An Air-Well sparging minifermenter system for high-throughput protein production.

BACKGROUND: Over the last few years High-Throughput Protein Production (HTPP) has played a crucial role for functional proteomics. High-quality, high yield and fast recombinant protein production are critical for new HTPP technologies. Escherichia coli is usually the expression system of choice in protein production thanks to its fast growth, ease of handling and high yields of protein produced. Even though shake-flask cultures are widely used, there is an increasing need for easy to handle, lab scale, high throughput systems. RESULTS: In this article we described a novel minifermenter system suitable for HTPP. The Air-Well minifermenter system is made by a homogeneous air sparging device that includes an air diffusion system, and a stainless steel 96 needle plate integrated with a 96 deep well plate where cultures take place. This system provides aeration to achieve higher optical density growth compared to classical shaking growth without the decrease in pH value and bacterial viability. Moreover the yield of recombinant protein is up to 3-fold higher with a considerable improvement in the amount of full length proteins. CONCLUSIONS: High throughput production of hundreds of proteins in parallel can be obtained sparging air in a continuous and controlled manner. The system used is modular and can be easily modified and scaled up to meet the demands for HTPP.

Determination by ICP-MS and multivariate data analysis of elemental urine excretion profile during the EDTA chelation therapy: A case study.

BACKGROUND: Based on the medical history and laboratory analytical tests, a patient presenting symptoms compatible with Chronic Fatigue Syndrome was suspected of metal intoxication; therefore, a chelating therapy was attempted. In parallel, the profile of elemental excretion in urine was determined. METHODS: Chelation therapy by CaNa2EDTA was administered every two weeks and urine samples were routinely collected for 17 months. The samples were mineralized with HNO3 69 % and analyzed by Inductively-Coupled Plasma - Mass Spectrometry. Data were processed by multivariate statistical methods. RESULTS: Most of the toxic elements showed a peak of excretion in 12-24 h after EDTA administration, which returned to basal level by 24-36 h after the treatment. Yet, the excretion of some trace elements persisted in the urine collected 26 h after the treatment. CONCLUSIONS: The analysis of excreted metals following the CaNa2EDTA infusion allowed to monitor dynamically the chelation therapy. The chelation therapy was effective in mobilizing and eliminating the principal heavy metals present from the body. However, since such clearance almost vanished 24 h after the treatment, a protocol with more frequent and low-dose administrations is advisable to improve the metal excretion.

Monitoring of water quality inflow and outflow of a farm in Italian Padana plain for rice cultivation: a case study of two years.

Rice cultivation requires a large use of pesticides and nutrients to control weed proliferation and improve production. The water quality of four neighboring rice fields located in the Lomellina area (Italian Padana plain) was monitored in this study along with the cultivation period (before, during, and after the period of planting), for two successive agricultural seasons (2015 and 2016). Two paddy fields were traditionally cultivated with wet-seeding and the other fields with dry-seeding. Eighteen sampling points were considered: eight points for surface water, two points for underground water, and eight points for porous cups with two different depths. In order to evaluate the goodness of the paddy field system to maintain unchanged the quality of the inflow with respect to the outflow water, three of the most used herbicides in Italian rice cultivation (imazamox, oxadiazon, and profoxydim) and other physical-chemical parameters were determined, namely biological oxygen demand after 5 days; chemical oxygen demand; total suspended solids; anionic surfactants; total hardness; total amount of phosphorus, nitrogen, and potassium; and heavy metal concentrations. In general, all the collected data confirmed that paddy fields did not contribute to worsen the environmental pollution. The different flooding techniques adopted in the fields did not highlight significant differences in concentrations of pesticides or metals. The pesticides reached their maximum concentration (of the magnitude order of few ng mL-1) on the day after the administration and on the day after the application in the adjacent field. A slight reduction of total As in grain was obtained adopting a dry period from steam elongation up to booting. From the collected data, it was possible to identify a general water flow direction in the paddy fields from north-west to south-east: this prevailing flow direction was useful to understand not only the diffusion of the pesticides and their degradation products in the fields but also that of the nutrients. Concerning nutrients, it was important not to activate a recirculation of the water in the field during the first 10 days from the administration, in order to avoid loss of nitrogen in the water vents or for percolation. Moreover, the monitoring of potassium concentration allowed to avoid the use of unnecessary potassic fertilization when there was already a high amount of this element in the paddy field derived from irrigation. However, all the investigated water quality parameters were under the limits fixed by the European regulation. In addition, the presence of seven unexpected compounds was identified by the nontarget approach in both campaigns in samples collected in the early summer period. Four of these emerging contaminants were identified as N,N-diethyl-meta-toluamide, tricyclazole, amidosulfuron, and one of the imazamox photodegradation products. Although the obtained low concentrations of oxadiazion, tricyclazole, and arsenic, in particular, justified a preexisting contamination of the water inflow or of the investigated paddy area, the obtained results supported the good quality of the paddy water outflow, confirming the rational use of the water resource and the correct use of agronomic practices.

