3Cell and Molecular Biology Laboratory, Department of Biotechnology, University of Pune, Pune 411007, India 4Department of Applied Physics, Defense Institute of Advanced Technology, Girinagar, Pune 411025, India 5Department of Chemical Engineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India 6Department of Microbiology, University of Pune, Pune 411007, India


Introduction
Recent advances in the field of nanotechnology encompass the development of safe and ecofriendly route towards the synthesis of nanoparticles for the benefit of human health.Gold nanoparticles (AuNPs) with unique physicochemical and optical properties are reported to have potent applications in photonics, chemical sensing as well as biomedical applications like photothermal therapy and drug delivery [1][2][3][4].Silver nanoparticles (AgNPs) are considered to be superior to other nanostructured inorganic metal particles due to their well-known electrical conductivity, optical properties, and oxidative catalysis [5,6].AgNPs are used in different fields in medicine due to their broad spectrum antimicrobial effect [7][8][9][10].Therefore, AgNPs have found their applications in various pharmaceutical products, such as water purification systems, burn dressings, and medical devices [11,12].Thus, combination of both gold and silver to synthesize bimetallic Au core Ag shell NPs is of utmost scientific rationale.However, till date there are no well-defined biological routes for synthesizing Au core Ag shell NPs with therapeutic potential.
Multidrug resistant (MDR) Gram negative and Gram positive bacteria like Acinetobacter baumannii and Staphylococcus aureus,respectively, have emerged as highly infectious nosocomial pathogens owing to their remarkable resistance to antibiotics, metal salts, desiccation, and disinfectants [24][25][26][27][28].Such type of pathogenesis among the critically ill and immune-compromised patients is characterized by the biofilm formation that comprise of a complex, organized bacterial community adhering to the surface forming microcolonies composed of exopolymeric matrix of carbohydrates, proteins, and nucleic acids [29][30][31][32].Hereby, the bacteria gain ability to evade the host defenses, resistance to antibiotic therapy and deliberate delocalization of planktonic bacteria resulting in implantation, and colonization at newer sites, causing nascent acute infections in the host [33].Currently, there are no reports on the application of bimetallic nanoparticles composed of gold and silver, in controlling the biofilm formation which can prove to be a powerful therapeutic strategy.
In view of the above background, we report for the first time the synthesis of Au core Ag shell NPs by D. bulbifera tuber extract (DBTE) followed by characterization using UV-visible spectroscopy, high resolution transmission electron microscopy (HRTEM), energy dispersive spectroscopy (EDS), and dynamic light scattering (DLS).Bioreduced Au core Ag shell NPs were checked for the biofilm inhibitory activity against A. baumannii, P. aeruginosa, E. coli, and S. aureus employing field emission scanning electron microscopy (FESEM) and atomic force microscopy (AFM).

Synthesis of Gold Core Silver Shell Nanoparticles
(Au  Ag ℎ NPs).DBTE was prepared as per our earlier report [7].In brief, fresh tubers of D. bulbifera were washed thoroughly and chopped into slices and shade dried followed by grinding into fine powder.5 g of the powder was boiled in 100 mL of distilled water for 5 min and decanted followed by filtration.Synthesis was initiated by addition of 5 mL of DBTE to 95 mL of aqueous solution with 1 mM HAuCl 4 and 0.7 mM of AgNO 3 followed by incubation at 50 ∘ C for 5 h.The progress of reduction was monitored by measuring the UV-visible spectra of the solution at regular intervals on a spectrophotometer (SpectraMax M5, Molecular Devices Corp, USA) operated at resolution of 1 nm.Bioreduced Au core Ag shell NPs synthesized by DBTE were centrifuged at 10,000 rpm for 15 min at room temperature, followed by redispersal of the pellet in sterile distilled water to remove any uncoordinated biological molecules.Repetition of alternate centrifugation and redispersion in sterile distilled water for three times ensured better separation of free entities from the nanoparticles which were used for all further biological activities.

