Characterisation of Grass Fibres

The elementary grass fibres were isolated from different grass and legumes sorts, i.e. Ryegrass (Lolium hybridum Gumpenstein), Wheat straw, Trefoil (Trifolium pratense) and Lucerne (Medicago sativa). The fibre-samples were obtained in a bio-refinery, after the liquid phase containing proteins and lactic acid was eliminated from the ensiled and green grasses, respectively. For the isolation of elementary grass fibres different processes were used. The morphological characteristics of stems and leaves of different grass species were microscopically observed. On the microscopical stem and leaves cross-section samples the quantification of fibres sclerenchyma cells was performed. The quantitative analysis was carried out in order to obtain basic quantitative data on grass fibres, such as area of the single fibre or group of fibres, diameter of a single fibre or group of fibres and distances between the most distant and least distant points on the area of the fibre. Measurements were made using a Carl Zeiss software KS 300, which runs on a computer connected to the image analysis equipment consisting of a microscope and a digital camera. In addition to, geometrical and mechanical properties of isolated fibres and fibre bundles were determined. Due to the grass history, i.e. deformations and damages caused by the treatment of grasses in the bio-refinery, maturity grade, grass or legumes type and conditions during grass growth, the plant structures vary considerable in their properties.     

Instantaneous phytosynthesis of gold nanoparticles via Persicaria salicifolia leaf extract,and their medical applications

In the current investigation, an instantaneous, single step, cost-effective, and environmentally safe biosynthesis of gold nanoparticles (AuNPs) using the aqueous extract of P. salicifolia leaves is reported for the first time. P. salicifolia-AuNPs were characterized via several techniques including UV–Visible spectroscopy, Zeta potential, Fourier Transform Infrared (FT-IR) spectroscopy, Energy Dispersive X-ray spectroscopy (EDX) attached to SEM, High-resolution Transmission electron microscope (HRTEM), X-ray diffraction (XRD), and Selected Area Electron Diffraction (SAED), EDX elemental mapping and X-ray Photoelectron Spectroscopy (XPS). Results demonstrated the formation of violet-colored, mostly spherical-shaped AuNPs with a diameter between 5 and 23 nm and a plasmon peak at 535 nm indicated the successful phytosynthesis of AuNPs using the aqueous extract of the investigated species. The cytotoxicity investigation of the phytosynthesized AuNPs using Sulphorhodamine-B (SRB) assay reflected superb cell capability in inhibiting the growth and proliferation of human breast cancer cells (MCF7 cell line) with IC₅₀ 2.24 μg/mL. Additionally, P. salicifolia-AuNPs were proved to be a potent antioxidant. These results offer a promising route of AuNPs synthesis using the extract of P. salicifolia.     

Biogenic Syntheses of Gold Nanoparticles Using Plant Extracts

A biogenic approach for the synthesis of gold nanoparticles (GNPs) by reducing chloroauric acid (HAuCl₄) with three different plant extracts (from Angelica, Hypericum, and Hamamelis) is described. The content of reducing compounds (polyphenols) in each plant extract was determined by photometric dosing. The formation of GNPs was tracked by ultraviolet-visible spectroscopy and their characterization was performed using transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging and FTIR spectra. GNPs, with diameters ranging from about 4 nm to 8 nm, were obtained at room temperature and pH value about 8. They present various shapes from spherical, to ovals, heart or polyhedral forms. Generally, the GNPs colloidal dispersions are rather stable in time, and the self assembly of GNPs is sparsely observed. However, at lower concentration of the plant extract the tendency to self aggregation of the GNPs increased. The plant extracts contain reducing agents, compounds with stabilizing effect on the GNPs, but also components which mediate their self assembly. The GNPs obtained by these biogenic syntheses have potential biological and medical applications, taking into account at least two characteristics, their size and aqueous stability.     

Electrothermal atomic absorption spectrometric determination of Cr(VI) during ferrochrome production

The level of the generation of hexavalent chromium during ferrochrome production was checked. The concentration of Cr(VI) increases with each stage of ferrochrome production, 7070microgg(-1) being the highest concentration encountered in the last stage of production (dust). This concentration exceeds the maximum acceptable total Cr concentration per 8h by a factor of more than 1000. It was further observed that there is a higher contamination of soil by this pollutant closer to the plant than further away. The highest concentrations of Cr(VI) in soil and grass were found to be 12.7 and 4.2microgg(-1), respectively. The results of the investigation indicate that the consumption of such grass by animals do not pose any health hazard, for concentrations of the toxic Cr species are very low. Therefore, the release of emissions, including dust, during ferrochrome production, is a major contributor to occupational diseases and death to people working in ferrochrome production plant or mine.     

