ABSTRACT
PURPOSE: Tooth eruption is a dynamic process. Appearance of any part of the cusp through gingiva may be a clinical marker of eruption. Early childhood caries (ECC) is a public health problem globally. This study aimed to assess the relationship between parent-reported timing of first tooth emergence and ECC in toddlers. METHODS: This study is a secondary data analysis of 627 toddlers involved in a case-control study on sleep-time feeding practises in children. The children were categorised into four groups based on the parent-reported timing of first primary tooth emergence (G1-when the first primary tooth emerged before 6 months of age, G2-between 7 and 9 months; G3-10 to 12 months and G4-when the first primary tooth emerged after 12 months of age). Univariate binary logistic regression analysis was performed to evaluate the association between timing of first tooth emergence and ECC. RESULTS: The mean age of the children was 24.4 ± 7.3 months (cases, that is children with ECC-25.4 ± 6.9 months, controls, that is children without ECC-23.6 ± 7.5 months). Of 60 children, whose first tooth erupted before 6 months of age, 35 (12%) were cases compared to 25(8%) controls. Amongst the cases, boys had more caries than girls (p < 0.05). Of the anterior teeth, 22% of the emerged teeth were decayed in the first group, followed by 19%, 16% and 10% in the second, third and fourth groups, respectively (p < 0.05). Analysis of the posterior teeth showed a lower percentage of decayed teeth with delayed emergence of the first primary tooth (p < 0.05). Children whose teeth emerged before 6 months of age had an odds ratio of 3.5 (95% CI 1.49, 8.42) (p = 0.004). CONCLUSION: This study concluded that the early emergence of the first primary tooth, as reported by the parent, was associated with an increased risk of developing ECC.
ABSTRACT
Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.
Subject(s)
Antigens, Protozoan/biosynthesis , Gene Expression , Pichia/genetics , Protozoan Proteins/biosynthesis , Ammonium Sulfate/chemistry , Antigens, Protozoan/genetics , Antigens, Protozoan/isolation & purification , Carbon/metabolism , Chromatography, Affinity , Cloning, Molecular , Culture Media/chemistry , Dialysis , Fractional Precipitation , Hydrogen-Ion Concentration , Methanol/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/isolation & purification , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Sensitivity and Specificity , Serologic Tests/methods , Temperature , Toxoplasmosis/diagnosisABSTRACT
Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.
ABSTRACT
GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZα A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the α-factor of yeast so that it could be excreted out of the P. pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen.
