Undoped and Mn2+ doped CoAl2O4 (MnxCo1-xAl2O4; x = 0.0 to 1.0) spinel nanoparticles were successfully synthesized by a microwave heating method using glycine as the fuel. X-ray powder diffraction (XRD) was confirmed the cubic spinel structure. The average crystallite size of the samples was found to be in the range of 16.46 nm to 20.25 nm calculated by Scherrer's formula. The nano-sized particle-like morphology of the samples was confirmed by high resolution scanning electron microscopy (HR-SEM) and transmission electron microscopy (HR-TEM) analysis. Energy dispersive X-ray (EDX) results showed the pure form of spinel aluminate structure. The band gap energy (Eg) of pure CoAl2O4 was estimated to be 3.68 eV from UV-Visible diffuse reflectance spectroscopy (DRS), and the Eg values increased with increase of Mn2+ ions, due to the smaller grain size. The magnetic hysteresis (M-H) loop showed the superparamagnetic nature, and the magnetization and coercivity values increased with increasing Mn2+ ions, which was confirmed by vibrating sample magnetometer (VSM). All compositions of the nano-catalysts were tested as catalyst successfully for the conversion of benzyl alcohol into benzaldehyde and observed good catalytic activity.
Ni-doped cobalt aluminate NixCo1-xAl2O4 (x = 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0) spinel nanoparticles were successfully synthesized by a simple microwave combustion method using urea as the fuel and as well as reducing agent. X-ray powder diffraction (XRD) was confirmed the formation of single phase, cubic spinel cobalt-nickel aluminate structure without any other impurities. Average crystallite sizes of the samples were found to be in the range of 18.93 nm to 21.47 nm by Scherrer's formula. Fourier transform infrared (FT-IR) spectral analysis was confirmed the corresponding functional groups of the M-O, Al-O and M-Al-O (M = Co and Ni) bonds of spinel NixCo1-xAl2O4 structure. Scanning electron microscope (SEM) and transmission electron microscope (TEM) images was confirmed the particle like nanostructured morphology. Energy band gap (Eg) value was calculated using UV-Visible diffuse reflectance spectra (DRS) and the Eg values increased with increasing Ni2+ dopant from x = 0.2 (3.58 eV) to x = 1.0 (4.15 eV). Vibrating sample magnetometer (VSM) measurements exposed that undoped and Ni-doped CoAl2O4 samples have superparamagnetic behavior and the magnetization (Ms) values were increased with increasing Ni2+ ions. Spinel NixCo1-xAl2O4 samples has been used for the catalytic oxidation of benzyl alcohol into benzaldehyde and was found that the sample Ni0.6Co0.4Al2O4 showed higher conversion 94.37% with 100% selectivity than other samples, which may be due to the smaller particle size and higher surface area.
Genetic engineering is a powerful tool for the improvement of plant traits. Despite reported successes in the plant kingdom, this technology has barely scratched the surface of the Melastomataceae family. Limited studies have led to some optimisation of parameters known to affect the transformation efficiency of these plants. The major finding of this study was to optimise the presence of selected enhancers [e.g., monosaccharides (D-glucose, D-galactose and D-fructose), tyrosine, aluminium chloride (AICI3) and ascorbic
acid] to improve the transformation efficiency of Tibouchina semidecandra. Agrobacterium tumefaciens strain LBA4404 harbouring the disarmed plasmid pCAMBIA1304 was used to transform shoots and nodes of T. semidecandra. Different concentrations of the transformation enhancers were tested by using green fluorescent protein (GFP) as a reporter. The results obtained were based on the percentage of GFP expression, which was observed 14 days post-transformation. A combination of 120 µM galactose and 100
µM tyrosine supplemented with 600 µM AICI3 in the presence of 15 mg/l ascorbic acid gave the highest percentage of positive transformants for T. semidecandra shoots. Whereas 60 µM galactose and 50 µM tyrosine with 200 µM AICI3 in the presence of 15 mg/l ascorbic acid was optimum for T. semidecandra nodes. The presence of the hygromycin phosphotransferase II (hptII) transgene in the genomic DNA of putative