Transmission Electron Microscopy (TEM), High Resolution Transmission Electron Microscopy (HRTEM), and Dynamic
Light Scattering (DLS) Measurements.Shape and size of the bioreduced Au core Ag shell NPs were determined using TEM (Tecnai 12 cryo TEM, FEI, Netherland) and was confirmed by JEOL-JEM-2100 higher resolution transmission electron microscope (HRTEM) coupled with elemental composition mapping under scanning transmission electron microscopic mode (STEM).Energy dispersive spectra of Au core Ag shell NPs, recorded in the energy dispersive spectrometer (EDS) equipped in JEOL JSM 6360A analytical scanning electron microscope at an energy range 0-20 keV confirmed the synthesis of Au core Ag shell NPs.Particle size was analyzed using the dynamic light scattering equipment (Zetasizer Nano-2590, Malvern Instruments Ltd, Worcestershire, UK) in polystyrene cuvette.
2.3.Antibiofilm Activity.5 L, OD 600 (0.05) adjusted overnight grown cultures of A. baumannii, P. aeruginosa, E. coli, and S. aureus were incubated in absence and in presence of Au core Ag shell NPs at various concentrations ranging from 1 to 1024 g/well in Mueller Hinton broth in 96 well microtitre plates.Negative controls (no cells) were included for each of the experimental preparations.The microtitre plates were then incubated overnight at 37 ∘ C under static conditions and processed thereafter.Nonadherent cells were removed from microtitre by aspiration.Wells containing biofilm matrices were washed thrice with sterile PBS followed by staining with 0.1% gentian violet (HiMedia, India) for 10 min at room temperature.Excess stain was removed by immersing in water trough followed by drying in laminar air flow.Finally, 200 L of absolute ethanol was added to each well and shaken at 1020 rpm for 10 sec.Absorbance at 570 nm was recorded in a multiplate reader.Biofilm indices were calculated after normalizing with appropriate controls.All biofilm assays were repeated thrice.

Visualization of Biofilm
Employing SEM and AFM.The biofilm inhibition was carried out on sterile grease free glass cover slips as per the above mentioned procedure with a final volume of 2 mL in 6 well plates.After 24 h, cover slips were washed with sterile PBS followed by fixation with glutaraldehyde which was further dehydrated sequentially using ethyl alcohol and analyzed using scanning electron microscope (SEM, JEOL JSM 6360A).

Atomic Force Microscopy.
Imaging of the cell surface was performed using JPK Nano-wizard II (Germany) AFM setup in intermittent contact (IC) mode in air.Images were recorded in height and deflection (amplitude) modes where deflection mode gives higher contrast in morphological details and height image provide sample surface topography.Silicon nitride tips were used with the tip radius < 10 nm, spring constant of 40 N/m and resonant frequency of 300 KHz for IC mode [34].Line rate of 0.5−1 Hz was used for topographical (height) and deflection images.Elastic properties like Young's Modulus of the treated and untreated biofilm samples were evaluated where the cantilever served as soft nanoindenter allowing local testing of small and inhomogeneous samples.Hertzian model of contact theory for indentation with spherical tips was used to evaluate Young's Modulus [35][36][37][38][39].The cantilever was modified by gluing (Dymax-OPV 30 UV curable glue) 20 M glass bead to the tip.The spring constant of the cantilever was determined using thermal noise analysis in liquid while glass was used as rigid surface to calibrate the photo detector sensitivity.Force curves were recorded at the rate of 5 M/sec with typical piezodisplacement of 1 M-10 M.Ensemble of 64 force curves was obtained on different locations of the cell surfaces.Analysis of the force curves was done using JPK data processing software.The mean values of Young's modulus were calculated as the average, weighted over the data obtained by analysis of 64 curves from each independent experiment.Young's modulus is calculated by the equation [36,38,40] where  is the force applied by the cantilever,  is Young's Modulus,  is the indentation depth,  is the radius of the bead (10 um),  is the contact radius, and ] is Poisson's ratio (0.5 for biological samples).

Results
3.1.UV-Visible Spectroscopy.DBTE mediated reduction of silver and gold ions in aqueous solution was confirmed by the gradual change in color from orange red to dark reddish violet as a function of reaction time.UV-vis spectra recorded from 0 to 5 h revealed a distinct and clear increase in intensity of the gold surface plasmon band, while a well-defined band corresponding to AgNPs was not observed in case of Au core Ag shell NPs (Figure 1(a)).Although no significant synthesis was observed from 0 to 0.5 h, but thereafter the rate of reduction took a rapid pace, almost completing the reduction within 4 h, with a slight increase in the intensity till 5 h.UVvis spectra confirmed the synthesis of Au core Ag shell NPs which showed a peak at 540 nm unlike that of individual AgNPs at 440 nm (Figure 1(b)).However, a decrease in the peak intensity was observed in case Au core Ag shell NPs as compared to that of individual AuNPs at 540 nm which might be attributed due to formation of the silver shell onto a gold core.

