Impact of neurite alignment on organelle motion

Intracellular transport is pivotal for cell growth and survival. Malfunctions in this process have been associated with devastating neurodegenerative diseases, highlighting the need for a deeper understanding of the mechanisms involved. Here, we use an experimental methodology that leads neurites of differentiated PC12 cells into either one of two configurations: a one-dimensional configuration, where the neurites align along lines, or a two-dimensional configuration, where the neurites adopt a random orientation and shape on a flat substrate. We subsequently monitored the motion of functional organelles, the lysosomes, inside the neurites. Implementing a time-resolved analysis of the mean-squared displacement, we quantitatively characterized distinct motion modes of the lysosomes. Our results indicate that neurite alignment gives rise to faster diffusive and super-diffusive lysosomal motion than the situation in which the neurites are randomly oriented. After inducing lysosome swelling through an osmotic challenge by sucrose, we confirmed the predicted slowdown in diffusive mobility. Surprisingly, we found that the swelling-induced mobility change affected each of the (sub-/super-)diffusive motion modes differently and depended on the alignment configuration of the neurites. Our findings imply that intracellular transport is significantly and robustly dependent on cell morphology, which might in part be controlled by the extracellular matrix.     

Mining the Wheat Grain Proteome

Bread wheat is the most widely cultivated crop worldwide, used in the production of food products and a feed source for animals. Selection tools that can be applied early in the breeding cycle are needed to accelerate genetic gain for increased wheat production while maintaining or improving grain quality if demand from human population growth is to be fulfilled. Proteomics screening assays of wheat flour can assist breeders to select the best performing breeding lines and discard the worst lines. In this study, we optimised a robust LC–MS shotgun quantitative proteomics method to screen thousands of wheat genotypes. Using 6 cultivars and 4 replicates, we tested 3 resuspension ratios (50, 25, and 17 µL/mg), 2 extraction buffers (with urea or guanidine-hydrochloride), 3 sets of proteases (chymotrypsin, Glu-C, and trypsin/Lys-C), and multiple LC settings. Protein identifications by LC–MS/MS were used to select the best parameters. A total 8738 wheat proteins were identified. The best method was validated on an independent set of 96 cultivars and peptides quantities were normalised using sample weights, an internal standard, and quality controls. Data mining tools found particularly useful to explore the flour proteome are presented (UniProt Retrieve/ID mapping tool, KEGG, AgriGO, REVIGO, and Pathway Tools).     

Phenotypic and PCR-based characterization of the microflora in Norvegia cheese during ripening

Microbiological sampling of Norvegia cheese from three cheese factories was done during ripening. The evolution of aerobic mesophilic bacteria, lactococci, lactobacilli, enterococci, presumptive leuconostoc and pediococci was investigated after 30, 90, 180 and 270 days of ripening. Isolates (135) of non-starter lactic acid bacteria (NSLAB) from nine Norvegia cheeses after 90, 180 and 270 days of ripening were examined. The isolates were tested by physiological and biochemical assays, species-specific PCR and 16S rDNA sequencing. After 90 days of ripening Leuconostoc spp., most probably from the starter, and the NSLAB specie Lactobacillus paracasei dominated among the isolates, however, after longer ripening Lb. paracasei dominated. The development and evolution of the microflora in Norvegia varied according to dairy and ripening time.     

Dietary Fish Oil Alters DNA Methylation of Genes Involved in Polyunsaturated Fatty Acid Biosynthesis in Muscle and Liver of Atlantic Salmon (Salmo salar)

Adequate dietary supply of eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) is required to maintain health and growth of Atlantic salmon (Salmo salar). However, salmon can also convert α-linolenic acid (18:3n-3) into eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3) by sequential desaturation and elongation reactions, which can be modified by 20:5n-3 and 22:6n-3 intake. In mammals, dietary 20:5n-3 + 22:6n-3 intake can modify Fads2 expression (Δ6 desaturase) via altered DNA methylation of its promoter. Decreasing dietary fish oil (FO) has been shown to increase Δ5fad expression in salmon liver. However, it is not known whether this is associated with changes in the DNA methylation of genes involved in polyunsaturated fatty acid synthesis. To address this, we investigated whether changing the proportions of dietary FO and vegetable oil altered the DNA methylation of Δ6fad_b, Δ5fad, Elovl2, and Elovl5_b promoters in liver and muscle from Atlantic salmon and whether any changes were associated with mRNA expression. Higher dietary FO content increased the proportions of 20:5n-3 and 22:6n-3 and decreased Δ6fad_b mRNA expression in liver, but there was no effect on Δ5fad, Elovl2, and Elovl5_b expression. There were significant differences between liver and skeletal muscle in the methylation of individual CpG loci in all four genes studied. Methylation of individual Δ6fad_b CpG loci was negatively related to its expression and to proportions of 20:5n-3 and 22:6n-3 in the liver. These findings suggest variations in dietary FO can induce gene-, CpG locus-, and tissue-related changes in DNA methylation in salmon.     