T. semidecandra transformants was verified by PCR amplification with specific primers.
Haemorrhagic septicaemia (HS) is a major disease in cattle and buffaloes, caused by certain serotypes of Pasteurella multocida, mainly B and E serotypes. Frequent HS outbreak has a major impact in many Asian countries, including Malaysia, where farmers encounter economic loss due to low milk production as well as death of their livestock. There are four types of vaccines available; broth bacterins, alum precipitated vaccine, aluminium hydroxide gel vaccine and oil adjuvant vaccine (OAV), but these vaccines can only provide short term immunity and therefore need to be administered annually. Hence, the development of a protein vaccine using recombinant antigen can be a potential candidate for the production of HS vaccine that would give longer immunity. We have successfully cloned the ABA392 gene fragment into a protein expression vector, pET-30a. The protein was expressed from our ABA392/pET30a clone and the immunogenicity of the protein has been tested on rats. This vaccine was able to trigger an immune response and therefore has the potential to be tested as suitable vaccine candidate in future studies. It is envisaged that this subunit vaccine will make a significant contribution in the management of HS among livestock in future.
In current work, the nutritional composition, bioactive compounds, total phenolic contents and anti-oxidant activity of young Malaysian ginger rhizome were investigated. Proximate analysis and high performance liquid chromatography (HPLC) recruited to determine nutritional composition and bioactive compounds. The total flavonoid (TF) and total phenolic contents (TPC) of ginger rhizome were determined by aluminium chloride calorimetric assay and Folin-Ciocalteau reagent, respectively. 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method were used to measure antioxidant capacity. The rhizome contained high moisture content and low level of carbohydrate and energy. 6-gingerol was the most abundant component in the selected ginger, and total flavonoid and phenolic content were reported to be 3.66±0.45 mg gallic acid/g and 10.22±0.87 mg quercetin/g of dry weight of rhizome, respectively. The rhizome also showed lower antioxidant activity than controls, with the IC 50 value of 46.5 vs. 15.5 for α-tocopherol and 22 for BHT. The results of this study predicted that the young ginger rhizome originated from Malaysia may exhibit anti-oxidative and anti-inflammatory potentials due to high levels of gingerols, total flavonoid and phenolic compounds and antioxidant capacity.
Water and ethanolic extracts of four Malaysian local herbs, Tenggek burung (Melicope Iunu-ankenda), Kesum (Polygonum minus), Curry leave (Murraya Koenigii) and Salam (Eugenia polyantha) were investigated for their total phenolic content (TPC), total flavonoids content (TFC) and antioxidant activities (AA). Total phenolic content (TPC) of the herbs was determined using Folin-Ciocalteu reagent assay while the total flavonoid content (TFC) was determined based on aluminium chloride-flavonoid assay. The determination of AA was done using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activitiy and β-carotene bleaching assays (BCB). Different extraction solvents significantly affected the TPC, TFC and AA of all herbs studied (p < 0.05). Both Tenggek burung and Kesum showed highest TPC, TFC and AA regardless of extraction solvents compared to Curry leave and Salam. All herbs showed strong positive correlation between TPC and DPPH assay. However, negative and low correlation between TFC and AA were obtained for all herbs studied. This showed that phenolic compounds of certain structures were responsible for the AA of all the herbs in this study. In conclusion, all herbs in this study except curry leave could be inexpensive sources of good natural antioxidants with nutraceutical potential in food industry.
The main objective of a root end filling material is to provide an apical seal that prevents the movement of bacteria and the diffusion of bacterial products from the root canal system into periapical tissues. The aim of this study was to compare the microleakage of three root end filling materials Mineral trioxide aggregate (MTA), Glass ionomer cement (GIC) and Silver GIC (Miracle Mix) using dye penetration technique under stereomicroscope. Forty-five extracted human maxillary central incisors were instrumented and obturated with gutta percha using lateral compaction technique. Following this, the teeth were stored in saline. After one week, teeth were apically resected at an angle of 90ï° to the long axis of the root and root end cavities were prepared. The teeth were divided into three groups of fifteen specimens each and were filled with Group I -MTA, Group II - GIC and Group III - Miracle Mix. The samples were coated with varnish and after drying, they were immersed in 1% methylene blue dye for 72 hours. The teeth were then rinsed, sectioned longitudinally and observed under stereomicroscope. The depth of dye penetration was measured in millimeters. Microleakage was found to be significantly less in MTA (0.83 mm) when compared to GIC (1.32 mm) (p < 0.001) and with Miracle Mix (1.39 mm) (p < 0.001) No significant difference was found when microleakage in Miracle Mix was compared to that of GIC (p = 0.752). Thus we concluded that MTA is a better material as root end filling material to prevent microleakage, in comparison to GIC and Miracle Mix.