TEM, HRTEM, and EDS Analysis
. TEM images confirmed the size, shape, and nature of bioreduced nanoparticles.TEM images distinguished the shell from the core with prominent boundary between the two layers (Figure 2(a)).
The diameter of the inner core was in a range between 5 and 11 nm while the entire particle showed a diameter of 13 to 19 nm.Particles were found to be almost monodispersed, the majority being spherical with a core represented by dark contrast surrounded by a shell with a lighter shade.Aggregation was not observed indicating the stable nature of the particles.HRTEM analysis was also in agreement with the TEM analysis where the diameter of the core and the entire particle was measured to be 6 to 10 nm and 13 to 18 nm, respectively (Figure 2(c)).Lattice fringes were clearly observed on the surface of the nanoparticles confirming the crystalline nature (Figure 2(c)).Additionally, elemental mapping clearly indicated the location of elemental silver and gold in the core shell nanoparticles (Figure 2(d)).It was distinctly evident that gold formed the core while silver formed the shell of the nanoparticles confirming the fact that the bioreduced nanoparticles were Au core Ag shell in nature.EDS results confirmed the presence of both elemental silver as well as elemental gold in the nanoparticles.Au core Ag shell NPs were comprised of 57.34 ± 1.01% gold and 42.66 ± 0.97% silver of the total mass (Figure 2(b)).Particle size analysis exhibited a variation mostly between 7 and 18 nm, the majority being 10 nm (Figure 2(a)).up to 83.68 ± 0.09% against A. baumannii (Figure 3).However, Au core Ag shell NPs showed a comparatively lower activity equivalent to 18.93 ± 1.94% against P. aeruginosa at the same concentration.Biofilm formation in case of E. coli was inhibited up to 22.33 ± 0.56% which was found to be superior to P. aeruginosa while inferior as compared to A. baumannii.It is important to note that the biofilm formation was inhibited up to 30.70 ± 1.33% even against Gram positive bacteria like S. aureus.

Scanning Electron Microscopy for Biofilm Inhibition.
Scanning electron micrographs revealed the effective inhibition of biofilm by Au core Ag shell NPs against A. baumannii.Untreated biofilm were represented by confluent lawns (Figure 4(a-i)) with intact cellular morphology.Treatment with Au core Ag shell NPs exhibited marked biofilm inhibition with discrete cells on the glass surface instead of lawns (Figure 4(a-ii)).The treated cells showed irregular morphology owing to cell damage by the Au core Ag shell NPs.A similar observation was achieved in case of P. aeruginosa as well.In case of the untreated cells a profound bacterial biofilm surrounded by exopolymeric slimy matrix was seen (Figure 4(bi)).Treated cells exhibited biofilm inhibition characterized by prominent membrane disintegration leading to efflux of cellular materials (Figure 4(b-ii)).Size of E. coli cells were found to increase abruptly followed by efficient biofilm inhibition on treatment with Au core Ag shell NPs (Figure 4(c-ii)).Surface attachment was greatly compromised on treatment unlike the untreated cells which formed a compact biofilm due to effective adherence onto the glass surface (Figure 4(c-i)).In case of S. aureus, the untreated biofilm was characterized by groups of closely packed spherical cells (Figure 4(d-i)) which on treatment showed discontinuous biofilm consisting of sparsely packed cells in discrete aggregates.The cells in treated biofilm showed surface invaginations and deformities (Figure 4(d-ii)).

