Low Concentrations Of Silver Nanoparticles Research Articles

Growth of New, Optically Active, Semi-Organic Single Crystals Glycine-Copper Sulphate Doped by Silver Nanoparticles

The purpose of this study is to modify all physicochemical properties of glycine–copper sulphate single crystals, such as crystal habits, molar mass, thermal stability, optical activity, and electrical properties. The novelty of this study is growth of glycine–copper sulphate single crystals doped by a low concentration of silver nanoparticles (SNPs) that improved both crystal habits and physicochemical properties. The originality of this work is that trace amounts of SNPs largely increased the crystal size. Crystals have molar stoichiometric formula [glycine]0.95, [CuSO4·5H2O]0.05 in the absence and presence of silver nanoparticles (SNPs) in different concentrations: 10 ppm, 20 ppm, and 30 ppm. The crystals’ names and abbreviations are: glycine–copper sulphate (GCS), glycine–copper sulphate doped by 10 ppm SNPs (GCSN1), glycine–copper sulphate doped by 20 ppm SNPs (GCSN2), and glycine–copper sulphate doped by 30 ppm SNPs (GCSN3). Dopant silver nanoparticles increased: crystallinity reflecting purity, transparency to UV-Vis. electromagnetic radiation, thermal stability, and melting point of glycine–copper sulphate single crystal. GCSN3 is a super conductor. High thermal conductivity of crystals ranging from 1.1 W·min−1·K−1 to 1.6 W·min−1·K−1 enabled attenuation of electromagnetic radiation and rapid heat dissipation due to good dielectric and polar properties. On rising temperature, AC electrical conductivity and dielectric properties of perfect crystal GCSN3 increased confirmed attenuation of thermal infrared radiation.

Specific study examining silver nanoparticle-induced adaptable physiological signal changes in Phanerochaete chrysosporium in a tetracycline hydrochloride-abundant system

Numerous studies have documented the toxicity of AgNPs to cells; however, few studies have attempted to explore the intrinsic mechanistic relationships between fungal cells and contaminants, particularly in the context of changes in physiological signaling in the presence of AgNPs. Therefore, we investigated the response of the model white-rot fungal species Phanerochaete chrysosporium (P. chrysosporium) to different concentrations of AgNPs in a tetracycline hydrochloride environment. The results revealed that tetracycline hydrochloride (TCH) enhanced the detoxification of P. chrysosporium at low concentrations of silver nanoparticles (AgNPs). In the TCH-abundant system, a relatively low concentration (1.0 μM) of AgNPs activated the antioxidant stress response of P. chrysosporium, enhanced the expression of H+-ATPase, K+-ATPase, and Ca2+-ATPase, increased the flow of cellular mobility signals (H+, K+, Ca2+, and O2 fluxes), and elevated cellular activity for detoxification. Higher concentrations (50.0 μM) of AgNPs inhibited cellular responses in regard to ATPase activity and mobile signals and induced high levels of reactive oxygen species (ROS), ultimately leading to cell death. The effect of AgNPs on P. chrysosporium genes in the TCH-abundant system was also investigated, and significant expression of genes related to transmembrane transport, stress response, and substance and energy metabolism was observed. This study examining the adaptive physiological response of P. chrysosporium to AgNPs in an antibiotic environment provides a new understanding of the intrinsic interaction between this fungus and contaminants.

The antimicrobial effect of 400nm femtosecond laser and silver nanoparticles on gram-positive and gram-negative bacteria.

Silver nanoparticles are well-known for their antimicrobial effect. However, they are potentially toxic in high doses. We explored the possibility of enhancing the bactericidal effect of low concentrations of silver nanoparticles with blue light femtosecond laser irradiation, since such concentrations are less toxic. The growth dynamics of Pseudomonas aeruginosa, Listeria monocytogenes and methicillin-resistant Staphylococcus aureus grown in pre-synthesized silver nanoparticles were measured with or without pre-irradiation with 50mW and 400nm femtosecond laser irradiation. With each bacterium, combined treatment with laser and silver nanoparticles significantly reduced bacterial growth, indicating that this form of treatment could be beneficial in the ongoing efforts to reduce the deleterious effects of antibiotic resistant Gram-positive and Gram-negative bacteria. The combined treatment was more antimicrobial than treatment with silver nanoparticles alone or photo-irradiation alone. P. aeruginosa and L. monocytogenes were more susceptible to the bactericidal effects of silver nanoparticles, and the combination of laser treatment and silver nanoparticles than MRSA.