The land area of Tanah Putih, Gua Musang, Kelantan (Malaysia) is well-known for its wealth in industrial mineral resources, especially aluminosilicate of feldspar and mica. Natural feldspar and mica were physicochemically characterized with regard to X-ray diffraction (XRD), nitrogen sorption analysis and transmission electron microscopy (TEM) techniques for qualitative and quantitative identification of feldspar and mica. They show a good crystallinity, high surface area and uniformity of mesoporous structures. For the purpose of this experiment, the aluminosilicate of feldspar was modified either by acid treatment, or grafting the silanol groups present with various functional groups including aminopropyl-, octyl-, vinyl-, mercapto- and glycidoxy-triethoxysilanes, or activation of pre-treated support with glutaraldehyde. These support derivatives were used for further utilization in the immobilization of lipase from Candida rugosa and resulted in various interaction mechanisms between enzyme and introduced supports. It seemed that the features of the functionalized feldspar surfaces provide a preferable environmental host to enable the adsorption of lipase via interfacial adsorption method. Lipase immobilization onto feldspar support were further confirmed by scanning electron microscopy (SEM) coupled with energy dispersive X-ray microanalysis (EDX), transmission electron microscopy (TEM) and infra-red spectroscopy (FTIR) techniques. Enhancement of protein loading (up to 8.22 mg protein/g support) and immobilization yield (up to 78%) were shown by modified feldspar-lipase derivatives compared to unmodified feldspar support.
In this present experimental study, geopolymer cement is developed using high calcium fly ash and used in the production of one-part alkali-activated binders. At 8-16 percent of the total precursor materials, the HCFA was activated with anhydrous sodium metasilicate powder and cured in ambient condition. Five mixtures of one-part geopolymer paste were intended at a steady w/b proportion. Density, flowability, setting time, compressive strength, splitting tensile strength and molar ratio impact were envisaged. It was observed that the setting time of the designed one-part geopolymer paste decreases with higher activator content. The experimental findings showed that the resistance of one-part geopolymer cement paste increases with comparatively greater activator content. However, raising the granular activator beyond 12 percent by fly ash weight decreases the strength and workability of the established one-part geopolymer cement. The optimum mix by weight of the fly ash was discovered to be 12 percent (i.e. 6 percent Na2O). At 28 days of curing, one-part alkali-activated paste recorded the greatest compressive strength of almost 50 MPa. The density of the one-part geopolymer paste is nearly the same regardless of the mixes. Microstructural assessment by FESEM, FTIR and XRD has shown that the established geopolymer paste includes quartz, pyrrhotite, aluminosilicate sodium and hydrate gels of calcium aluminosilicate. Based on the experimental information acquired, it can be deduced that the strength growth of one-part geopolymer cement is similar to that of Portland cement.
Due to the increase in price of petroleum and environmental concerns, the search for alternative fuels has gained importance. In this work, biodiesel production by transesterification of palm oil with methanol has been studied in a heterogeneous system using sodium hydroxide loaded on alumina (NaOH/Al2O3). NaOH/Al2O3 catalyst was prepared by impregnation of alumina with different amount of an aqueous solution of sodium hydroxide followed by calcination in air for 3 h. The prepared catalysts were then characterized by using x-ray diffraction (XRD), Fourier transform infrared spectrometer (FT-IR), Brunner-Emmett-Teller surface area measurement (BET), scanning electron microscopy (SEM) and temperature-programmed desorption of CO2 (CO2-TPD). Moreover, the dependence of the conversion of palm oil on the reactions variables such as the molar ratio of methanol/oil, the amount of catalysts used, reaction temperatures and reaction times were performed. The conversion of 99% was achieved under the optimum reaction conditions. The biodiesel obtained was characterized by FT-IR and the pour point was measured.
