Biogenic synthesis of gold nanoparticles by marine endophytic fungus-Cladosporium cladosporioides isolated from seaweed and evaluation of their antioxidant and antimicrobial properties

Marine endophytes are the most untapped group of microorganisms having enormous applications in pharmaceutical and cosmetra id="spar0060">Marine endophytes are the most untapped group of microorganisms having enormous applications in pharmaceutical and cosmetic industries. In the present study, we have optimized a method for biogenic synthesis of gold nanoparticles (AuNPs) from Cladosporium cladosporioides, an endophytic fungus of the seaweed, Sargassumwightii. The identity of the fungus was established by the 18 s rRNA and ITS sequence. The AuNPs synthesized using C. cladosporioides were characterized by UV–vis spectrophotometer, Field Emission Scanning Electron Microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, Dynamic light scattering, Atomic force microscopy, and Energy dispersive X-ray spectroscopic studies. They were tested for free radical scavenging activity (DPPH and FRAP assay) and antimicrobial activity against a panel of pathogenic microorganisms. The AuNps were within 100 nm as confirmed by the above methods. An attempt was made to understand the mechanism of the gold nanoparticle synthesis using the fungal extract. The present study shows the involvement of NADPH-dependent reductase and phenolic compounds in the bioreduction of the gold metal salts to nanoparticles. The AuNPs showed significant antioxidant as well as the antimicrobial activity. Hence, this study has shown a great potential for the development of a cost effective antimicrobial treatment utilizing biogenic gold nanoparticles.     

Synthesis and characterization of pHLIP® coated gold nanoparticles

Novel approaches in synthesis of spherical and multispiked gold nanoparticles coated with polyethylene glycol (PEG) and pH Low Insertion Peptide (pHLIP^®) were introduced. The presence of a tumor-targeting pHLIP^® peptide in the nanoparticle coating enhances the stability of particles in solution and promotes a pH-dependent cellular uptake. The spherical particles were prepared with sodium citrate as a gold reducing agent to form particles of 7.0±2.5 nm in mean metallic core diameter and ～43 nm in mean hydrodynamic diameter. The particles that were injected into tumors in mice (21 µg of gold) were homogeneously distributed within a tumor mass with no staining of the muscle tissue adjacent to the tumor. Up to 30% of the injected gold dose remained within the tumor one hour post-injection. The multispiked gold nanoparticles with a mean metallic core diameter of 146.0±50.4 nm and a mean hydrodynamic size of ~161 nm were prepared using ascorbic acid as a reducing agent and disk-like bicelles as a template. Only the presence of a soft template, like bicelles, ensured the appearance of spiked nanoparticles with resonance in the near infrared region. The irradiation of spiked gold nanoparticles by an 805 nm laser led to the time- and concentration-dependent increase of temperature. Both pHLIP^® and PEG coated gold spherical and multispiked nanoparticles might find application in radiation and thermal therapies of tumors.     

Extracellular biosynthesis of silver nanoparticles using Rhizopus stolonifer

Synthesis of silver nanoparticles (AgNPs) has become a necessary field of applied science. Biological method for synthesis of AgNPs by Rhizopus stolonifer aqueous mycelial extract was used. The AgNPs were identified by UV–visible spectrometry, X-ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier transform infrared spectrometry (FT-IR). The presence of surface plasmon band around 420 nm indicates AgNPs formation. The characteristic of the AgNPs within the face-centered cubic (fcc) structure are indicated by the peaks of the X-ray diffraction (XRD) pattern corresponding to (1 1 1), (2 0 0) and (2 2 0) planes. Spherical, mono-dispersed and stable AgNPs with diameter around 9.47 nm were prepared and affirmed by high-resolution transmission electron microscopy (HR-TEM). Fourier Transform Infrared (FTIR) shows peaks at 1426 and 1684 cm⁻¹ that affirm the presence of coat covering protein the AgNPs which is known as capping proteins. Parameter optimization showed the smallest size of AgNPs (2.86 ± 0.3 nm) was obtained with 10⁻² M AgNO₃ at 40 °C. The present study provides the proof that the molecules within aqueous mycelial extract of R. stolonifer facilitate synthesis of AgNPs and highlight on value-added from R. stolonifer for cost effectiveness. Also, eco-friendly medical and nanotechnology-based industries could also be provided. Size of prepared AgNPs could be controlled by temperature and AgNO₃ concentration. Further studies are required to study effect of more parameters on size and morphology of AgNPs as this will help in the control of large scale production of biogenic AgNPs.     