Synthesis and antibacterial activity of stable bio-conjugated nanoparticles mediated by walnut (Juglans regia) green husk extract

Nanoparticles have gained significant attention in recent years due to their numerous applications in various aspects of human life. A variety of methods have been investigated for synthesis of nanoparticles among which, biogenic approaches are considered as both simple and eco-friendly. Here, a new single-step biological approach was employed for synthesis of silver chloride nanoparticles (AgCl-NPs) at room temperature, using walnut green husk extract. Macromolecules present in the plant extract, which might act as bio-reductants and/or stabilisers of nanoparticles were characterised by Fourier transform Infrared spectroscopy. X-ray diffraction pattern and transmission electron microscopy revealed that 1 mM of AgNO3 produced mostly spherical nanoparticles in a range of 4–30 nm in diameter with an average of 16 nm. Interestingly, the synthesised nanoparticles showed significant inhibitory effects against Escherichia coli and Staphylococcus aureus clinical isolates. Altogether, these data suggest a new encouraging application of a medicinal plant bound with synthesised AgCl nanoparticles.     

Biogenic nano silver: Synthesis,characterization, antibacterial,antibiofilms, and enzymatic activity

Powder technology covers many fields from medicine to material science. Especially easy synthesis of nano- and micro-sized particles and low-cost synthesis with non-toxic chemicals are essential. The ability to design nanoparticles according to the application area and desired features is an advantage for many applications. Nanoparticles biosynthesis is under research due to broad biomedical practise. To synthesize of silver nanoparticle from the plant extract, Corylus avellana (Hazelnut) leaves were utilized for bioreduction of silver nitrate. The plant extract is thoroughly mixed with silver nitrate solution, studied the synthesis of nanoparticles using UV–Vis spectroscopy. The nanoparticles were characterized by SEM and XRD-Pattern. XRD analysis proved that the size range of the nanoparticles was 32 nm. Scanning microscope images also demonstrate nanoparticles having spherical shape and a mean size between 9 nm and 50 nm. The antibacterial effect of AgNPs obtained from hazelnut leaves demonstrated antibacterial effect against all tested bacteria strains. The results demonstrated that the plant extract acts as a good bioreductant for the production of silver nanoparticles. This compound was good inhibitor of the α-glycosidase, human carbonic anhydrase I and II isoforms, and cholinesterase enzymes. The results were obtained with Ki values of 12.84 ± 0.41 µM for α-glycosidase, 27.94 ± 3.77 µM for hCA I, 36.12 ± 6.03 µM for hCA II, 47.05 ± 6.83 µM for BChE, and 53.94 ± 7.55 µM for AChE, respectively.     

Optical chloride sensor based on dimer-monomer equilibrium of indium(III) octaethylporphyrin in polymeric film

A novel transduction chemistry for preparing optical anion-selective polymeric films that respond reversibly and selectively to chloride ion activity is demonstrated. The chloride sensors are prepared by casting thin (5-10 microm) plasticized PVC films containing indium(III) octaethylporphyrin hydroxide, along with optimized levels of a lipophilic tetraphenylborate salt, onto glass slides. When bathed in low-pH buffered solutions void of chloride, the porphyrin species spontaneously forms a hydroxide ion-bridged dimer, with the added lipophilic borate species serving as the counteranion for this complex. The maximum for the Soret absorption band of this dimeric species is shifted to 390 nm, from 410 nm for the initial monomeric porphyrin. Increases in chloride ion levels in the bathing solution results in chloride extraction and ligation to the In(III) center, and concomitant breaking of the dimer into monomeric porphyrin species, yielding a decrease in absorbance at 390 nm and an increase in optical signal at 410 nm. Under optimized conditions, optical selectivity coefficients toward chloride over a wide range of other anions (NO3-, ClO4-, SCN-, SO4(2-), F-, Br-, H2PO4-) are measured to be < 10(-3). Of all anions tested, only salicylate yields a slightly greater response than chloride. This selectivity is shown to be adequate for reversible and accurate sensing of chloride levels in diluted serum samples.     

