Prediction of type 1 diabetes with machine learning algorithms based on FTIR spectral data in peripheral blood mononuclear cells.

The incidence of autoimmunity is increasing, to ensure timely and comprehensive treatment, there must be a diagnostic method or markers that would be available to the general public. Fourier-transform infrared spectroscopy (FTIR) is a relatively inexpensive and accurate method for determining metabolic fingerprint. The metabolism, molecular composition and function of blood cells vary according to individual physiological and pathological conditions. Thus, by obtaining autoimmune disease-specific metabolic fingerprint markers in peripheral blood mononuclear cells (PBMC) and subsequently using machine learning algorithms, it might be possible to create a tool that will allow the diagnosis of autoimmune diseases. In this preliminary study, it was found that the peak shift at 1545 cm-1 could be considered specific for autoimmune disease type 1 diabetes (T1D), while the shifts at 1070 and 1417 cm-1 could be more attributed to the autoimmune condition per se. The prediction of T1D, despite the small number of participants in the study, showed an inverse AUC = 0.33 ± 0.096, n = 15, indicating a stable trend in the prediction of T1D based on FTIR metabolic fingerprint data in the PBMC.

Kinetic and Stoichiometric Modeling-Based Analysis of Docosahexaenoic Acid (DHA) Production Potential by Crypthecodinium cohnii from Glycerol, Glucose and Ethanol.

Docosahexaenoic acid (DHA) is one of the most important long-chain polyunsaturated fatty acids (LC-PUFAs), with numerous health benefits. Crypthecodinium cohnii, a marine heterotrophic dinoflagellate, is successfully used for the industrial production of DHA because it can accumulate DHA at high concentrations within the cells. Glycerol is an interesting renewable substrate for DHA production since it is a by-product of biodiesel production and other industries, and is globally generated in large quantities. The DHA production potential from glycerol, ethanol and glucose is compared by combining fermentation experiments with the pathway-scale kinetic modeling and constraint-based stoichiometric modeling of C. cohnii metabolism. Glycerol has the slowest biomass growth rate among the tested substrates. This is partially compensated by the highest PUFAs fraction, where DHA is dominant. Mathematical modeling reveals that glycerol has the best experimentally observed carbon transformation rate into biomass, reaching the closest values to the theoretical upper limit. In addition to our observations, the published experimental evidence indicates that crude glycerol is readily consumed by C. cohnii, making glycerol an attractive substrate for DHA production.

Crypthecodinium cohnii Growth and Omega Fatty Acid Production in Mediums Supplemented with Extract from Recycled Biomass.

Crypthecodinium cohnii is a marine heterotrophic dinoflagellate that can accumulate high amounts of omega-3 polyunsaturated fatty acids (PUFAs), and thus has the potential to replace conventional PUFAs production with eco-friendlier technology. So far, C. cohnii cultivation has been mainly carried out with the use of yeast extract (YE) as a nitrogen source. In the present study, alternative carbon and nitrogen sources were studied: the extraction ethanol (EE), remaining after lipid extraction, as a carbon source, and dinoflagellate extract (DE) from recycled algae biomass C. cohnii as a source of carbon, nitrogen, and vitamins. In mediums with glucose and DE, the highest specific biomass growth rate reached a maximum of 1.012 h-1, while the biomass yield from substrate reached 0.601 g·g-1. EE as the carbon source, in comparison to pure ethanol, showed good results in terms of stimulating the biomass growth rate (an 18.5% increase in specific biomass growth rate was observed). DE supplement to the EE-based mediums promoted both the biomass growth (the specific growth rate reached 0.701 h-1) and yield from the substrate (0.234 g·g-1). The FTIR spectroscopy data showed that mediums supplemented with EE or DE promoted the accumulation of PUFAs/docosahexaenoic acid (DHA), when compared to mediums containing glucose and commercial YE.

Syntrophy of Crypthecodinium cohnii and immobilized Zymomonas mobilis for docosahexaenoic acid production from sucrose-containing substrates.

