Gliadin proteins from wheat flour: the optimal determination conditions by ELISA

Introduction. The number of people with celiac disease is rapidly increasing. Gluten, is one of the most common food allergens, consists of two fractions: gliadins and glutenins. The research objective was to determine the optimal conditions for estimating gliadins by using enzyme-linked immunosorbent assay (ELISA). Study objects and methods. The experiment involved wheat flour samples (0.10; 0.20, 0.25, 0.50, and 1.0 g) suspended in different solvents (ethanol, methanol, 1-propanol, and isopropanol) of different concentrations (40, 50, 60, 70, 80, and 90% v/v). The samples were diluted with Tris buffer in ratios of 1:50, 1:100, 1:150, and 1:200. The gliadin test was performed using a Gliadin/Gluten Biotech commercial ELISA kit (Immunolab). Results and discussion. The optimal conditions for determining gliadin proteins that provided the highest gliadin concentration were: solvent – 70% v/v ethanol, extract:Tris buffer ratio – 1:50, and sample weight – 1.0 g. Conclusion. The obtained results can be of great importance to determine gliadin/gluten concentrations in food products by rapid analysis methods.

Synthesis of 225Ac-PSMA-617 for preclinical use

Background: The recent approval of radiopharmaceuticals for diagnosis and treatment of cancer is ushering nuclear medicine into a new era of theranostics, and alpha therapy using radiopharmaceuticals labeled with 225Ac is showing remarkable results in clinical trials. As such, reliable methods for the synthesis and quality control of 225Ac-radiopharmaceuticals are needed. Objective: 225Ac-PSMA-617 is being used for targeted alpha therapy in patients with prostate cancer, and we aimed to synthesize the agent for preclinical use. However, technology transfer proved cumbersome owing to the paucity of information available on synthesizing and analyzing 225Ac-radiotherapeutics. To address this need, we describe a straightforward synthesis of 225Ac-PSMA-617 as well as suitable approaches for quality control analysis using standard equipment in a modern PET Center. Methods: PSMA-617 precursor was dissolved in 25 μL metal-free water (0.67 mg/mL), and combined with 500 μL 0.05M Tris buffer, pH 9. Actinium stock solution (~65 μCi in 15 µL) was added and the reaction was heated at 120˚C for 40-50 min. The reaction was cooled and 0.6 mL gentisic acid solution (4 mg/mL in 0.2 M NH4OAc) was added. To formulate the dose for injection, sterile saline, USP (8 mL) was added and the pH was adjusted by addition of 100 μL 0.05 M Tris buffer (pH 9) to achieve a final pH of ~7.2. The final solution was filtered using a 0.22 µm GV sterile filter into a sterile dose vial. Radiochemical purity was determined by radio-TLC (eluent: 50mM Sodium Citrate, pH 5) and plates were analyzed using an AR2000 scanner. Results: The method provided 225Ac-PSMA-617 in high radiochemical yield (57 ± 3 µCi, >99%) and radiochemical purity (98 ± 1%), formulated for preclinical studies (9 mL, pH = 7.2), n=3. Conclusion: A straightforward synthesis of 225Ac-PSMA-617 is described that will facilitate production for (pre)clinical studies. The approach could also be applicable to the synthesis of other alpha radiotherapeutics incorporating 225Ac.

Thermodynamic Origin of Differential Excipient-Lysozyme Interactions

Understanding the intricate interplay of interactions between proteins, excipients, ions and water is important to achieve the effective purification and stable formulation of protein therapeutics. The free energy of lysozyme interacting with two kinds of polyanionic excipients, citrate and tripolyphosphate, together with sodium chloride and TRIS-buffer, are analysed in multiple-walker metadynamics simulations to understand why tripolyphosphate causes lysozyme to precipitate but citrate does not. The resulting multiscale decomposition of energy and entropy components for water, sodium chloride, excipients and lysozyme reveals that lysozyme is more stabilised by the interaction of tripolyphosphate with basic residues. This is accompanied by more sodium ions being released into solution from tripolyphosphate than for citrate, whilst the latter instead has more water molecules released into solution. Even though lysozyme aggregation is not directly probed in this study, these different mechanisms are suspected to drive the cross-linking between lysozyme molecules with vacant basic residues, ultimately leading to precipitation.