Atomic Force Microscopy
. AFM images were recorded to confirm the effect of Au core Ag shell NPs treatment on bacterial biofilms which illustrated a packed lawn in case of untreated biofilms.The interrupted biofilms without packed morphology owing to compromised cell adhering capability in treated biofilm (Figure 5(a-ii)) were readily differentiated from the untreated bacterial biofilms (Figure 5(a-i)).A. baumannii showed complete elimination of biofilm characterized by discrete single cells attaching onto the glass surface.The measurements of elastic moduli showed that the treated cells (104.59KPa) were stiffer as compared to untreated cells (0.82 KPa).P. aeruginosa biofilm exhibited cells embedded in the exopolymeric matrix while in case of treated biofilm, discrete cells were observed (Figures 5(b-i) and 5(b-ii)).Similar trend in the increase of stiffness in the treated cells (75.72 KPa) was observed compared to untreated cells (40.02 KPa).Untreated E. coli biofilm was characterized by evenly distributed uninterrupted confluent bacterial lawns while biofilms treated with Au core Ag shell NPs showed uneven cellular distribution with discrete solitary cells on the glass surface at large distances (Figures 5(c-i) and 5(c-ii)).Stiffness (57.64 KPa) in the treated biofilm increased as compared to untreated biofilm (22.84 KPa).Inhibition of S. aureus biofilms was clearly evident from the AFM micrographs showing the irregularly clumped cellular masses separated by large valleys unlike the untreated biofilm which exhibited profound uniform lawn (Figures 5(d-i) and 5(d-ii)).Stiffness in treated biofilm increased up to 197.45 KPa which was significantly higher compared to 48.63 KPa found for the untreated biofilm.

Discussion
D. bulbifera is considered as most important medicinal plant in both Indian and Chinese traditional medicine due to its multidimensional therapeutic potential owing to diverse phytochemicals.In this study, we found a route for rapid and efficient synthesis of Au core Ag shell NPs by DBTE within 5 h.In our earlier report, AuNPs were found to be formed rapidly within 90 min using D. bulbifera; however, synthesis of AgNPs was found to be completed at 5 h [7,23].Thus, the rapidly synthesized AuNPs acts as nanocrystalline seeds on which slowly formed AgNPs are uniformly deposited forming a complete coat on 9 nm Au core.Our results are in close correlation with the recent report on 11 nm Au seed mediated chemical synthesis of 13 to 50 nm core shell nanoparticles using ascorbic acid as reductant and cetyltrimethylammonium chloride (CTAC) as capping agent [42].Hereby, it is immensely important to note the promises of DBTE mediated rapid synthesis of exotic Au core Ag shell NPs as a novel route without involvement of any toxic chemicals.Phenolics and flavonoids might be the key players in the rapid synthesis while starch might stabilize the bioreduced nanoparticles which were composed of a gold core and a silver shell.Similarly citric acid and diosgenin may be responsible for the shape evolution.D. bulbifera tubers are reported to possess a rich phenolic and citric acid contents supporting the claim that fresh yam tubers are rich source of antioxidant that play a key role in prevention of oxidative stress mediated emergence of cancer and cardiovascular diseases [7,43,44].Diosgenin and flavonoids and are also reported in D. bulbifera to promote antitumor and anticancer activity, respectively, which strongly support the potential of DBTE to synthesize Au core Ag shell NPs [19,22,45].Synthesis of Au core Ag shell NPs was characterized by a peak at 540 nm which was similar to the report of Salunke et al. on synthesis of bimetallic nanoparticles (AgAuNPs) by Plumbago zeylanica indicating Au core and Ag shell [46].DBTE synthesized Au core Ag shell NPs showed concentric spherical morphology strikingly different from both the polyhedral particles synthesized by polyol process and blunt ended nanopolygonal particles formed in preliminary reports on biological synthesis [46][47][48].
Bioreduced nanoparticles have found profuse utility in clinical studies and for designing novel therapeutic strategies.Adhesion and colonization lead to most emerging problem of biofilm associated microbial infections.Chronicity of nosocomial infections owing to enhanced persistence is a result of protective biofilms [49].Hereby, DBTE synthesized Au core Ag shell NPs, exhibiting potent bacterial biofilm inhibition is a very significant finding towards development of new therapeutic strategy against microbial infections.This is the first report on antibiofilm activity of Au core Ag shell NPs, synthesized by DBTE against A. baumannii which was found to be considerably superior as compared to bimetallic nanoparticles synthesized by P. zeylanica [46].Enhanced antibiofilm activity can be attributed by high surface to volume ratio [10,50,51].Our results are concurrent with earlier reports which showed, in nanoparticulate form, metallic silver exhibits antibiofilm activity against A. baumannii at concentrations as high as 1024 g/well [9,46].However, Au core Ag shell NPs inhibited the biofilm at a lower concentration of 100 g/mL against A. baumannii which is one of the emerging most potent nosocomial multidrug-resistant pathogen.It poses threat due to its frequent outbreak causing urinary tract infection, meningitis, bacteremia, wound infections, and pneumonia.The ability of the pathogen to survive in diverse ecological niche and environmental conditions forming resistant biofilm facilitates its persistence on both inanimate and animate surfaces for extended periods of time [29,52,53].
Similarly it has been reported to gain resistance against multiple metal and metal ions as well in addition to antibiotics leading to compromised drug sensitivity [26-28, 54, 55].Multidrug resistance associated bacteremia, osteomyelitis, and respiratory infections in patients suffering from traumatic injuries have high mortality rate in ICU, ranging from 26% to 68% [52,56].It is speculated that the mortality can be attributed to antimicrobial resistance, lower effectiveness of empirical therapy and scarcity of available therapeutic options.Hereby, the observed selectivity of the bioreduced Au core Ag shell NPs towards inhibition of A. baumannii biofilm is an immensely significant perspective which can be further exploited for designing powerful therapeutic strategy against A. baumannii infections.
Au core Ag shell NPs showed antibiofilm activity against P. aeruginosa, E. coli and S. aureus leading to damage of cell wall causing efflux of cellular materials.This may be attributed to the property of silver component to adhere to cell surface of bacteria, further generating reactive oxygen species leading to catalytic oxidation of cell membrane causing deformation and damage to the cell surface [7,57].Although, AgNPs are reported to inhibit the biofilms formed by P. aeruginosa, this is the first report on inhibition by spherical Au core Ag shell NPs synthesized by DBTE [58].It is important to note that abrupt increase in bacterial cell length of E. coli was observed on treatment with Au core Ag shell NPs which is well in agreement with earlier report justifying gold to be responsible for the observed phenomenon [59].Au core Ag shell NPs inhibited the biofilm formation in Gram positive S. aureus as well.Potential of the bioreduced nanoparticles might be attributed due to mechanisms like binding to cellular enzymes and proteins, specifically to SH-groups, interference with bacterial cell integrity and production and conservation of energy, as reported earlier against Staphylococcus epidermidis [8,[60][61][62][63].DBTE synthesized Au core Ag shell NPs showed antileishmanial activity against L. donovani leading to complete disintegration of cellular morphology probably due to the silver component of the nanoparticles.Allahverdiyev et al. reported that AgNPs are leishmanicidal due to their capacity to produce ROS, owing to their large surface areas, smaller size, and ability to bind sulfur-and phosphor-containing groups [64].

