IADR Abstract Archives
Mechanism of Gold-Titanates Action on S. Mutans
Objectives: To examine effects of gold-titanate on various S. mutans mRNA expressions that are relate to different bacterial gene functions.
Methods: S. mutans were grown aerobically overnight. Low, medium, and high concentrations (10, 200, and 400 mg/L) of nMST-Au(III) were tested. Untreated bacteria were used as controls. S. mutans overnight cultures (108 CFUs/mL) were exposed to nMST-Au(III) at time=0 and samples were collected from each culture at 6 hand 10 h. Bacterial samples were centrifuged, washed and bacterial RNA were extracted by using TRIzol® Max™ Bacterial RNA Isolation Kit according to manufacturer’s protocol. Complementary DNA (cDNA) was prepared by standard procedures.
QRT-PCR was conducted using an aliquot of total 15 ng cDNA with SYBR® Select Master Mix for CFX and CFX96 Touch™ Real-Time PCR Detection System. The reactions were set up in 96-well plates with a total volume of 20 μl containing 10 μl of 2X SYBR Green Select Mix, 1 μL of 0.3 μg cDNA, 1 μL of each 10 μM primer, and 7 μL RNase-free water. The amplification was run under the following conditions: 50 °C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 59-64 °C (depending on primer sets for each gene) for 60 s. Melt-curve analysis was done at the end to verify that the detected signal was that of the expected amplification product and not possible primer-dimers. For each gene, QRT-PCR was performed in triplicate and normalized to the 16s rRNA reference gene. The relative fold changes in mRNA expression related to untreated group were analyzed by using the 2 -ΔΔCt Method. All experiments were repeated three times independently.
Results: Four genes evaluated, the two components signal transduction systems (CiaH), controlling growth/ protein repair (htrA2), peroxidase resistance protein (dpr), and Sortase A showed similar response to gold-titanate NPs. High and medium concentrations (200 and 400 mg/L) of gold-titanate NPs are likely to up regulated all four gene expressions at both 6 and 10 h, while no significant difference of relative fold change was observed in group of low concentration when compared to control.
Conclusions: Genes playing roles in ATP synthase, ribosomal protein, repressor of sugar transport, ABC transporter and heat shock protein were slightly changed by nMST-Au(III).
Methods: S. mutans were grown aerobically overnight. Low, medium, and high concentrations (10, 200, and 400 mg/L) of nMST-Au(III) were tested. Untreated bacteria were used as controls. S. mutans overnight cultures (108 CFUs/mL) were exposed to nMST-Au(III) at time=0 and samples were collected from each culture at 6 hand 10 h. Bacterial samples were centrifuged, washed and bacterial RNA were extracted by using TRIzol® Max™ Bacterial RNA Isolation Kit according to manufacturer’s protocol. Complementary DNA (cDNA) was prepared by standard procedures.
QRT-PCR was conducted using an aliquot of total 15 ng cDNA with SYBR® Select Master Mix for CFX and CFX96 Touch™ Real-Time PCR Detection System. The reactions were set up in 96-well plates with a total volume of 20 μl containing 10 μl of 2X SYBR Green Select Mix, 1 μL of 0.3 μg cDNA, 1 μL of each 10 μM primer, and 7 μL RNase-free water. The amplification was run under the following conditions: 50 °C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 59-64 °C (depending on primer sets for each gene) for 60 s. Melt-curve analysis was done at the end to verify that the detected signal was that of the expected amplification product and not possible primer-dimers. For each gene, QRT-PCR was performed in triplicate and normalized to the 16s rRNA reference gene. The relative fold changes in mRNA expression related to untreated group were analyzed by using the 2 -ΔΔCt Method. All experiments were repeated three times independently.
Results: Four genes evaluated, the two components signal transduction systems (CiaH), controlling growth/ protein repair (htrA2), peroxidase resistance protein (dpr), and Sortase A showed similar response to gold-titanate NPs. High and medium concentrations (200 and 400 mg/L) of gold-titanate NPs are likely to up regulated all four gene expressions at both 6 and 10 h, while no significant difference of relative fold change was observed in group of low concentration when compared to control.
Conclusions: Genes playing roles in ATP synthase, ribosomal protein, repressor of sugar transport, ABC transporter and heat shock protein were slightly changed by nMST-Au(III).
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