Kinetics parameter estimation for the binding process of salicylic acid to human serum albumin (HSA) with capacitive sensing technique

A novel method for real-time investigating the binding interaction between human serum albumin (HSA) and salicylic acid with capacitive sensing technique was successfully proposed. HSA was immobilized on the surface of a gold electrode modified with an insulating poly (o-phenylenediamine) (o-PD) film and colloid Au nanoparticles layers. The bioactivity of HSA was remained and major binding sites were available because of the excellent biocompatibility of gold nanoparticles. The capacitance and interfacial electron resistance of the sensor were altered, owing to the binding of HSA to salicylic acid. The time courses of the capacitance change were acquired with capacitive sensing technique during the binding process. Based on the capacitance response curves with time, the response model for the binding was derived in theory and the corresponding regression parameters were determined by fitting the real-time experimental data to the model. The binding and the dissociation rate constants (k₁ and k_− 1) were estimated to be 54.8 (mol l^− 1)^− 1 s^− 1 and 2.9 × 10^− 3 s^− 1, respectively. And the binding equilibrium constant (K_a) was calculated to be 1.89 × 10⁴ (mol l^− 1)^− 1.     

Excited state properties of dinaphthyl ditelluride

Di-1-naphthyl ditelluride (Te₂naphthyl₂) is characterized by two low-energy excited states. The corresponding electronic transitions nTe → σ¹ Te–Te and nTe → π¹ naphthyl CT give rise to absorptions at λ_max = 403 and 311 nm, respectively. In solution nTe → σ¹ excitation leads to the cleavage of the Te–Te bond. In contrast to Te₂naphthyl₂ in the dissolved state the solid compound shows a luminescence (λ_max = 576 nm) which originates from nTe → π¹ naphthyl CT triplet.     

Biosynthesis of ethyl caproate and other short ethyl esters catalyzed by cutinase in organic solvent

The main objective of this work was to study the enzymatic synthesis of short chain ethyl esters, a group of relevant aroma molecules, by Fusarium solani pisi cutinase in an organic solvent media (iso-octane), and to assess the influence of different parameters on the reaction yield.Cutinase displayed high initial esterification rates in iso-octane, which amounted to 1.15 μmol min⁻¹ mg⁻¹ for ethyl butyrate (C₄ acid chain) and 1.06 μmol min⁻¹ mg⁻¹ for ethyl valerate (C₅ acid chain). High product yields, 84% for ethyl butyrate and 96% for ethyl valerate, were observed after 6 h of reaction, for an initial equimolar concentration of substrates (0.1 M).The highest product yield (97%) was observed for ethyl caproate (C₆) synthesis, a compound which is a part of natural apple and pineapple flavour, for an alcohol:acid molar ratio of 2 (0.2 M ethanol concentration).Cutinase affinity for short chain length carboxylic acids (C₄–C₆) in ester synthesis in iso-octane confirmed previous observations in reversed micellar system.     

Design of a new fluorescent probe: Pyrrole/imidazole hairpin polyamides with pyrene conjugation at their γ-turn

Fluorophores that are conjugated with N-methylpyrrole-N-methylimidazole (Py–Im) polyamides postulates versatile applications in biological and physicochemical studies. Here, we show the design and synthesis of new types of pyrene-conjugated hairpin Py–Im polyamides (1–5). We evaluated the steady state fluorescence of the synthesized conjugates (1–5) in the presence and absence of oligodeoxynucleotides 5′-CGTATGGACTCGG-3′ (ODN 1) and 5′-CCGAGTCCATACG-3′ (ODN 2) and observed a distinct increase in emission at 386 nm with conjugates 4 and 5. Notably, conjugate 5 that contains a β-alanine linker had a stronger binding affinity (K_D = 1.73 × 10^?8 M) than that of conjugate 4 (K_D = 1.74 × 10^?6 M). Our data suggests that Py–Im polyamides containing pyrene fluorophore with a β-alanine linker at the γ-turn NH₂ position can be developed as the competent fluorescent DNA-binding probes.     