Performance improvement of Si solar cell via down - Conversion and plasmonic processes using Eu3+ doped TeO2-GeO2-PbO glasses with silver nanoparticles as cover layer

We report Si solar cell efficiency enhancement covering it with a layer of Eu3+ doped TeO2–GeO2 - PbO glasses with silver nanoparticles. The glass samples were prepared using the well-known melt quenching technique and thermally treated to nucleate metallic particles by thermal reduction. Different concentrations of Eu2O3 and AgNO3 were investigated and the influence of the down -conversion process and the plasmonic effects of silver nanoparticles on Si solar cell performance was demonstrated. The best performance of the photovoltaic device was reached when it was covered with samples that exhibit the highest luminescence combined with considerable transmittance in the visible range. We highlight the best results in which the efficiency of Si solar cell was increased in 11.42% and 11.81%, covering it with samples doped with 1.0% and 5.0% of Eu2O3 containing low concentration of silver nanoparticles, respectively. On the other hand, the effect of large concentration of silver nanoparticles was explored. In this case, quenching of the luminescence promoted the decreasing of Si solar cell performance.

Silver Nanoparticles Conjugated with Contact Lens Solutions May Reduce the Risk of Acanthamoeba Keratitis.

Acanthamoeba keratitis (AK), a severe sight-threatening corneal infection, has become a significant medical problem, especially among contact lens wearers. The disease manifests as eye pain, congestion, blurred vision, lachrymation, and ring-shaped infiltrates of the cornea, and can lead to permanent blindness. Inappropriate habits of contact lens users may result in an increased risk of AK infection. The anti-amoebic efficiency of popular multipurpose contact lens solutions is insufficient to reduce this risk. An effective and non-toxic therapy against AK has not yet been developed. The prevention of AK is crucial to reduce the number of AK infections. Nanoparticles are known to be active agents against bacteria, viruses, and fungi and were also recently tested against protozoa, including Acanthamoeba spp. In our previous studies, we proved the anti-amoebic and anti-adhesive activity of silver nanoparticles against Acanthamoeba castellanii. The aim of this study is to evaluate the activity, cytotoxicity, and anti-adhesive properties of silver nanoparticles conjugated with five commonly used multipurpose contact lens solutions against the Acanthamoeba castellanii NEFF strain. The obtained results show a significant increase in anti-amoebic activity, without increasing the overall cytotoxicity, of Solo Care Aqua and Opti Free conjugated with nanoparticles. The adhesion of Acanthamoeba trophozoites to the contact lens surface is also significantly reduced. We conclude that low concentrations of silver nanoparticles can be used as an ingredient in contact lens solutions to decrease the risk of Acanthamoeba keratitis infection.

Impacts of Continuous Inflow of Low Concentrations of Silver Nanoparticles on Biological Performance and Microbial Communities of Aerobic Heterotrophic Wastewater Biofilm.

Attached-growth wastewater processes are currently used in water resource recovery facilities (WRRFs) for required upgrades due to an increase in influent loading or to reach more stringent discharge criteria. Yet, the distribution and long-term inhibitory effects of silver nanoparticles (AgNPs) in attached-growth biological wastewater processes and their impact on involved microbial communities are poorly understood at relevant, low concentrations. Retention, distribution, and long-term inhibitory effect of polyvinylpyrrolidone (PVP)-coated AgNPs were evaluated in bench-scale moving bed biofilm reactors (MBBRs), achieving soluble organic matter removal, over a 64 day exposure to nominal concentrations of 10 and 100 μg/L. Distributions of continuously added AgNPs were characterized in the influent, bioreactor, and effluent of MBBRs using single particle inductively coupled plasma mass spectroscopy (spICP-MS). Aerobic heterotrophic biofilms in MBBRs demonstrated limited retention capacity for AgNPs over long-term exposure, with release of AgNPs, and Ag-rich biofilm sloughed from the carriers. Continuous exposure to both influent AgNP concentrations significantly decreased soluble chemical oxygen demand (SCOD) removal efficiency (11% to 31%) and reduced biofilm viability (8% to 30%). Specific activities of both intracellular dehydrogenase (DHA) and extracellular α-glucosidase (α-Glu) and protease (PRO) enzymes were significantly inhibited (8% to 39%) with an observed NP dose-dependent intracellular reactive oxygen species (ROS) production and shift in biofilm microbial community composition by day 64. Our results indicated that long-term exposure to AgNPs in biofilm processes at environmentally relevant concentrations can impact the treatment process stability and the quality of the discharged effluent.