Hazardous impact and translocation of vanadium (V) species from soil to different vegetables and grasses grown in the vicinity of thermal power plant

The distribution of vanadium (V) species in soil (test soil), vegetables and grasses, collected from the vicinity of a thermal power plant has been studied. For comparison purpose soil (control soil), same vegetable and grass samples were collected from agricultural land devoid of any industrial area. A simple and efficient ultrasonic assisted extraction method has been developed for the extraction of V(5+) species from soil, vegetable and grass samples using Na(2)CO(3) in the range of 0.1-0.5 mol/L. For comparison purpose same sub samples were also extracted by conventional heating method. The total and V species were determined by electrothermal atomic absorption spectrometry using different modifiers. The validity of V(5+) and V(4+) determination had been confirmed by the spike recovery and total amount of V by the analysis of CRM 1570 (spinach leave) and sub samples of agricultural soil. The concentration of total V was found in the range of 90-215 and 11.4-42.3 μg/g in test and control soil samples, respectively. The contents of V(5+) and total V in vegetables and grasses grown around the thermal power plant were found in the range of 2.9-5.25 and 8.74-14.9 μg/g, respectively, which were significantly higher than those values obtained from vegetables and fodders grown in non exposed agricultural site (P<0.01). Statistical evaluations indicate that the sum of concentrations of V(5+) and V(4+) species was not significantly different from total concentration of V in same sub samples of vegetable, grass and soil of both origins, at 95% level of confidence.     

Biomimetic synthesis of platinum nanoparticles utilizing a terrestrial weed Antigonon leptopus

In this work, we report for the first time a new bio-inspired method for platinum nanoparticle (PtNP) synthesis utilizing a highly invasive terrestrial weed coral vine (Antigonon leptopus) as the main bioagent. Extracts of all three basic components of the plant – leaves, stem and root – were explored and were found to be suitable in effecting the PtNP synthesis. The electron micrographs of the synthesized PtNPs revealed the presence of particles of monodispersed spherical and polydispersed shapes in sizes ranging from 5 to 190?nm. The presence of Pt atoms was confirmed from the energy dispersive X-ray (EDAX) and X-ray diffraction (XRD) studies. The FTIR spectral study indicated that the polysaccharides and proteins in the plant extract could have been responsible for the reduction of Pt ions to PtNPs and the latter’s stabilization.     

A small-angle X-ray scattering study of aqueous solutions of uranium(IV) complexes with lacunar heteropolytungstates

The behavior of aqueous and acid solutions of U(IV) complexes with lacunar heteropolyanions was studied by small-angle X-ray scattering. The polytungstate forms in solutions and in crystals are generally similar. The polyanionic complexes in solutions can exhibit ordering with a period of approximately 5.6–7.8 nm. Such species are probably precursors of the crystalline form.     

Characterization of woody and herbaceous biomasses lignin composition with 1064 nm dispersive multichannel Raman spectroscopy

Biomass representing different classes of bioenergy feedstocks, including woody and herbaceous species, was measured with 1064 nm Raman spectroscopy. Pine, oak, poplar, kenaf, miscanthus, pampas grass, switchgrass, alfalfa, orchard grass, and red clover were included in this study. Spectral differences have been identified with an emphasis on lignin guaiacyl and syringyl monomer content and carotenoid compounds. The interpretation of the Raman spectra was correlated with (13)C-nuclear magnetic resonance cross-polarization/magic-angle spinning spectra of select biomass samples. Thioacidolysis quantification of guaiacyl and syringyl monomer composition and the library of Raman spectra were used as a training set to develop a principal component analysis model for classifying plant samples and a principal component regression model for quantifying lignin guaiacyl and syringyl composition. Raman spectroscopy with 1064 nm excitation offers advantages over alternative techniques for biomass characterization, including low spectral backgrounds, higher spectral resolution, short analysis times, and nondestructive analyses.     