Liquefaction,Saccharification and Isomerization of Starches from Sources Other than Maize

Examination of the suitability of a number of starches for the production of fructose syrup is described. In addition to maize starch, starch from wheat, potatoes and tapioca has been examined. Liquefaction, saccharification and purification were performed in pilot scale, whereas the produced syrups were isomerized in laboratory scale. The liquefactions were performed with Termamyl® in a jet cooker process. Saccharification with Amyloglucosidase Novo 150 L under standard conditions gave good DX values. After purification the syrups were isomerized in laboratory columns with Sweetzyme®. The results - activity and productivity of Sweetzyme - were for all syrups as good as or better than those obtained with dextrose solutions.     

Growth and metabolism of selected strains of probiotic bacteria in milk

Growth and metabolism of five probiotic strains with well-documented health effects were studied in ultra-high temperature (UHT) treated milk, supplemented with 0.5% (w/v) tryptone or 0.75% (w/v) fructose. The probiotic strains were Lactobacillus acidophilus La5, Lb. acidophilus 1748, Lactobacillus rhamnosus GG, Lactobacillus reuteri SD 2112 and Bifidobacterium animalis BB12. Fermentation was followed for 72 h at 37 degrees C and the samples were analysed for pH, log cfu ml(-1), volatile compounds, organic acids and carbon dioxide. The strains reduced pH from 6.7 to between 3.9 and 4.4 after 24 h of incubation. All strains attained viable cell counts above 8.7-9.18 log cfu ml(-1) after 6-16 h of incubation. The two Lb. acidophilus strains showed a stable level of viable cells during 12-72 h of incubation but the three other strains showed a reduction of 0.4-1.1 log cfu ml(-1) from 24 to 72 h of incubation. However, all strains showed cell levels between 7.8 and 8.7 log cfu ml(-1) after 72 h of incubation. After 48 h of incubation, the amount of lactic acid produced varied according to strain from 6949 to 14,000 mg kg(-1) and acetic acid produced varied from 0 to 6901 mg kg(-1). Three of the strains metabolised citrate but only low amounts of diacetyl and acetoin were detected within strains, 0.2-0.8 and 6.5-10 mg kg(-1), respectively. Carbon dioxide produced varied from 221 to 3942 mg kg(-1) and was connected to the citrate-fermenting ability of the strain used and their carbohydrate fermentation pathway. Three of the strains produced detectable levels of acetaldehyde and the concentration varied from 9.4 to 12.6 mg kg(-1) after 24 h of incubation. All five probiotic strains showed very different profiles of metabolites during fermentation; however, the two Lb. acidophilus strains were the most alike. Our findings show the importance of controlling the fermentation time since the probiotic strains produced different amounts of metabolic products according to fermentation time.     

Combination of nuclear magnetic resonance spectroscopy and nonlinear methods to analyze the copolymerization of phosphonic acid derivatives

We demonstrate in this study that the combination of modern inline monitoring methods [here: inline nuclear magnetic resonance (NMR)] with simulations gains more exact and profound kinetic results than previously used methods like linearization without that combination. The ¹H-NMR spectroscopic data (more than 100 data points) are used to construct the copolymerization diagram. The reactivity ratios are obtained applying the van Herks nonlinear least square method. The examination of the radical copolymerization of 2-hydroxyethyl methacrylate (HEMA) with (2-{[2-(ethoxycarbonyl)prop-2-en-1-yl]oxy}ethyl) phosphonic acid (ECPPA) as important adhesive monomer used in dentistry yields reactivity ratios of r_HEMA = 1.83; r_ECPPA = 0.42. The copolymerization diagram reflects nonideal, non-azeotropic copolymerization. The sequence distribution of the obtained by Monte Carlo simulation indicates the generation of statistical copolymers. As an important finding, it is demonstrated that the repeating units responsible for etching and adhesion are arranged over the whole polymer chain, which is necessary to achieve proper functionality. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019 , 136, 48256.