Coal combustion and the disposal of combustion wastes emit enormous quantities of nano-sized particles that pose significant health concerns on exposure, particularly in unindustrialized countries. Samples of fresh and weathered class F fly ash were analysed through various techniques including X-ray fluorescence (XRF), X-ray diffraction (XRD), focused ion beam scanning electron microscopy (FIB-SEM), field-emission gun scanning electron microscopy (FE-SEM), high-resolution transmission electron microscopy (HR-TEM) coupled with energy dispersive x-ray spectroscopy (EDS), and Raman Spectroscopy. The imaging techniques showed that the fresh and weathered coal fly ash nanoparticles (CFA-NPs) are mostly spherical shaped. The crystalline phases detected were quartz, mullite, ettringite, calcite, maghemite, hematite, gypsum, magnetite, clay residues, and sulphides. The most abundant crystalline phases were quartz mixed with Al-Fe-Si-K-Ti-O-amorphous phases whereas mullite was detected in several amorphous phases of Al, Fe, Ca, Si, O, K, Mg, Mn, and P. The analyses revealed that CFA-NPs are 5-500 nm in diameter and encapsulate several potentially hazardous elements (PHEs). The carbon species were detected as 5-50 nm carbon nanoballs of graphitic layers and massive fullerenes. Lastly, the aspects of health risks related to exposure to some detected ambient nanoparticles are also discussed.
Catalytic hydrogenation of carbon dioxide (CO2) to methanol is an attractive way to recycle and utilize CO2. A series of Cu/ZnO/Al2O3/ZrO2 catalysts (CZAZ) containing different molar ratios of Cu/Zn were prepared by the co-precipitation method. The catalysts were characterized by temperature-programmed reduction (TPR), field emission scanning electron microscopy-energy dispersive x-ray analysis (FESEM-EDX) and X-ray diffraction (XRD). Higher surface area, SABET values (42.6-59.9 m2/g) were recorded at low (1) and high (5) Cu/Zn ratios with the minimum value of 35.71 m2/g was found for a Cu/Zn of 3. The reducibility of the metal oxides formed after calcination of catalyst samples was also affected due to change in metal-support interaction. At a reaction temperature of 443 K, total gas pressure of 3.0 MPa and 0.1 g/mL of the CZAZ catalyst, the selectivity to methanol decreased as the Cu/Zn molar ratio increased, and the maximum selectivity of 93.9 was achieved at Cu/Zn molar ratio of 0.33. With a reaction time of 3h, the best performing catalyst was CZAZ75 with Cu/Zn molar ratio of 5 giving methanol yield of 6.4%.
In all-ceramic systems, a high incidence of veneer chip-off has been reported in clinical studies. Coefficient of thermal expansion (CTE) behaviour is one of the factors that may increase residual stress in the interface and influence the veneer/core bond strength. Therefore, this study aimed to evaluate the effect of sodalite zeolite-infiltration on the CTE behaviour and bond strength of different all-ceramic prostheses. The case-study groups were synthesized sodalite zeolite-infiltrated alumina (IA-SOD) and synthesized sodalite zeolite-infiltrated zirconia-toughened alumina (ZTA) (IZ-SOD), while the control groups were glass-infiltrated alumina (IA-glass) and glass-infiltrated ZTA (IZ-glass). Forty cylindrical-shaped samples measuring 5 mm in diameter and 10 mm in height were tested for CTE using a thermo-mechanical analyser machine, and forty disc-shaped ceramic samples measuring 12 mm in diameter and 1.2 ± 0.2 mm in thickness were prepared using specially designed stainless steel split mould and veneered by cylinder-shaped (2 mm high × 2 mm diameter) low-fusing porcelain (Vita VM7). The veneer/core samples were sintered and tested for shear bond strength using a high precision universal testing machine. Scanning electron microscope, stereo microscope, atomic force microscope, and energy-dispersive X-ray spectroscopy were used to investigate the structural characteristics of samples at the fracture surface. The collected data were analyzed with a one-way ANOVA and Tukey HSD test (α=.05). IZ-SOD revealed highest CTE and shear bond strength values, while the IA-glass revealed the lowest values than the other groups. There was no significant difference in CTE and bond strength among IZ-SOD, IA-SOD and IZ-glass samples (p>0.05). The experimental SOD zeolite-infiltrated samples revealed higher CTE mismatch and bond strength along with a more favourable mode of failure than did the commercial glass-infiltrated samples. Sandblast technique is considered as effective conditioning procedure for enhancing the surface roughness of SOD zeolite-infiltrated frameworks which subsequently improving the bond strength.