Synthesis and characterization of human transferrin-stabilized gold nanoclusters

Human transferrin has been biolabelled with gold nanoclusters (Au NCs) using a simple, fast and non-toxic method. These nanocrystals (<2 nm) are stabilized in the protein via sulfur groups and have a high fluorescence emission in the near infrared region (QY=4.3%; λem=695 nm). Structural investigation and photophysical measurements show a high population of clusters formed of 22-33 gold atoms covalently bound to the transferrin. In solutions with pH ranging from 5 to 10 and in buffer solutions (PBS, HEPES), those biolabelled proteins exhibit a good stability. No significant quenching effect of the fluorescent transferrin has been detected after iron loading of iron-free transferrin (apoTf) and in the presence of a specific polyclonal antibody. Additionally, antibody-induced agglomeration demonstrates no alteration in the protein activity and the receptor target ability. MTT and Vialight? Plus tests show no cytotoxicity of these labelled proteins in cells (1 μg ml(-1)-1 mg ml(-1)). Cell line experiments (A549) indicate also an uptake of the iron loaded fluorescent proteins inside cells. These remarkable data highlight the potential of a new type of non-toxic fluorescent transferrin for imaging and targeting.     

Investigation of aqueous stability of taxol in different release media

In the present study, the aqueous stability of taxol in different aqueous media and immiscible aqueous/organic systems at 37?°C was investigated. The aqueous media included phosphate buffered saline (PBS) and PBS containing 10% methanol, 10% ethanol, 10% hydroxypropyl β-cyclodextrin (HP-βCD), 1% sodium citrate and 1% Tween 80. The immiscible systems consisted of PBS/octanol, PBS/dichloromethane, PBS/chloroform and PBS/ethyl acetate. The concentrations of taxol and related derivatives in each of the media were determined through the high-performance liquid chromatography assay. Results showed that hydrolysis and epimerization were two major types of degradation for taxol in the aqueous media starting from the initial hours of contact (6 hours). Addition of Tween 80 to PBS moderately increased the aqueous stability of taxol. As well, using PBS containing 10% HP-βCD inhibited the taxol hydrolysis, while epimerization still in process. In the case of immiscible systems, except for PBS/ethyl acetate system, no evidences of taxol hydrolysis were observed. Meanwhile, epimerization of taxol in PBS/dichloromethane and PBS/chloroform systems underwent due to the ability of C–Cl bonds to form hydrogen bonding with the hydroxyl group of C7 of taxol.     

Unveiling the cytotoxicity of phytosynthesised silver nanoparticles using Tinospora cordifolia leaves against human lung adenocarcinoma A549 cell line

Biosynthesis of silver nanoparticles (AgNPs) using plant extract is a cheap, easily accessible and natural process in which the phyto‐constituents of the plants act as capping, stabilising and reducing agent. The present study explored the biosynthesis of AgNPs using aqueous leaf extract of Tinospora cordifolia and characterised via various techniques such as Fourier transform infrared, scanning electron microscopy, transmission electron microscopy (TEM), energy dispersive X‐ray analysis and X‐ray diffraction. Here, TEM confirmed the spherical morphology with 25–50 nm size of synthesised AgNPs. Further, anticancer efficiency of AgNPs synthesised using T. cordifolia leaves were evaluated against human lung adenocarcinoma cell line A549 by MTT, trypan blue assay, apoptotic morphological changes using Annexin V‐FITC and Propidium iodide (PI), nuclear morphological changes by DAPI (4, 6‐diamidino‐2‐phenylindole dihydrochloride) staining, reactive oxygen species generation and mitochondrial membrane potential determination. Results confirmed the AgNPs synthesised using T. cordifolia leaves are found to be highly toxic against human lung adenocarcinoma cell line A549.Inspec keywords: toxicology, cellular biophysics, cancer, silver, biomembranes, drugs, nanofabrication, nanoparticles, transmission electron microscopy, drug delivery systems, nanomedicine, lung, biomedical materials, antibacterial activity, X‐ray diffraction, Fourier transform infrared spectra, scanning‐transmission electron microscopyOther keywords: cytotoxicity, phytosynthesised silver nanoparticles, A549 cell line, biosynthesis, aqueous leaf, transmission electron microscopy, TEM, X‐ray analysis, X‐ray diffraction, spherical morphology, human lung adenocarcinoma cell line, nuclear morphological changes, 4, 6‐diamidino‐2‐phenylindole dihydrochloride, Tinospora cordifolia leaves, scanning electron microscopy, Fourier transform infrared, energy dispersive X‐ray analysis, Ag, size 25.0 nm to 50.0 nm, anticancer efficiency, trypan blue assay, propidium iodide, Annexin V‐FITC, DAPI staining, reactive oxygen species generation, mitochondrial membrane potential determination     