Conclusion
This is the first report on synthesis of spherical Au core Ag shell NPs using DBTE which is a novel biological    route for synthesis of nanoparticles with unique morphology with concentric Au core and Ag shell using medicinal plant, D. bulbifera.It showed efficient biofilm inhibition against Gram negative bacteria, particularly A. baumannii and Gram positive bacteria, S. aureus.The mechanism behind the biofilm inhibition was observed to be intense damage to the cells leading to disintegration of cell wall causing efflux of cellular materials.Similarly, it showed effective antileishmanial activity against L. donovani.Hence, the present study provides a strong scientific rationale for the use of DBTE synthesized Au core Ag shell NPs as potent antibiofilm and antileishmanial agent.

Figure 1 :
Figure 1: Ultraviolet-visible characterization.(a) UV-vis spectra recorded as a function of reaction time for synthesis of Au core Ag shell NPs by DBTE at 40 ∘ C. (b) Comparative ultraviolet-visible spectra recorded for AuNPs, AgNPs and Au core Ag shell NPs after complete bioreduction by DBTE for 5 h at 40 ∘ C.

Figure 2 :
Figure 2: Micrographs of representative Au core Ag shell NPs synthesized by DBTE.(a) Transmission electron micrograph showing core shell nanoparticles.Inset represents the histogram for particle size distribution.(b) Representative spot EDS profile confirming the presence of both elemental Ag and Au in Au core Ag shell NPs synthesized by DBTE.(c) High resolution transmission electron micrographs of Au core Ag shell NPs with lattice fringes synthesized by DBTE.(d) Elemental mapping showing silver shell (green) and gold core (inset figure), with scale bar indicating 100 nm.