Fast classification of hazelnut cultivars through portable infrared spectroscopy and chemometrics.

The authentication and traceability of hazelnuts is very important for both the consumer and the food industry, to safeguard the protected varieties and the food quality. This study investigates the use of a portable FTIR spectrometer coupled to multivariate statistical analysis for the classification of raw hazelnuts. The method discriminates hazelnuts from different origins/cultivars based on differences of the signal intensities of their IR spectra. The multivariate classification methods, namely principal component analysis (PCA) followed by linear discriminant analysis (LDA) and partial least square discriminant analysis (PLS-DA), with or without variable selection, allowed a very good discrimination among the groups, with PLS-DA coupled to variable selection providing the best results. Due to the fast analysis, high sensitivity, simplicity and no sample preparation, the proposed analytical methodology could be successfully used to verify the cultivar of hazelnuts, and the analysis can be performed quickly and directly on site.

Chemical profiling and multivariate data fusion methods for the identification of the botanical origin of honey.

The characterization of 72 Italian honey samples from 8 botanical varieties was carried out by a comprehensive approach exploiting data fusion of IR, NIR and Raman spectroscopies, Proton Transfer Reaction - Time of Flight - Mass Spectrometry (PTR-MS) and electronic nose. High-, mid- and low-level data fusion approaches were tested to verify if the combination of several analytical sources can improve the classification ability of honeys from different botanical origins. Classification was performed on the fused data by Partial Least Squares - Discriminant Analysis; a strict validation protocol was used to estimate the predictive performances of the models. The best results were obtained with high-level data fusion combining Raman and NIR spectroscopy and PTR-MS, with classification performances better than those obtained on single analytical sources (accuracy of 99% and 100% on test and training samples respectively). The combination of just three analytical sources assures a limited time of analysis.

Image Pretreatment Tools II: Normalization Techniques for 2-DE and 2-D DIGE.

Gel electrophoresis is usually applied to identify different protein expression profiles in biological samples (e.g., control vs. pathological, control vs. treated). Information about the effect to be investigated (a pathology, a drug, a ripening effect, etc.) is however generally confounded with experimental variability that is quite large in 2-DE and may arise from small variations in the sample preparation, reagents, sample loading, electrophoretic conditions, staining and image acquisition. Obtaining valid quantitative estimates of protein abundances in each map, before the differential analysis, is therefore fundamental to provide robust candidate biomarkers. Normalization procedures are applied to reduce experimental noise and make the images comparable, improving the accuracy of differential analysis. Certainly, they may deeply influence the final results, and to this respect they have to be applied with care. Here, the most widespread normalization procedures are described both for what regards the applications to 2-DE and 2D Difference Gel-electrophoresis (2-D DIGE) maps.

Differential Analysis of 2-D Maps by Pixel-Based Approaches.

Two approaches to the analysis of 2-D maps are available: the first one involves a step of spot detection on each gel image; the second one is based instead on the direct differential analysis of 2-D map images, following a pixel-based procedure. Both approaches strongly depend on the proper alignment of the gel images, but the pixel-based approach allows to solve important drawbacks of the spot-volume procedure, i.e., the problem of missing data and of overlapping spots. However, this approach is quite computationally intensive and requires the use of algorithms able to separate the information (i.e., spot-related information) from the background. Here, the most recent pixel-based approaches are described.