Plastics have been one of the highly valued materials and it plays an significant role in human's life such as in food packaging and biomedical applications. Bioplastic materials can gradually work as a substitute for various materials based on fossil oil. The issue like sustainability and environmental challenges which occur due to manufacturing and disposal of synthetic plastics can be conquering by bio-based plastics. Feathers are among the most inexpensive abundant, and renewable protein sources. Feathers disposal to the landfills leads to environmental pollutions and it results into wastage of 90% of protein raw material. Keratin is non-burning hydrophilic, and biodegradable due to which it can be applicable in various ways via chemical processing. Main objective of this research is to synthesis bioplastic using keratin from chicken feathers. Extracted keratin solution mixed with different concentration of glycerol (2 to 10%) to produce plastic films. The mixture was stirred under constant magnetic stirring at 60 °C for 5 h. The mixtures are then poured into aluminum weighing boat and dried in an oven at 60 °C for 24 h. The mechanical properties of the samples were tested and the physic-chemical properties of the bioplastic were studied. According to the results, Scanning Electron Microscopy test showed good compatible morphologies without holes, cavity and edge. The difference in chemical composition was analyzed using Fourier transform infrared spectroscopy (FTIR). The samples were also characterized by thermo gravimetric analysis (TGA), differential scanning calorimetry (DSC), X-Ray diffraction (XRD) to check the thermal and crystallinity properties. Other than that, bioplastic made up from keratin with 2% of glycerol has the best mechanical and thermal properties. According to biodegradability test, all bioplastic produced are proven biodegradable. Therefore, the results showed possible application of the film as an alternative to fossil oil based materials which are harmful to the environment.
A voltammetric immunosensor was developed for detection of porcine serum albumin (PSA) to identify raw meat products adulterated with pork. A novel strategy to fabricate multiple individual nanoporous alumina (NPA) millirods (length, 5.0 mm; diameter, 1.0 mm) as the biorecognition platform is described. Each NPA millirod was covalently bioconjugated with anti-PSA capturing antibodies (α-PSAC). Following immunocapture, the PSA bound to the α-PSAC/NPA millirod bioconjugate were tagged with gold nanoparticles (AuNPs) functionalized with anti-PSA detection antibodies as the signaling probe. Subsequently, the AuNPs were voltammetrically analyzed to quantify the target PSA. The immunosensor exhibited 100 % specificity and high sensitivity to PSA with a limit of detection (LoD) of 50 (range, 0-1000) pg/mL (R2 = 0.9907). Real-world applicability was successfully validated using pork/beef adulterated mixtures with a LoD of 0.05 % (w/w). Overall, the detection performance of the proposed immunosensor was excellent and, thus, is suitable for surveillance of food safety and quality.
Red mud as industrial waste from bauxite was utilized as a precursor for the synthesis of mesoporous ZSM-5. A high concentration of iron oxide in red mud was successfully removed using alkali fusion treatment. Mesoporous ZSM-5 was synthesized using cetyltrimethylammonium bromide (CTABr) as a template via dual-hydrothermal method, and the effect of crystallization time was investigated towards the formation of mesopores. Characterization using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), N2 adsorption-desorption, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) indicated the formation of cubic crystallite ZSM-5 with high surface area and mesopore volume within 6 h of crystallization. Increasing the crystallization time revealed the evolution of highly crystalline ZSM-5; however, the surface area and mesoporosity were significantly reduced. The effect of mesoporosity was investigated on the adsorption of methylene blue (MB). Kinetic and thermodynamic analysis of MB adsorption on mesoporous ZSM-5 was carried out at a variation of adsorption parameters such as the concentration of MB solution, the temperatures of solution, and the amount of adsorbent. Finally, methanol, 1-butanol, acetone, hydrochloric acid (HCl), and acetonitrile were used as desorbing agents to investigate the reusability and stability of mesoporous ZSM-5 as an adsorbent for MB removal.