Synthesis and characterization of silver nanoparticles and their antimicrobial efficacy

An efficient protocol for synthesis of silver nanoparticles (AgNPs) using the combination of aqueous extract of Tinospora cordifolia leaves and 5 mM silver nitrate (AgNO₃) solution was developed. This study revealed that bioactive compounds present in the extract function as stabilizing and capping agent for AgNPs. Scanning electron microscope and transmission electron microscope studies confirm the structure and surface morphology of the AgNPs. The size of synthesized AgNPs was in the range of 30–50 nm having spherical morphology. The crystalline nature of NPs was defined by the X-ray diffraction pattern. The AgNPs were found to be toxic against pathogenic bacteria such as Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), and Staphylococcus aureus (ATCC 29213) and against plant pathogenic fungi Fusarium oxysporum (MTCC 8608) and Sclerotinia sclerotiorum (MTCC 8785). The use of AgNPs as antibacterial and antifungal agent is advantageous over other methods for control of pathogenic microorganisms, and it can be of great importance in developing novel drugs for curing many lethal diseases.     

黄芩提取物/PBS复合材料的界面作用及其性能

从天然植物黄芩(SBG)中提取有效成分(SBGE),并与可生物降解材料聚丁二酸丁二醇酯(PBS)复合。为提高两者相容性,合成了2种PBS改性产物:PBS—OH和PBS—COOH。探讨了SBGE/PBS复合材料的界面作用,研究了SBGE对不同PBS基体性能的影响。结果表明:—OH和—COOH的引入改善了基体与亲水性提取物的相容性,加强了相互间的氢键作用,增强了界面结合强度。SBGE的添加对PBS的晶型没有影响。1%SBGE/PBS、3%SBGE/PBS—OH和1%SBGE/PBS—COOH热性能最优,且均高于基体本身;7%SBGE/PBS、3%SBGE/PBS—OH和1%SBGE/PBS—COOH的力学性能最佳;抗菌性能的变化均证明了相互作用的存在。SBGE/PBS—OH和SBGE/PBS—COOH的综合性能优于SBGE/PBS。     

Quantitative analysis of liquids from aerosols and microdrops using laser induced breakdown spectroscopy

Laser induced breakdown spectroscopy (LIBS) is shown to be capable of low volume (90 pL) quantitative elemental analysis of picogram amounts of dissolved metals in solutions. Single-pulse and collinear double-pulse LIBS were investigated using a 532 nm dual head laser coupled to a spectrometer with an intensified charge coupled device (CCD) detector. Aerosols were produced using a micronebulizer, conditioned inside a concentric spray chamber, and released through an injector tube with a diameter of 1 mm such that a LIBS plasma could be formed ~2 mm from the exit of the tube. The emissions from both the aerosols and a single microdrop were then collected with a broadband high resolution spectrometer. Multielement calibration solutions were prepared, and continuing calibration verification (CCV) standards were analyzed for both aerosol and microdrop systems to calculate the precision, accuracy, and limits of detection for each system. The calibration curves produced correlation coefficients with R(2) values > 0.99 for both systems. The precision, accuracy, and limit of detection (LOD) determined for aerosol LIBS were averaged and determined for the emission lines of Sr II (421.55 nm), Mg II (279.80 nm), Ba II (493.41 nm), and Ca II (396.84 nm) to be ~3.8% RSD, 3.1% bias, 0.7 μg/mL, respectively. A microdrop dispenser was used to deliver single drops containing 90 pL into the space where a LIBS plasma was generated with a focused laser pulse. In the single drop microdrop LIBS experiment, the analysis of a single drop, containing a total mass of 45 pg, resulted in a precision of 13% RSD and a bias of 1% for the Al I (394.40 nm) emission line. The absolute limits of detection of single drop microdrop LIBS for the emission lines Al I (394.40 nm) and Sr II (421.5 nm) were approximately 1 pg, and Ba II (493.41 nm) produced an absolute detection limit of approximately 3 pg. Overall, the precision, accuracy, and absolute LOD determined for single microdrop LIBS resulted in a typical performance of ~14% RSD, 6% bias, and 1 pg for the elements Sr II (421.55 nm), Al I(394.40 nm), Mg II (279.80), and Ba II(493.41 nm).     