Antibacterial effects and cytotoxicity of an adhesive containing low concentration of silver nanoparticles

ObjectivesTo evaluate the antibacterial effects, cytotoxicity and microtensile bond strength of an adhesive containing low concentrations of silver nanoparticles (NAg). MethodsVarious concentrations of NAg (50, 100, 150, 200 and 250 ppm) were incorporated into the primer of the Scotchbond Multi-Purpose adhesive system (SBMP). Antibacterial activity was examined using a broth microdilution assay to determine minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), agar diffusion assay and the MTT assay was used to examine the biofilm metabolic activity (S. mutans). The Microtensile Bond Test (μTBS) was performed after 24 h, followed by 6-months storage in distilled water. Cytotoxicity was assessed with an MTT reduction assay in human dental pulp stem cells viability after exposure to Nag-conditioned culture media during 0, 24, 48, and 72 h. The results were statistically analyzed (α ≤ 0.05). ResultsMIC was found between NAg 25 and 50 ppm MBC was determined at 50 ppm of NAg. Bacterial activity inhibition was higher than control in all NAg groups compared to control in agar diffusion assay. Biofilm inhibition was statistically higher in 250 ppm NAg than control. All NAg groups and SBMP presented similar cytotoxicity in each period. Adhesives with NAg 200 and 250 ppm and SBMP (control) presented the highest μTBS values, similar to that of SBMP control, in both instances (24 h and 6 months) (p > 0.05). ConclusionsThe commercial primer containing NAg 250 ppm showed both antibacterial effect and reliable bond strength with no cytotoxicity increase. The addition of NAg to primers seems promising for the improvement of conventional dental adhesives efficacy.Clinical Significance: The addition of low concentrations of NAg (250 ppm) to primers were effective to improve antibacterial effect preserving the bond strength and the biocompatibility of the commercial product. NAg/primer association could protect the tooth-adhesive interface increasing dental restoration longevity.

Newborn Neurons Are Damaged In Vitro by a Low Concentration of Silver Nanoparticles Through the Inflammatory Oxidative Stress Pathway.

With increasing applications of nanomaterials, including silver nanoparticles (AgNPs), unknown potential risks are present against humans and the environment, especially to the fetus and neonates, which are more sensitive to the cytotoxicity of such agents. This study focused on the effects of AgNP exposure on newborn neurons differentiated from neural stem cells (NSCs) in vitro. We isolated NSCs from fetal rat hippocampus and incubated them in neural differentiation medium for 3-7 days to form newborn neurons and networks. After exposure to 2 μg/mL AgNPs, cell viability was reduced, and early neuronal processes and extensions were fragmented. Furthermore, AgNP treatment increased cellular superoxide dismutase activity and decreased the mitochondrial membrane potential, leading to neuronal death. AgNPs also increased the expression of FOXO3 and decreased nuclear factor-erythroid 2-related factor-2, as well as stimulated the formation of autophagosomes. Therefore, even a low concentration of AgNPs can interrupt early neuronal processes, and facilitate neuron apoptosis by increased cellular oxidative stress and mitochondrial disruption. Thus, it is necessary to note the daily exposure of nanomaterials (e.g., AgNPs) to pregnant women and infants, which may cause neurodevelopmental disorders.