Synthesis and characterization of a fluorescent probe for α-tocopherol suitable for fluorescence microscopy

Previously prepared fluorescent derivatives of α-tocopherol have shown tremendous utility in both in vitro exploration of the mechanism of ligand transfer by the α-tocopherol transfer protein (α-TTP) and the intracellular transport of α-tocopherol in cells and tissues. We report here the synthesis of a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) containing α-tocopherol analog having extended conjugation with an alkenyl thiophene group that extends the absorption and emission maxima to longer wavelengths (λ_ex = 571 nm and λ_em = 583 nm). The final fluorophore thienyl-ene-BODIPY-α-tocopherol, 2, binds to recombinant human α-TTP with a K_d = 8.7 ± 1.1 nM and is a suitable probe for monitoring the secretion of α-tocopherol from cultured Mcf7#189 cells.     

Detoxification of Indigo carmine using a combined treatment via a novel trimeric thermostable laccase and microbial consortium

For the first time, the investigation of Indigo carmine decolorization was done using an atypical Scytalidium thermophilum laccase. Crude and purified laccases required high temperatures and slight acidic pH to achieve maximum Indigo decolorization. Kinetic parameters (K_m and k_cat) of the homotrimeric laccase toward Indigo carmine were determined and laccase efficacy toward repeated dye decolorization process was studied. For the first time, 5 g l⁻¹ as initial Indigo carmine concentration were efficiently transformed up to 50% within 6 h of incubation using 0.1 U ml⁻¹ of laccase and without presence of any mediators. In this study, we showed that the atypical laccase transformed the indigoid dye structure, confirmed by the color changing from blue to red. This intermediate (red) was a subject to an efficient microbial consortium treatment monitored by measuring the decrease in optical density and the total organic carbon removal efficiencies. Toxicological studies via micro-toxicity test showed that the released enzymatic and adapted consortium degradation products were both non-toxic while the initial product was toxic.     

Effects of Zn2+ and niflumic acid on photosynthesis in Glycine soja and Glycine max seedlings under NaCl stress

The effects of two anion/Cl^? channel inhibitors, Zn²⁺ and niflumic acid (NA), on seedling photosynthetic and fluorescent parameters of two Glycine soja populations (salt-tolerant BB52; salt-sensitive N23227) and Glycine max cultivar (salt-tolerant Lee68) were studied and compared under salt stress. Treatments with Zn²⁺ and NA only (10, 20 μmol L^?1) were also imposed for comparisons. Results showed that, there were non-toxic and non-nutritional effects of Zn²⁺ and NA treatments alone on seed germination and seedling growth of soybeans. Under 150 mmol L^?1 NaCl for 6 d, leaf chlorophyll and carotenoid contents, net photosynthetic rate (P_n), stomatal conductance (G_s), intercellular CO₂ concentration (C_i), transpiration rate (Tr), and the maximum photochemical efficiency of photosystem II (PS II) (F_v/F_m) except the stomatal limitation (Ls) significantly decreased in three kinds of soybean seedlings when compared with their control plants. The NaCl stress plus additional 20 μmol L^?1 Zn showed an obvious enhancement of leaf chlorophyll and carotenoid contents, P_n, G_s, C_i and Tr, especially for the G. max cultivar Lee68, but the supplementation of 20 μmol L^?1 NA showed the reverse effects.     

Amperometric hydrogen peroxide biosensor based on the immobilization of HRP on nano-Au/Thi/poly (p-aminobenzene sulfonic acid)-modified glassy carbon electrode

A novel hydrogen peroxide biosensor was fabricated for the determination of H₂O₂. The precursor film was first electropolymerized on the glassy carbon electrode with p-aminobenzene sulfonic acid (p-ABSA) by cyclic voltammetry (CV). Then thionine (Thi) was adsorbed to the film to form a composite membrane, which yielded an interface containing amine groups to assemble gold nanoparticles (nano-Au) layer for immobilization of horseradish peroxidase (HRP). The electrochemical characteristics of the biosensor were studied by CV and chronoamperometry. The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor responded to H₂O₂ in the linear range from 2.6 × 10^− 6 mol/L to 8.8 × 10^− 3 mol/L with a detection limit of 6.4 × 10^− 7 mol/L. Moreover, the studied biosensor exhibited good accuracy and high sensitivity. The proposed method was economical and efficient, making it potentially attractive for the application to real sample analysis.     