A rapidly-dissolving silica-silver bioactive glass for cariostatic applications

In this study, bioactive glasses were designed for the purposes of reducing the incidence of dental caries and lesion formation by supplying the teeth with therapeutic ions which may support remineralization and provide an antibacterial effect against oral cavity bacteria. Three glasses were synthesised through the melt quench method: Si-Control (SiO2-CaO-P2O5-Na2O), Si-02 and Si-05, where 0.2% and 0.5% Ag2O were substituted, respectively, for SiO2 in the control glass. The glasses were then ground, characterized and dissolved in tris buffer solution (pH=7.30) for 6, 12 and 24 hours, with the pH rise of the solution being recorded (7.48 for Si-Control, and 7.66 for both Si-02 and Si-05 after 24 hours) and the ions that were released into the tris buffer solution quantified. Samples of each glass were subsequently embedded into non-fluoridated toothpaste and samples of the paste were used to brush resin-mounted lamb molars after a 1.0M HCl overnight demineralization challenge. Knoop microhardness measurements were recorded before and after brushing to determine the presence of remineralization on the surface of the teeth (Percent Surface Hardness Loss of 37%, 35% and 34% for Si-Control, Si-02 and Si-05 respectively after 24 hours). Four oral cavity bacterial strains were isolated through swabs of the inner cheek, gums and teeth surfaces of three volunteers, and placed on agar discs. 0.5g of each glass were placed onto the discs and the resultant inhibition zones were measured after 6, 12 and 24 hours. Si-05 consistently performed better than Si-02 on all strains and timeframes, while Si-Control exhibited no antibacterial effect at any time point.

A rapidly-dissolving silica-silver bioactive glass for cariostatic applications

In this study, bioactive glasses were designed for the purposes of reducing the incidence of dental caries and lesion formation by supplying the teeth with therapeutic ions which may support remineralization and provide an antibacterial effect against oral cavity bacteria. Three glasses were synthesised through the melt quench method: Si-Control (SiO2-CaO-P2O5-Na2O), Si-02 and Si-05, where 0.2% and 0.5% Ag2O were substituted, respectively, for SiO2 in the control glass. The glasses were then ground, characterized and dissolved in tris buffer solution (pH=7.30) for 6, 12 and 24 hours, with the pH rise of the solution being recorded (7.48 for Si-Control, and 7.66 for both Si-02 and Si-05 after 24 hours) and the ions that were released into the tris buffer solution quantified. Samples of each glass were subsequently embedded into non-fluoridated toothpaste and samples of the paste were used to brush resin-mounted lamb molars after a 1.0M HCl overnight demineralization challenge. Knoop microhardness measurements were recorded before and after brushing to determine the presence of remineralization on the surface of the teeth (Percent Surface Hardness Loss of 37%, 35% and 34% for Si-Control, Si-02 and Si-05 respectively after 24 hours). Four oral cavity bacterial strains were isolated through swabs of the inner cheek, gums and teeth surfaces of three volunteers, and placed on agar discs. 0.5g of each glass were placed onto the discs and the resultant inhibition zones were measured after 6, 12 and 24 hours. Si-05 consistently performed better than Si-02 on all strains and timeframes, while Si-Control exhibited no antibacterial effect at any time point.