The Effects of Low Concentrations of Silver Nanoparticles on Wheat Growth, Seed Quality, and Soil Microbial Communities

The growing demand for and production of commercial silver nanoparticles (AgNPs) inevitably increases the risk for their environmental release and soil accumulation, which could have deleterious effects on plant growth and soil microorganism communities. However, to date, little is known about how AgNPs impact plant growth, seed quality, and soil microbial communities. We therefore evaluated wheat growth and seed quality after exposure to low concentration of AgNPs while characterizing the composition of the associated soil microbial community by high-throughput sequencing of 16S rRNA genes. Our results showed that low concentration of AgNPs (1 mg/kg in fresh soil) neither inhibited wheat seedling growth nor changed the amino acid content in wheat seeds. Interestingly, the soil microorganisms in the wheat-planted group had more diversity and richness than those in the bulk-soil group. The structure of the bacterial community was affected by AgNP exposure, most significantly during the transition from the seedling to the vegetative stage of the wheat, but recovered to normal level after 49 days of treatment. In conclusion, the results from this study highlight that the environmental risks associated with low concentration of AgNPs, which have clear bioeffects on soil microorganisms, warrant further investigation.

Low concentrations of silver nanoparticles have a beneficial effect on wound healing in vitro

Silver has been used in medical application for its antibacterial, antifungal, and anti-inflammatory effects. Silver nanoparticles (AgNPs) are currently in the spotlight. It was shown that their application can be useful in the management of wounds. Our study was conducted to determine whether AgNPs (average size 10.43 ± 4.74 nm) and ionic silver (Ag-I) could affect the wound healing in the in vitro model of normal human dermal fibroblasts (NHDF). We evaluated their effect on reactive oxygen species (ROS) generation and the expression of key transcription factors that coordinate the cellular response to oxidative stress [nuclear factor (erythroid-derived 2)-like 2 (Nrf2)] and inflammation [nuclear factor-κB (NF-κB)], expression of heme oxygenase-1 (HO-1), and interleukin-6 (IL-6) level. Isolated primary NHDF were scratched, heated (1 h; 42 °C), and cultured with AgNPs (0.25, 2.5, and 25 μg/ml) and Ag-I (0.025, 0.1, and 0.25 μg/ml) for 8 or 24 h. The ROS generation, Nrf2, NF-κB, and HO-1 protein expression and IL-6 protein level were then evaluated by standard methods. Non-cytotoxic concentrations of AgNPs (0.25 and 2.5 μg/ml) did not affect the ROS generation but activated the Nrf2/HO-1 pathway and decreased the NF-κB expression and IL-6 level in the in vitro wound healing model. AgNPs at concentrations of 0.25 and 2.5 μg/ml seem to be suitable for the intended application as a topical agent for wound healing, although the gene silencing technique, chemical inhibitors, and detailed time- and concentration-dependent experiments are needed for a comprehensive study of signaling pathway regulation. Further investigation is also necessary to exclude any possible adverse effects.

Synthesis and Characterization of Composite Hydroxyapatite-Silver Nanoparticles

Hydroxyapatite (HAp) is commonly used as bone implant coating recently; however, the material has disadvantage such as lack of antibacterial properties, that can cause an bacterial infection. Addition of silver nanoparticles is expected to be able to provide antibacterial properties. Silver nanoparticles was obtained by reduction of AgNO3 using glucose monohydrate with microwave heating at 100p for 4 minutes. The composite of hydroxyapatite-silver nanoparticles was synthesized using chemical methods by coprecipitation suspension of Ca(OH)2 with (NH4)HPO4, followed by adding silver nanoparticles solution. The size of the synthesized silver nanoparticles was 30-50 nm and exhibited good antibacterial activity. Nevertheless, when it was composited with HAp to form HAp-AgNPs, there was no antibacterial activity due to very low concentration of silver nanoparticles. This was indicated by the absence of silver nanoparticles diffraction patterns. Infrared spectra indicated the presence of chemical shift and the results of scanning electron microscope showed size of the HAp-AgNPs composite was smaller than that of the HAp. This showed the interaction between HAp and the silver nanoparticles.

Soil microbial community responses to contamination with silver, aluminium oxide and silicon dioxide nanoparticles.