Incense sticks ash is one of the most unexplored by-products generated at religious places and houses obtained after the combustion of incense sticks. Every year, tonnes of incense sticks ash is produced at religious places in India which are disposed of into the rivers and water bodies. The presence of heavy metals and high content of alkali metals challenges a potential threat to the living organism after the disposal in the river. The leaching of heavy metals and alkali metals may lead to water pollution. Besides this, incense sticks also have a high amount of calcium, silica, alumina, and ferrous along with traces of rutile and other oxides either in crystalline or amorphous phases. The incense sticks ash, heavy metals, and alkali metals can be extracted by water, mineral acids, and alkali. Ferrous can be extracted by magnetic separation, while calcium by HCl, alumina by sulfuric acid treatment, and silica by strong hydroxides like NaOH. The recovery of such elements by using acids and bases will eliminate their toxic heavy metals at the same time recovering major value-added minerals from it. Here, in the present research work, the effect on the elemental composition, morphology, crystallinity, and size of incense sticks ash particles was observed by extracting ferrous, followed by extraction of calcium by HCl and alumina by H2SO4 at 90-95 °C for 90 min. The final residue was treated with 4 M NaOH, in order to extract leachable silica at 90 °C for 90 min along with continuous stirring. The transformation of various minerals phases and microstructures of incense sticks ash (ISA) and other residues during ferrous, extraction, calcium, and alumina and silica extraction was studied using Fourier transform infrared (FTIR), dynamic light scattering (DLS), X-ray fluorescence (XRF), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), and inductively coupled plasma-optical emission spectroscopy (ICP-OES). DLS was used for analyzing the size during the experiments while FTIR helped in the confirmation of the formation of new products during the treatments. From the various instrumental analyses, it was found that the toxic metals present in the initial incense sticks ash got eliminated. Besides this, the major alkali metals, i.e., Ca and Mg, got reduced during these successive treatments. Initially, there were mainly irregular shaped, micron-sized particles that were dominant in the incense sticks ash particles. Besides this, there were plenty of carbon particles left unburned during combustion. In the final residue, nanosized flowers shaped along with cuboidal micron-sized particles were dominant. present in If, such sequential techniques will be applied by the industries based on recycling of incense sticks ash, then not only the solid waste pollution will be reduced but also numerous value-added minerals like ferrous, silica, alumina calcium oxides and carbonates can be recovered from such waste. The value-added minerals could act as an economical and sustainable source of adsorbent for wastewater treatment in future.
The aim of this study was to assess the effects of four enamel preparation techniques on shear bond strength (SBS) of brackets bonded with a resin-modified glass ionomer cement (RMGIC). Adhesive Remnant Index (ARI) and enamel surface roughness (Ra) were also investigated after cement removal.
The intercalation of perindopril erbumine into Zn/Al-NO(3)-layered double hydroxide resulted in the formation of a host-guest type of material. By virtue of the ion-exchange properties of layered double hydroxide, perindopril erbumine was released in a sustained manner. Therefore, this intercalated material can be used as a controlled-release formulation.
A bioflocculant-producing bacterial strain with highly mucoid and ropy colony morphological characteristics identified as Bacillus spp. UPMB13 was found to be a potential bioflocculant-producing bacterium. The effect of cation dependency, pH tolerance and dosage requirement on flocculating ability of the strain was determined by flocculation assay with kaolin as the suspended particle. The flocculating activity was measured as optical density and by flocs formation. A synergistic effect was observed with the addition of monovalent and divalent cations, namely, Na⁺, Ca²⁺, and Mg²⁺, while Fe²⁺ and Al³⁺ produced inhibiting effects on flocculating activity. Divalent cations were conclusively demonstrated as the best cation source to enhance flocculation. The bioflocculant works in a wide pH range, from 4.0 to 8.0 with significantly different performances (P < 0.05), respectively. It best performs at pH 5.0 and pH 6.0 with flocculating performance of above 90%. A much lower or higher pH would inhibit flocculation. Low dosage requirements were needed for both the cation and bioflocculant, with only an input of 50 mL/L for 0.1% (w/v) CaCl₂ and 5 mL/L for culture broth, respectively. These results are comparable to other bioflocculants produced by various microorganisms with higher dosage requirements.