Green synthesis,characterisation and biological evaluation of AgNPs using Agave americana,Mentha spicata and Mangifera indica aqueous leaves extract

The current study was performed to synthesize stable, eco‐friendly and bio‐compatible silver nano‐particles (AgNPs) of Agave americana, Mentha spicata and Mangifera indica leaves and to screen them for biological activities. The ultraviolet‐visible spectroscopic analysis revealed that λ‐max for AgNPs range from 350–500 nm. All AgNPs possessed polycrystalline structure as notified as intense graphical peaks in complete spectrum of 20 values ranging from 10–80° in X‐ray diffraction measurements and supported by scanning electron microscopy data. The size of the nano‐particles was confirmed by transmission electron microscopy (30–150 nm). Mass loss at variable temperatures was evaluated by simultaneous thermogravimetric and differential thermal analysis revealed reduction in mass and activity of compounds was notified by temperature increase from 200 to 800 °C, thus concluding it as thermally sensitive compounds. A. americana AgNPs showed significant (96%) activity against Methicillin resistant Staphylococcus aureus, Escherichia coli (95%) and Fusarium oxysporum (89%). Good antioxidant activity was shown by M. spicata AgNPs at 300 µl (79%). M. indica AgNPs showed significant phytotoxic activity (88%) at highest concentration. No haemagglutination reaction was observed for the test samples. The above results revealed that AgNPs synthesized from selected plant species possesses significant antimicrobial and phytotoxic effect.Inspec keywords: silver, nanoparticles, nanofabrication, X‐ray diffraction, transmission electron microscopy, scanning electron microscopy, differential thermal analysis, microorganisms, antibacterial activity, nanomedicine, particle size, toxicologyOther keywords: green synthesis, biological evaluation, Agave americana aqueous leave extract, Mentha spicata aqueous leave extract, Mangifera indica aqueous leave extract, stable ecofriendly biocompatible silver nanoparticles, ultraviolet‐visible spectroscopy, polycrystalline structure, X‐ray diffraction, scanning electron microscopy, nanoparticle size, transmission electron microscopy, thermogravimetric analysis, differential thermal analysis, mass loss, thermally sensitive compounds, Methicillin resistant Staphylococcus aureus, Escherichia coli, Fusarium oxysporum, antioxidant activity, phytotoxic activity, plant species, antimicrobial effect, temperature 200 degC to 800 degC, Ag     

Effect of polymers and media type on extending the dissolution of amorphous pioglitazone and inhibiting the recrystallization from a supersaturated state

Amorphous forms of crystalline drug are widely utilized for bioavailability enhancement of low solubility drugs in the pharmaceutical industry. Polymers have been found to be effective crystallization inhibitors for amorphous forms in solid states during storage or in liquid states during dissolution process. The dissolution and crystallization behaviors of these amorphous forms in the presence or absence of polymers are still far from adequately understood especially in different dissolution environments. The objective of this study was to investigate the effects of polymers and media type on extending the dissolution of amorphous pioglitazone and inhibiting the recrystallization from a supersaturated state. Polyvinylpyrrolidone K30 (PVPK30), polyvinylpyrrolidone K90 (PVPK90), polyethylene glycol 6000 (PEG6000), polyethylene-polypropylene glycol 188 (F-68), hydroxypropylmethylcellulose (HPMC) and beta-cyclodextrin (β-CD) were employed to understand these behaviors changes because these polymers were used widely. Three solutions including neutral water and phosphate buffer solutions (PBS, pH6.8 and pH7.4) were adopted as dissolution media to determine the behaviors changes comprehensively. In the presence of polymers, dissolution and solubility were extended to different degrees in three media. Polymers can delay the crystallization routes dependently of the medium type. Buffer salts in media reduced the dissolution and accelerated the crystallization process. Crystallization inhibition of these polymers was strongly dependent on the type and pH of media. HPMC displayed the strongest crystallization inhibition effects, resulting in the greatest degree of maintaining a supersaturated state that can sustain most effectively for biologically relevant timeframes.