Green synthesized doxorubicin loaded zinc oxide nanoparticles regulates the Bax and Bcl-2 expression in breast and colon carcinoma

The green synthesis of zinc oxide nanoparticles (ZnONPs) using Borassus flabellifer fruit extract was characterized by UV–visible spectroscopy, FT-IR, XRD, TEM, Zeta potential and EDS analysis. The UV–visible spectrum showed an absorption peak at 368 nm that reflects surface Plasmon resonance (SPR) ZnONPs. TEM photograph showed that the green synthesized ZnONPs were porous in nature and rod like structure with an average size of 55 nm. The Zeta potential value of −21.5 mV revealed the surface charge of green synthesized ZnONPs. In this study, we examined the synthesized DOX-ZnONPs exhibited a dose-dependent cytotoxicity against MCF-7 and HT-29. The inhibitory concentration (IC₅₀) was found to be 0.125 μg mL⁻¹ for MCF-7 and HT-29 cells. An induction of apoptosis was evidenced by nuclear stain Hoechst 33258. In vivo toxicity assessment showed that DOX-ZnONPs have low systemic toxicity in murine model system. The results prove that the DOX-ZnONPs has low toxicity and high therapy efficacy, which provides convincing evidence for the green biosynthesized ZnO as a promising candidate for a drug delivery system.     

Application of liquid chromatography-tandem mass spectrometry (LC–MS/MS) for the analysis of stable isotope enrichments of phenylalanine and tyrosine

Whole-body protein synthesis and breakdown are measured by a combined tracer infusion protocol with the stable isotope amino acids l-[ring-²H₅]-phenylalanine, l-[ring-²H₂]-tyrosine and l-[ring-²H₄]-tyrosine that enable the measurement of the phenylalanine to tyrosine conversion rate. We describe a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the measurement of very low tracer–tracee ratios (TTR) of the amino acids l-phenylalanine and l-tyrosine in human plasma. TTR calibration curves of the tracers l-[ring-²H₅]-phenylalanine, l-[ring-²H₂]-tyrosine and l-[ring-²H₄]-tyrosine were linear (r² > 0.99) in the range between 0.01% and 5.0% TTR and lowest measurable TTR for the tracers was 0.01% at a physiological concentration of 60 μM. The method was applied successfully to plasma samples from a clinical study reaching a steady state enrichment plateau (mean ± SD) of 3.33 ± 0.19% for l-[ring-²H₅]-phenylalanine, 2.40 ± 0.43% for l-[ring-²H₂]-tyrosine and 0.29 ± 0.07% for l-[ring-²H₄]-tyrosine, respectively. The LC–MS/MS method can be applied for measurement of very low plasma enrichments of phenylalanine and tyrosine for the determination of whole-body protein synthesis and breakdown rates in humans.     

Graft copolymerization of acrylamide on chitosan-co-chitin and its application for immobilization of Aspergillus sp. RL2Ct cutinase

The synthesis of chitosan (Chs) and chitin (Chi) copolymer and grafting of acrylamide (AAm) onto the synthesized copolymer have been carried out by chemical methods. The grafted copolymer was characterized by FTIR, SEM and XRD. The extracellular cutinase of Aspergillus sp. RL2Ct (E.C. 3.1.1.3) was purified to 4.46 fold with 16.1% yield using acetone precipitation and DEAE sepharose ion exchange chromatography. It was immobilized by adsorption on the grafted copolymer. The immobilized enzyme was found to be more stable then the free enzyme and has a good binding efficiency (78.8%) with the grafted copolymer. The kinetic parameters K_M and V_max for free and immobilized cutinase were found to be 0.55 mM and 1410 μmol min⁻¹ mg⁻¹ protein, 2.99 mM and 996 μmol min⁻¹ mg⁻¹ protein, respectively. The immobilized cutinase was recycled 64 times without considerable loss of activity. The matrix (Chs-co-Chi-g-poly(AAm)) prepared and cutinase immobilized on the matrix have potential applications in enzyme immobilization and organic synthesis respectively.     