Marine heterotrophic dinoflagellate Crypthecodinium cohnii is an aerobic oleaginous microorganism that accumulates intracellular lipid with high content of 4,7,10,13,16,19-docosahexaenoic acid (DHA), a polyunsaturated ω-3 (22:6) fatty acid with multiple health benefits. C. cohnii can grow on glucose and ethanol, but not on sucrose or fructose. For conversion of sucrose-containing renewables to C. cohnii DHA, we investigated a syntrophic process, involving immobilized cells of ethanologenic bacterium Zymomonas mobilis for fermenting sucrose to ethanol. The non-respiring, NADH dehydrogenase-deficient Z. mobilis strain Zm6-ndh, with high ethanol yield both under anaerobic and aerobic conditions, was taken as the genetic background for inactivation of levansucrase (sacB). SacB mutation eliminated the levan-forming activity on sucrose. The double mutant Zm6-ndh-sacB cells were immobilized in Ca alginate, and applied for syntrophic conversion of sucrose to DHA of C. cohnii, either taking the ethanol-containing fermentation medium from the immobilized Z. mobilis for feeding to the C. cohnii fed-batch culture, or directly coculturing the immobilized Zm6-ndh-sacB with C. cohnii on sucrose. Both modes of cultivation produced C. cohnii CCMP 316 biomass with DHA content around 2-3 % of cell dry weight, corresponding to previously reported results for this strain on glucose.

Purine Auxotrophic Starvation Evokes Phenotype Similar to Stationary Phase Cells in Budding Yeast.

Purine auxotrophy is an abundant trait among eukaryotic parasites and a typical marker for many budding yeast strains. Supplementation with an additional purine source (such as adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We explored purine starvation effects in a model organism, a budding yeast Saccharomyces cerevisiae ade8 knockout, at the level of cellular morphology, central carbon metabolism, and global transcriptome. We observed that purine-starved cells stopped their cycle in G1/G0 state and accumulated trehalose, and the intracellular concentration of AXP decreased, but adenylate charge remained stable. Cells became tolerant to severe environmental stresses. Intracellular RNA concentration decreased, and massive downregulation of ribosomal biosynthesis genes occurred. We proved that the expression of new proteins during purine starvation is critical for cells to attain stress tolerance phenotype Msn2/4p targets are upregulated in purine-starved cells when compared to cells cultivated in purine-rich media. The overall transcriptomic response to purine starvation resembles that of stationary phase cells. Our results demonstrate that the induction of a strong stress resistance phenotype in budding yeast can be caused not only by natural starvation, but also starvation for metabolic intermediates, such as purines.

Spectroscopic and electrochemical study of interactions between DNA and different salts of 1,4-dihydropyridine AV-153.

1,4-dihydropyridines (1,4-DHP) possess important biochemical and pharmacological properties, including antimutagenic and DNA-binding activity. The latter activity was first described for water-soluble 1,4-DHP with carboxylic group in position 4, the sodium salt of the 1,4-DHP derivative AV-153 among others. Some data show the modification of physicochemical properties and biological activities of organic compounds by metal ions that form the salts. We demonstrated the different affinity to DNA and DNA-protecting capacity of AV-153 salts, depending on the salt-forming ion (Na, K, Li, Rb, Ca, Mg). This study aimed to use different approaches to collate data on the DNA-binding mode of AV-153-Na and five other AV-153 salts. All the AV-153 salts in this study quenched the ethidium bromide and DNA complex fluorescence, which points to an intercalation binding mode. For some of them, the intercalation binding was confirmed using cyclic voltammetry and circular dichroism spectroscopy. It was shown that in vitro all AV-153 salts can interact with four DNA bases. The FTIR spectroscopy data showed the interaction of AV-153 salts with both DNA bases and phosphate groups. A preference for base interaction was observed as the AV-153 salts interacted mostly with G and C bases. However, the highest differences were detected in the spectral region assigned to phosphate groups, which might indicate either conformational changes of DNA molecule (B form to A or H form) or partial denaturation of the molecule. According to the UV/VIS spectroscopy data, the salts also interact with the human telomere repeat, both in guanine quadruplex (G4) and single-stranded form; Na and K salts manifested higher affinity to G4, Li and Rb -to single-stranded DNA.

