Preparative SDS PAGE as an Alternative to His–Tag Purification of Recombinant Amelogenin

Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens and in the development of therapeutics. Recombinant proteins are typically engineered with “tags” that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralisation researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond

Pseudomonas aeruginosa and the Oropharyngeal Ecosystem of Tube-Fed Patients

We evaluated whether elderly patients fed with nasogastric tubes (NGT) are predisposed to Pseudomonas aeruginosa colonization in the oropharynx. Fifty-three patients on NGT feeding and 50 orally fed controls with similar clinical characteristics were studied. The tongue dorsum was swabbed and cultured. P. aeruginosa was isolated in 18 (34%) of the NGT-fed group but in no controls (p<0.001). Other gram-negative bacteria were cultured from 34 (64%) of NGT-fed patients as compared with 4 (8%) of controls (p<0.001). Antibiotic susceptibility of the oropharyngeal P. aeruginosa isolates was compared with that of isolates from sputum cultures obtained from our hospital’s bacteriologic laboratory. The oropharyngeal isolates showed a higher rate of resistance; differences were significant for amikacin (p<0.03). Scanning electron microscope studies showed a biofilm containing P. aeruginosa organisms. The pulsed-field gel electrophoresis profile of these organisms was similar to that of P. aeruginosa isolates from the oropharynx. NGT-fed patients may serve as vectors of resistant P. aeruginosa strains

The Associations of Maternal and Neonatal Vitamin D with Dental Development in Childhood

Background: Vitamin D influences the formation and mineralization of teeth. Objective: To investigate the association of maternal and neonatal vitamin D concentrations with the dental development of 10-y-old children, in a population-based prospective cohort study among 3,770 mothers and children in the Netherlands. Methods: Maternal venous blood samples were collected in the second trimester (median 20.4 weeks of gestation; range: 18.5-23.2 wk) whereas umbilical cord blood samples were collected immediately after delivery (median 40.1 weeks of gestation; range 35.9-42.3 wk). Dental development was defined using the Demirjian method. Multivariate regression models were built to analyze the studied associations. Results: High concentrations of 25-hydroxyvitamin D [25(OH)D] during midpregnancy (beta: -0.04; 95% CI: -0.08, -0.01) and at birth (beta: -0.06; 95% CI: -0.10, -0.02) were associated with a lower dental age in children. The children of mothers with severe vitamin D deficiency [25(OH)D /=75.0 nmol/L). Children with vitamin D deficiency [25(OH)D 25.0-49.9 nmol/L] at birth exhibited a higher dental age (beta: 0.11; 95% CI: 0.01, 0.20), higher developmental stages of the mandibular second premolar (beta: 0.27; 95% CI: 0.02, 0.51), and higher developmental stages of the mandibular second molar (beta: 0.24; 95% CI: 0.00, 0.48) compared with children with sufficient-to-optimal values of 25(OH)D (>/=50.0 nmol/L) at birth. Conclusion: Higher maternal and neonatal 25(OH)D concentrations are associated with decelerated dental development in childhood. The lower the vitamin D level during midpregnancy or at birth, the higher the dental age of children, and the higher the developmental stages of the mandibular teeth

Caries-preventing effect of a hydroxyapatite-toothpaste in adults: a 18-month double-blinded randomized clinical trial

BackgroundDental caries is a worldwide challenge for public health. The aim of this 18-month double-blinded, randomized, clinical trial was to compare the caries-preventing effect of a fluoride-free, hydroxyapatite toothpaste (test) and a toothpaste with sodium fluoride (1450 ppm fluoride; positive control) in adults.MethodsThe primary endpoint was the percentage of subjects showing no increase in overall Decayed Missing Filled Surfaces (DMFS) index. The study was designed as non-inferiority trial. Non-inferiority was claimed if the upper limit of the exact one-sided 95% confidence interval for the difference of the primary endpoint DMFS between test and control toothpaste was less than the predefined margin of non-inferiority (Δ ≤ 20%).ResultsIn total, 189 adults were included in the intention-to-treat (ITT) analysis; 171 subjects finished the study per protocol (PP). According to the PP analysis, no increase in DMFS index was observed in 89.3% of subjects of the hydroxyapatite group and 87.4% of the subjects of the fluoride group. The hydroxyapatite toothpaste was not statistically inferior to a fluoride toothpaste with regard to the primary endpoint.ConclusionHydroxyapatite was proven to be a safe and efficient anticaries agent in oral care.Clinical trial registrationNCT04756557

Microabrasion in tooth enamel discoloration defects: three cases with long-term follow-ups

Superficial irregularities and certain intrinsic stains on the dental enamel surfaces can be resolved by enamel microabrasion, however, treatment for such defects need to be confined to the outermost regions of the enamel surface. Dental bleaching and resin-based composite repair are also often useful for certain situations for tooth color corrections. This article presented and discussed the indications and limitations of enamel microabrasion treatment. Three case reports treated by enamel microabrasion were also presented after 11, 20 and 23 years of follow-ups

Focus on collagen: in vitro systems to study fibrogenesis and antifibrosis _ state of the art

Fibrosis represents a major global disease burden, yet a potent antifibrotic compound is still not in sight. Part of the explanation for this situation is the difficulties that both academic laboratories and research and development departments in the pharmaceutical industry have been facing in re-enacting the fibrotic process in vitro for screening procedures prior to animal testing. Effective in vitro characterization of antifibrotic compounds has been hampered by cell culture settings that are lacking crucial cofactors or are not holistic representations of the biosynthetic and depositional pathway leading to the formation of an insoluble pericellular collagen matrix. In order to appreciate the task which in vitro screening of antifibrotics is up against, we will first review the fibrotic process by categorizing it into events that are upstream of collagen biosynthesis and the actual biosynthetic and depositional cascade of collagen I. We point out oversights such as the omission of vitamin C, a vital cofactor for the production of stable procollagen molecules, as well as the little known in vitro tardy procollagen processing by collagen C-proteinase/BMP-1, another reason for minimal collagen deposition in cell culture. We review current methods of cell culture and collagen quantitation vis-à-vis the high content options and requirements for normalization against cell number for meaningful data retrieval. Only when collagen has formed a fibrillar matrix that becomes cross-linked, invested with ligands, and can be remodelled and resorbed, the complete picture of fibrogenesis can be reflected in vitro. We show here how this can be achieved. A well thought-out in vitro fibrogenesis system represents the missing link between brute force chemical library screens and rational animal experimentation, thus providing both cost-effectiveness and streamlined procedures towards the development of better antifibrotic drugs