A novel MODIS algorithm to estimate chlorophyll a concentration in eutrophic turbid lakes

A novel approach was developed to estimate phytoplankton biomass in eutrophic turbid lakes, using MODIS bands designed for land and atmospheric studies. The Baseline Normalized Difference Bloom Index (BNDBI) uses the difference of remote-sensing reflectance (Rrs, sr⁻¹) at 555 nm (band 4) and 645 nm (band 1) after baseline correction using bands at 469 nm and 859 nm: (Rrs′(555) − Rrs′(645))/(Rrs′(555) + Rrs′(645)). BNDBI takes advantage of the Chl-a’s absorption minimum near 572 nm and absorption maximum near 667 nm. Using data from Lake Chaohu, the index showed a strong relationship with Chl-a concentrations in conditions that would normally saturate more sensitive ocean-color sensors. Extensive field measurements were used to calibrate and validate the algorithm with unbiased root-mean-square-error (URMSE) of 47.9% when compared to in situ Rrs data. A reduced sensitivity to atmospheric effects was accomplished by using a baseline correction approach, anchored at 469 nm and 859 nm to correct the radiances at 555 nm and 645 nm. Radiative transfer simulations showed that the algorithm can be applied directly to MODIS Rayleigh-corrected reflectance (Rrc) after adjusting algorithm coefficients (URMSE uncertainty of 56.4% for MODIS Rrc data) for Chl-a concentrations <1000 μg L⁻¹. Comparative analyses showed that the index was resistant to changes in turbidity and organic matter concentrations. Theoretical simulations, image comparisons and spectral analyses demonstrated that the index was robust in a range of complex atmospheric and surface conditions, with different aerosol types, optical thickness (τ_a555), solar/viewing geometry, sun glint and thin clouds. A comparison with other MODIS and MERIS Chl-a algorithms for turbid waters showed that BNDBI provided consistent results with the advantage of using MODIS wavebands that remain unsaturated in high turbidity conditions. The BNDBI opens new possibilities to explore bio-optical dynamics in turbid eutrophic lakes using data from a range of satellite sources.     

Near native binding of a fluorescent serotonin conjugate to serotonin type 3 receptors

Described is the synthesis of 5-hydroxytryptamine-tetramethylrhodamine (5HT¹); an indole nitrogen linked fluorescent conjugate of serotonin. Through a series fluorescence quenching experiments and experiments in the presence of a known competitive antagonist (Granisetron), it was shown that 5HT¹ specifically binds to purified homo-pentameric type-3 human serotonin receptors (5HT_3A). The measured dissociation constant and Hill coefficient are K_d = 83 ± 3 nM and n = 3.1 ± 0.3, respectively which is indicative of multi-ligand binding and cooperativity similar to that of unconjugated serotonin.     

Does UV-B influence biomass growth in lichens deficient in sun-screening pigments?

This study aimed to assess biomass growth as a response variable in lichens during short-term laboratory experiments. To do this, we studied the influence of UV-B and temperature on lichen performance including the synthesis of solar radiation screening cortical compounds. The pioneer lichen Xanthoria aureola from exposed sea cliffs and the old forest lichen Lobaria pulmonaria were cultivated for 15 days in the laboratory in a factorial experiments with temperature (12 and 21 °C) and UV-B (0, 0.1, 0.3 and 1.0 W m^?2) as treatments. Prior to the experiment, the cortical pigment parietin was non-destructively extracted from X. aureola, whereas the sampled shade-adapted thalli of L. pulmonaria lacked cortical melanic compounds. Therefore both lichens were deficient in cortical sun-screening compounds when the UV-B exposure started. At 12 °C, the relative growth rate was 7.2 ± 0.6 and 3.0 ± 0.8 mg g^?1 day^?1 in L. pulmonaria and X. aureola, respectively, reduced to 1.8 ± 0.5 and ?2.6 ± 0.9 mg g^?1 day^?1, at 21 °C. These figures showed that lichen growth is a useful response variable in short-term laboratory experiments. Growth was not influenced by UV-B alone in these pigment-deficient transplants, suggesting that UV-B had little adverse effects on either of the lichen bionts. The cortical sun screens (parietin and melanic compounds) were synthesized in the presence of UV-B, and increased statistically significantly with increasing UV-B at both cultivation temperatures. However, in X. aureola the synthesis was highest at the lowest temperature (12 °C). At 12 °C, changes in chlorophylls, F_v/F_m and NPQ during cultivation were consistent with a substantial level of acclimation to the growth chamber conditions for both species, whereas strong reductions in photosynthetic pigments, F_v/F_m and Ф_II at 21 °C indicated serious damage and chlorophyll degradation at high temperature. In conclusion, lichen growth and the synthesis of protective compounds are highly responsive lichen processes in short-term experiments.     