Soil microorganisms are key contributors to nutrient cycling and are essential for the maintenance of healthy soils and sustainable agriculture. Although the antimicrobial effects of a broad range of nanoparticulate substances have been characterised in vitro, little is known about the impact of these compounds on microbial communities in environments such as soil. In this study, the effect of three widely used nanoparticulates (silver, silicon dioxide and aluminium oxide) on bacterial and fungal communities in an agricultural pastureland soil was examined in a microcosm-based experiment using a combination of enzyme analysis, molecular fingerprinting and amplicon sequencing. A relatively low concentration of silver nanoparticles (AgNPs) significantly reduced total soil dehydrogenase and urease activity, while Al2O3 and SiO2 nanoparticles had no effect. Amplicon sequencing revealed substantial shifts in bacterial community composition in soils amended with AgNPs, with significant decreases in the relative abundance of Acidobacteria and Verrucomicrobia and an increase in Proteobacteria. In particular, the relative abundance of the Proteobacterial genus Dyella significantly increased in AgNP amended soil. The effects of Al2O3 and SiO2 NPs on bacterial community composition were less pronounced. AgNPs significantly reduced bacterial and archaeal amoA gene abundance in soil, with the archaea more susceptible than bacteria. AgNPs also significantly impacted soil fungal community structure, while Al2O3 and SiO2 NPs had no effect. Several fungal ribotypes increased in soil amended with AgNPs, compared to control soil. This study highlights the need to consider the effects of individual nanoparticles on soil microbial communities when assessing their environmental impact.

Biosynthesized Silver Nanoparticles Used in Preservative Solutions forChrysanthemumcv. Puma

The use of pulse solutions containing antimicrobials has been reported, but more research is necessary. To increase vase life and to study their effect on opening inflorescences, silver nanoparticles were used in vase solutions for cv. PumaChrysanthemumstems. The nanoparticles were synthesized biologically usingChenopodium ambrosioidesL. applied at concentrations of 0.01, 0.05, 0.1, 0.5, 1, and 5 mM and compared with a control. Treatments were replicated five times. The stems were cut to 50 cm and observed until the end of their vase life. Low concentrations of silver nanoparticles promoted inflorescence opening and leaf yellowing, while the control leaves remained green, but there was a lower degree of inflorescence opening. High concentrations of silver nanoparticles (0.5, 1, and 5 mM) caused senescence due to low water uptake through the stems. Statistical differences in inflorescence opening and diameter, bacterial growth (CFU mL−1) in vase solutions, fresh weight, water uptake, and vase life were found among treatments. Longer vase life and less weight loss were observed in the stems exposed to low concentrations of silver nanoparticles. Low concentrations of silver nanoparticles promoted inflorescence opening and increased vase life ofChrysanthemumcv. Puma.

Silver nanoparticles as active ingredient used for alcohol-free mouthwash.

We developed an effective and non-irritant mouthwash that is alcohol-free and has a low concentration of silver nanoparticles (SNP) in order to be used for preventing oral cavity infections in immunocompromised oncologic patients. We studied antimicrobial effects of silver nanoparticles (SNP) in the range of (50–0.024 µg/ml) and 3% of ethanol (30,000 µg/ml) in mouthwash. Antimicrobial effects of two treatments were studied by doing challenge test on microorganisms such as Streptococcus mutans, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans and measuring MIC and MBC (MFC) values of SNP toward mentioned microorganisms. These values of SNP respectively were in the range of (0.78–3.12) and (1.56–12.5 µg/ml). Results showed that SNP in the MIC and the lower concentrations killed all of the used microorganisms. No difference was observed between the antimicrobial effect of ethanol-free mouthwash containing SNP and mouthwash containing SNP and ethanol (30,000 µg/ml). SNP has high antimicrobial effects at low concentrations and it can be a good alternative for ethanol (30,000 µg/ml) because ethanol is also irritating, especially to sensitive or inflamed mucosa.

Low Concentrations of Silver Nanoparticles in Biosolids Cause Adverse Ecosystem Responses under Realistic Field Scenario

A large fraction of engineered nanomaterials in consumer and commercial products will reach natural ecosystems. To date, research on the biological impacts of environmental nanomaterial exposures has largely focused on high-concentration exposures in mechanistic lab studies with single strains of model organisms. These results are difficult to extrapolate to ecosystems, where exposures will likely be at low-concentrations and which are inhabited by a diversity of organisms. Here we show adverse responses of plants and microorganisms in a replicated long-term terrestrial mesocosm field experiment following a single low dose of silver nanoparticles (0.14 mg Ag kg−1 soil) applied via a likely route of exposure, sewage biosolid application. While total aboveground plant biomass did not differ between treatments receiving biosolids, one plant species, Microstegium vimeneum, had 32 % less biomass in the Slurry+AgNP treatment relative to the Slurry only treatment. Microorganisms were also affected by AgNP treatment, which gave a significantly different community composition of bacteria in the Slurry+AgNPs as opposed to the Slurry treatment one day after addition as analyzed by T-RFLP analysis of 16S-rRNA genes. After eight days, N2O flux was 4.5 fold higher in the Slurry+AgNPs treatment than the Slurry treatment. After fifty days, community composition and N2O flux of the Slurry+AgNPs treatment converged with the Slurry. However, the soil microbial extracellular enzymes leucine amino peptidase and phosphatase had 52 and 27% lower activities, respectively, while microbial biomass was 35% lower than the Slurry. We also show that the magnitude of these responses was in all cases as large as or larger than the positive control, AgNO3, added at 4-fold the Ag concentration of the silver nanoparticles.