Figure 3 :
Figure 3: Inhibition of the growth of biofilm after treatment with Au core Ag shell NPs.

Figure 4 :
Figure 4: Scanning electron micrographs showing the architecture of untreated bacterial biofilm and biofilm treated with Au core Ag shell NPs.(a-i) Untreated biofilm of A. baumannii (inset scale bar representing 1 m); (a-ii) A. baumannii biofilm treated with Au core Ag shell NPs (inset scale bar representing 1 m); (b-i) untreated biofilm of P. aeruginosa (inset scale bar representing 1 m); (b-ii) P. aeruginosa biofilm treated with Au core Ag shell NPs (inset scale bar representing 1 m); (c-i) untreated biofilm of E. coli (inset scale bar representing 100 nm); (c-ii) E. coli biofilm treated with Au core Ag shell NPs (inset scale bar representing 1 m); (d-i) untreated biofilm of S. aureus (inset scale bar representing 1 m); (d-ii) S. aureus biofilm treated with Au core Ag shell NPs (inset scale bar representing 1 m).

Figure 5 :
Figure 5: Representative three-dimensional (3D) atomic force micrographs of untreated bacterial biofilm and biofilm treated with Au core Ag shell NPs.(a-i) Untreated biofilm of A. baumannii; (a-ii) A. baumannii biofilm treated with Au core Ag shell NPs; (b-i) untreated biofilm of P. aeruginosa; (b-ii) P. aeruginosa biofilm treated with Au core Ag shell NPs; (c-i) untreated biofilm of E. coli; (c-ii) E. coli biofilm treated with Au core Ag shell NPs; (d-i) untreated biofilm of S. aureus; (d-ii) S. aureus biofilm treated with Au core Ag shell NPs.

Figure 6 :
Figure 6: Morphological and molecular analysis for antileishmanial activity.(a) Representative scanning electron micrographs of untreated L. donovani; (b) SEM image of L. donovani after treatment with Au core Ag shell NPs (inset scale bar corresponds to 1 m); (c) representative atomic force micrographs of untreated L. donovani; (d) AFM image of L. donovani after treatment with Au core Ag shell NPs; (e) induction of internucleosomal DNA fragmentation in L. donovani in response to treatment with Au core Ag shell NPs.Lane 1-100 bp ladder; Lane 2untreated; Lane 3-Au core Ag shell NPs treated.
g/L) treatment was given for 3 h at 37 ∘ C followed by precipitation of DNA with 1/2 volume 10 M ammonium acetate and 2.5 volume ethanol.The DNA was dissolved in TE buffer (Tris 10 mM, EDTA 1 mM, pH 8.0) and analyzed by agarose gel electrophoresis.
[41]vity.Promastigotes of Leishmania donovani (MHOM/IN/83/Ag83) were cultured in Medium 199 (Sigma, St. Louis, MO) supplemented with 10% heat inactivated foetal bovine serum (FBS) (Sigma, St. Louis, MO) at 25 ∘ C. The parasites were maintained in vitro by subpassaging them every fifth day.Antileishmanial activity of Au core Ag shell NPs was investigated using MTT (3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay.Logarithmic phase L. donovani promastigotes (2 × 10 5 parasites/mL) were seeded in a 96-well microtitre plate and treated with the nanoparticles followed by incubation at 25 ∘ C for 72 h.2.7.SEM and AFM Analysis.72htreatedL.donovanipromastigotes were adhered to poly-L-lysine coated cover slips and fixed with 4% paraformaldehyde for 15 min at room temperature which were subjected to SEM and AFM analysis as per the procedure described in the earlier section.2.8.DNA Fragmentation Assay.The assay was carried as described in earlier report by Herrmann et al., 1994 with a slight modification[41].Briefly, L. donovani promastigotes were treated with nanoparticles for 72 h.The pellets were treated with lysis buffer (1% NP-40 in 2 mM EDTA, 50 mM Tris-HCl, pH 7.5) followed by centrifugation at 1600 ×g for 5 min.The supernatant was brought to 1% SDS and treated with RNAase (5 g/L) for 2 h at 56 ∘ C. Proteinase K(2.5