Gold nanoparticles functionalized by gadolinium–DTPA conjugate of cysteine as a multimodal bioimaging agent

The synthesis and characterization of gold nanoparticles coated with Gd-chelate (Au@GdL), where L is a conjugate of DTPA and cysteine, is described. These particles are obtained by the replacement of citrate from the gold nanoparticle surfaces with gadolinium chelate (GdL). The average size of Au@GdL is 14 nm with a loading of GdL reaching up to 2.9 × 10³ per particles, and they demonstrate very high R1 relaxivity (～10⁵ mM^?1 s^?1) as well as X-ray attenuation. The R1 relaxivity per [Gd] is 17.9 mM^?1 s^?1. The present system also exhibits macrophage-specific property, as demonstrated by histological and TEM images as well as CT and MR, rendering itself as a new class of T1 multimodal CT/MR contrast agent.     

Study of the interaction of human serum albumin with Alstonia scholaris leaf extract-mediated silver nanoparticles having bactericidal property

This study investigates the green synthesis of AgNPs from 1 mM aqueous AgNO₃ using 10% leaf extract of Alstonia scholaris (Chhatim) for its wide antibacterial and medicinal properties. The synthesized AgNPs were duly characterized by UV–vis (UV–vis) spectrophotometry, dynamic light scattering, field emission scanning electron microscopy, transmission electron microscopy, energy-dispersive analysis of X-rays spectroscopy, and fourier transform infrared spectrophotometry. Their antibacterial property was tested against Escherichia coli (ATCC 25922), and minimum inhibitory concentrations of 0.08 nM of AgNPs were obtained, which suggests improved therapeutic efficacy. We report the interaction of human serum albumin (HSA) with this nanoparticle, and this interaction was studied by UV–vis, fluorescence, and circular dichroism spectroscopies and zeta potential measurement at room temperature. It was found that the AgNPs form a complex with HSA, which may cause the slightest change in the conformation of HSA. The calculated values of Stern-Volmer quenching constant, binding constant, and binding distance were 1.82 × 10⁷ M⁻¹, 1.58 × 10⁷ M⁻¹, and 3.68 nm, respectively. Therefore, in future, the present study may provide useful information to design a better antibacterial compound by using green synthesized nanoparticles with fewer side effects.     

Structural characterization of thermostable,solvent tolerant,cytosafe tannase from Bacillus subtilis PAB2

Tannase production by Bacillus subtilis PAB2, was investigated under solid state fermentation using tamarind seed as sole carbon source and it was found as the highest titer (73.44 U/gds). The enzyme was purified to homogeneity, which showed the molecular mass around 52 kDa (K_m = 0.445 mM, V_max = 125.8 mM/mg/min and K_cat = 2.88 min^–1). The enzyme was found stable in a range of pH (3.0–8.0) and temperature (30–70 °C) with an optimal activity at pH 5.0, pI of 4.4 and at 40 °C temperature. It exhibited half-life (t_1/2) of 4.5 h at 60 °C. The enzyme comprised a typical secondary structure containing α-helix (9.3%), β-pleated sheet (33.6%) and β-turn (17.2%). The native conformation of the enzyme was alike a 44 nm spherical nanoparticle upon aggregation. Thermodynamic parameters of tannase revealed that it was stable at 40 °C and showed Q₁₀, ΔG_d and ΔS_d values of 2.08, 99.37 KJ/mol and 252.38 J mol⁻¹ K⁻¹, respectively. Organic solvents were stimulatory with regard to enzyme activity. Moreover, the altered enzyme activity was determined to be correlated with the changes in structural conformation in presence of inducer and inhibitor. Tannase was explored to have no cytotoxicity on Vero cell line as well as rat model study.