First Report of Bacterial Soft Rot Disease on Taro Caused by Dickeya fangzhongdai in China

Taro [Colocasia esculenta (L.) Schott.] is an important root crop in the world with great economic value. In recent years, outbreaks of soft rot were observed on taro plants in several plantation areas located in Shaoguan, Guangdong Province, China (25°7'57" N, 113°19'5" E). Root tubers of taro (Paodan variety) infected by soft rot had water-soaked lesions with a dark brown-black margin including a rotten smell, they also had internal rot that was also found in root tubers with no external symptoms. In some areas, the incidence of soft rot can reach up to 30%. To isolate the causal agent, ten pieces of taro root tubers with typical symptoms were surface-sterilized with 75% ethanol and 0.1% HgCl2 solution and then washed thrice with sterile water. The tuber slices were soaked in 50 ml sterile water and shaken at 28°C, 200 rpm for 2 h, and 100 µl was streaked onto the modified Yeast Extract Beef (YEB) agar medium (1% peptone, 0.5% yeast extract, 0.5% sucrose, 0.5% NaCl, 1 Mmol/L MgSO4•7H2O, 1.5% agar, pH 7.0) plates (Zhou et al. 2011) and incubated at 28°C for 24 h. Single colonies grown on YEB were selected for preliminary inoculation onto healthy taro (Paodan variety) slices. Two of the Gram-negative bacteria, named as ZXC1 and MPC2, developed symptoms consistent in rotted decay inside the root tubers after incubation for 24h at 30°C. ZXC1 and MPC2 were biochemically profiled using a Biolog Gen III MicroPlate (Microlog 3, 5.2) (Shen et al. 2019) and resulted Dickeya sp. (SIM 0.856 and 0.704). To determine the species of the Dickeya isolates, 16S rRNA sequences were amplified by primers 27F and 1492R (Hauben et al. 1998). Housekeeping genes including gyrB, atpD, rpoB, and infB were also amplified using degenerate primers (Brady et al. 2008). Results from the BLASTn analysis of the 16S rRNA (GenBank accession numbers MN853405, MN853406), gyrB (GenBank accession numbers MN866299, MN866303), atpD (GenBank accession numbers MN866298, MN866302), rpoB (GenBank accession numbers MN866301, MN866305), and infB (GenBank accession numbers MN866300, MN866304) genes in the isolates ZXC1 and MPC2 showed 99% identities to those of the previously reported D. fangzhongdai isolates from Phalaenopsis (Zhang et al. 2018). Multilocus sequence analysis (MLSA) by MEGA 7.0 performed with four housekeeping genes (gyrB, atpD, rpoB, infB) showed that they clustered with D. fangzhongdai isolates. Analyses using scanning and transmission electron microscopy showed that ZXC1 and MPC2 bacteria were rod-shaped, 0.5-1.0 μm × 1.0-3.0 µm, with peritrichous flagella. Pathogenicity tests were performed thrice using surface-sterilized 2-month-old taro seedlings (Paodan variety). Six individual seedlings were inoculated using a sterile syringe with ten microliters of bacterial suspension (108 CFU/ml) in Tris buffer (0.1 mol/L Tris and 0.1 mol/L HCl, pH 7.4). Taro seedlings injected with sterile Tris buffer were used as the negative control. These taro seedlings were grown in the greenhouse (30 ± 2°C, 90 ± 5% relative humidity). At the 25th day post inoculation, soft rot symptoms were observed in inoculated taro, while all control taro plants remained symptom-free. Small and pale yellow with irregular margins colonies consistent with morphological characteristics of those of D. fangzhongdai were re-isolated from symptomatic taro tubers and the housekeeping genes presence was verified by sequencing as described above, fulfilling Koch’s postulates. D. fangzhongdai is a newly emerging bacterial pathogen, which causes bleeding cankers in pear trees (Tian et al. 2016), and soft rot of Phalaenopsis (Zhang et al. 2018). This is the first report of D. fangzhongdai causing soft rot disease in taro. Considering the high incidence of soft rot, this pathogen might pose a significant threat to taro and other economically important crops. Therefore, further researches are needed to investigate host range of the pathogen and develop appropriate integrated management to contain this disease spreading.