Low concentration of silver nanoparticles not only enhances the activity of horseradish peroxidase but alter the structure also.

Chemical synthesis of Ag-NPs was carried out using reduction method. The reduction mechanistic approach of silver ions was found to be a basic clue for the formation of the Ag-NPs. The nanoparticles were characterized by UV-vis, FT-IR and TEM analysis. We had designed some experiments in support of our hypothesis, “low concentrations of novel nanoparticles (silver and gold) increases the activity of plant peroxidases and alter their structure also”, we had used Ag-NPs and HRP as models. The immobilization/interaction experiment had demonstrated the specific concentration range of the Ag-NPs and within this range, an increase in HRP activity was reported. At 0.08 mM concentration of Ag-NPs, 50% increase in the activity yield was found. The U.V-vis spectra had demonstrated the increase in the absorbance of HRP within the reported concentration range (0.06–0.12 mM). Above and below this concentration range there was a decrease in the activity of HRP. The results that we had found from the fluorescence spectra were also in favor of our hypothesis. There was a maximum increase in ellipticity and α-helix contents in the presence of 0.08 mM concentration of Ag-NPs, demonstrated by circular dichroism (CD) spectra. Finally, incubation of a plant peroxidase, HRP with Ag-NPs, within the reported concentration range not only enhances the activity but also alter the structure.

Antibacterial activity and toxicity of silver – nanosilver versus ionic silver

The in vitro study of antibacterial activity of silver nanoparticles (NPs), prepared via modified Tollens process, revealed high antibacterial activity even at very low concentrations around several units of mg/L. These concentrations are comparable with concentrations of ionic silver revealing same antibacterial effect. However, such low concentrations of silver NPs did not show acute cytotoxicity to mammalian cells - this occurs at concentrations higher than 60 mg/L of silver, while the cytotoxic level of ionic silver is much more lower (approx. 1 mg/L). Moreover, the silver NPs exhibit lower acute ecotoxicity against the eukaryotic organisms such as Paramecium caudatum, Monoraphidium sp. and D. melanogaster. The silver NPs are toxic to these organisms at the concentrations higher than 30 mg/L of silver. On contrary, ionic silver retains its cytoxicity and ecotoxicity even at the concentration equal to 1 mg/L. The performed experiments demonstrate significantly lower toxicity of silver NPs against the eukaryotic organisms than against the prokaryotic organisms.

Effects of Colloidal Silver Nanoparticles on Sclerotium-Forming Phytopathogenic Fungi

Effects of silver nanoparticles on the phytopathogenic fungal growth were investigated. Fungal phytopathogens, especially for sclerotium-forming species Rhizoctonia solani, Sclerotinia sclerotiorum and S. minor, were selected due to their important roles in survival and disease cycle. Tests for the fungal hyphal growth revealed that silver nanoparticles remarkably inhibit the hyphal growth in a dose-dependent manner. Different antimicrobial efficiency of the silver nanoparticle was observed among the fungi on their hyphal growth in the following order, R. solani > S. sclerotiorum > S. minor. Tests for the sclerotial germination growth revealed that the nanoparticles showed significant inhibition effectiveness. In particular, the sclerotial germination growth of S. sclerotiorum was most effectively inhibited at low concentrations of silver nanoparticles. A microscopic observation revealed that hyphae exposed to silver nanoparticles were severely damaged, resulting in the separation of layers of hyphal wall and collapse of hyphae. This study suggests the possibility to use silver nanoparticles as an alternative to pesticides for scleotium-forming phytopathogenic fungal controls.