Modeling the mobility of glyphosate from two contrasting agricultural soils in laboratory column experiments.

Glyphosate (GLP) currently is one of the most widely used herbicides worldwide. The persistence of GLP and its major metabolite, aminomethylphosphonic acid (AMPA) in the environment has been described by other authors. This study was aimed at comparing the GLP and AMPA behavior in sandy and loamy sand soils after spiking with enhanced (445 µg g-1) concentrations of GLP in herbicide KLINIK® (Nufarm, Austria) and bioaugmentation followed by 40 days weathering and a consistent three-stage leaching in a laboratory column experiment. Soil samples were obtained from mineral topsoil (0-10 cm) within former agricultural lands where soil parent material was formed by glacigenic deposits. The total amount of GLP and AMPA collected during three leaching stages was significantly (p<.05) higher from columns with sandy soil, compared to loamy sand soil. Bioaugmentation resulted in considerably lower concentrations of AMPA in leachates, especially in the sets with sandy soil (p=.01). Leachates were tested using FTIR spectroscopy and Daphnia magna. Statistical analysis of the changes in Ntot, Ctot, K+, Mg2+, Al3+, Ca2+, Mn2+ and Fe3+ concentrations in soils after the leaching experiment revealed that the loamy sand soil was likely to be more sensitive to the addition of GLP and bioaugmentation than sandy soil.

Miniature diamond-anvil cells for FTIR-microspectroscopy of small quantities of biosamples.

Fourier transform infrared (FTIR) spectroscopy techniques and data analyses have become widely available, are easy to use, and are convenient for studies of various biosamples, especially in biomedical science. Yet, cultivation of cells and purification of cell components are costly, often methodically challenging, and time and labor consuming. Therefore, reduction of the sample amount is of high value. Here we propose a novel method for the analysis of small quantities of biosamples by FTIR-microscopy of dry films using a diamond-anvil cell (DAC). This approach allows us to decrease the sample volume at least a hundred times compared to that for a high-throughput screening device (HTS-XT, Bruker, Germany), while still obtaining homogeneous films, acquiring qualitative spectra, and using a conventional 15× objective instead of an ATR-objective. Both FTIR methods were applied for analyses of human colorectal cancer cell lines SW480 and SW620 cultured under hypoxic conditions to estimate the strengths and weaknesses of each approach. FTIR absorption spectra acquired by both methods were compared and no significant spectral differences were detected. It was shown that FTIR-microscopy of films on the DAC can be used for evaluation, screening, discrimination and identification of biochemical markers in biosamples like cells. We conclude that the DAC can be transferred to other biosamples like tissues, biofluids, their components and extracellular matrix, and is especially valuable when the available quantities of biosamples are limited.

Drying enhances immunoactivity of spent brewer's yeast cell wall ß-D-glucans.

Due to immunological activity, microbial cell wall polysaccharides are defined as 'biological response modifiers' (BRM). Cell walls of spent brewer's yeast also have some BRM activity. However, up to date there is no consensus on the use of spent brewer's yeast D-glucan as specific BRM in humans or animals. The aim of this paper is to demonstrate the potential of spent brewer's yeast ß-D-glucans as BRM, and drying as an efficient pretreatment to increase ß-D-glucan's immunogenic activity. Our results revealed that drying does not change spent brewer's yeast biomass carbohydrate content as well as the chemical structure of purified ß-D-glucan. However, drying increased purified ß-D-glucan TNF-α induction activity in the murine macrophage model. We presume drying pretreatment enhances purity of extracted ß-D-glucan. This is corroborated with FT-IR analyses of the ß-D-glucan spectra. Based on our results, we suggest that dry spent brewer's yeast biomass can be used as a cheap source for high-quality ß-D-glucan extraction. Drying in combination with carboxylmethylation (CM), endows spent brewer's yeast ß-D-glucan with the immunoactivity similar or exceeding that of a well-characterized fungal BRM pleuran.