Effects of feed composition,protein denaturation and storage of milk serum protein/lactose powders on lactosylation

During whey powder production, the feed is subjected to several heat treatments which can cause lactosylation of proteins. In this study, lactosylation of whey proteins was evaluated in spray-dried powders before and after storage by varying the native protein fraction as well as the serum protein/lactose ratio in the powders. The lactosylation of native α-lactalbumin and β-lactoglobulin in the powders before storage was not affected to a large extent by the protein denaturation or if the feed had been heat treated in a high or low lactose environment. After storage (relative humidity of 23.5%, 30 °C, 25 days), the kinetic of lactosylation tended to increase with increasing native protein fraction and bulk protein content in the powders. An explanation could be that proteins dissolved in the lactose glassy structure might have a lower reactivity, while proteins present in the protein glassy structure with dissolved lactose may display higher lactosylation reactivity.     

Discovery of GPR183 Agonists Based on an Antagonist Scaffold

The G protein-coupled receptor GPR183/EBI2, which is activated by oxysterols, is a therapeutic target for inflammatory and metabolic diseases where both antagonists and agonists are of potential interest. Using the piperazine diamide core of the known GPR183 antagonist (E)-3-(4-bromophenyl)-1-(4-(4-methoxybenzoyl)piperazin-1-yl)prop-2-en-1-one (NIBR189) as starting point, we identified and sourced 79 structurally related compounds that were commercially available. In vitro screening of this compound collection using a Ca²⁺ mobilization assay resulted in the identification of 10 compounds with agonist properties. To enable establishment of initial structure-activity relationship trends, these were supplemented with five in-house compounds, two of which were also shown to be GPR183 agonists. Taken together, our findings suggest that the agonist activity of this compound series is dictated by the substitution pattern of one of the two distal phenyl rings, which functions as a molecular efficacy-switch.     

Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L⁻¹). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.     

Mixture ratio modeling of dynamic systems

Any knowledge extraction relies (possibly implicitly) on a hypothesis about the modelled-data dependence. The extracted knowledge ultimately serves to a decision-making (DM). DM always faces uncertainty and this makes probabilistic modelling adequate. The inspected black-box modeling deals with “universal” approximators of the relevant probabilistic model. Finite mixtures with components in the exponential family are often exploited. Their attractiveness stems from their flexibility, the cluster interpretability of components and the existence of algorithms for processing high-dimensional data streams. They are even used in dynamic cases with mutually dependent data records while regression and auto-regression mixture components serve to the dependence modeling. These dynamic models, however, mostly assume data-independent component weights, that is, memoryless transitions between dynamic mixture components. Such mixtures are not universal approximators of dynamic probabilistic models. Formally, this follows from the fact that the set of finite probabilistic mixtures is not closed with respect to the conditioning, which is the key estimation and predictive operation. The paper overcomes this drawback by using ratios of finite mixtures as universally approximating dynamic parametric models. The paper motivates them, elaborates their approximate Bayesian recursive estimation and reveals their application potential.     

Effects of breed and age at slaughter on degradation of muscle lipids during processing of dry‐cured hams

Hams from Landrace, Duroc and Hampshire pigs slaughtered at ages 6, 7.5 and 9 months were processed to generate Norwegian Parma‐style hams. Lipid contents and the compositions of fatty acid classes (ΣSFA, ΣMUFA, ΣPUFA) within neutral lipids, phospholipids and free fatty acids were determined. Small differences in lipid degradation and composition of the classes were revealed. However, significant sensory differences related to lipids were observed. Breed was more important than age. Dry‐cured Hampshire hams gave a more intense mature odour that may be associated with higher overall lipid degradation. Unexpectedly, these hams also demonstrated high juiciness and tenderness, which could be related to the melting characteristics of the fat. Dry‐cured Duroc hams showed a higher susceptibility towards rancidity, presumably associated with preferential oxidation of n‐6 fatty acids relative to C18:1 n‐9. Dry‐cured Landrace hams showed the lowest juiciness and tenderness, likely due to their lower fat content (marbling).     

Design and Characterization of a New pVII Combinatorial Phage Display Peptide Library for Protease Substrate Mining Using Factor VII Activating Protease (FSAP) as Model

We describe a novel, easy and efficient combinatorial phage display peptide substrate-mining method to map the substrate specificity of proteases. The peptide library is displayed on the pVII capsid of the M13 bacteriophage, which renders pIII necessary for infectivity and efficient retrieval, in an unmodified state. As capture module, the 3XFLAG was chosen due to its very high binding efficiency to anti-FLAG mAbs and its independency of any post-translational modification. This library was tested with Factor-VII activating protease (WT-FSAP) and its single-nucleotide polymorphism variant Marburg-I (MI)-FSAP. The WT-FSAP results confirmed the previously reported Arg/Lys centered FSAP cleavage site consensus as dominant, as well as reinforcing MI-FSAP as a loss-of-function mutant. Surprisingly, rare substrate clones devoid of basic amino acids were also identified. Indeed one of these peptides was cleaved as free peptide, thus suggesting a broader range of WT-FSAP substrates than previously anticipated.     

Hit to Leads with Cytotoxic Effect in Leukemic Cells: Total Synthesis Intermediates as a Molecule Treasure Chest

A previously designed and developed 12-step total synthesis that includes [1,1′-biphenyl]-2-amine and carbazole intermediates and that ultimately produces the carbazole alkaloid carbazomycin G was exploited as a screening compound library with the goal of identifying potential lead compound(s) with cytotoxic effect. These compounds were investigated by using in-vitro tests involving the two human cell lines HL-60 and MOLM-13, which both model acute myeloid leukaemia (AML). The in-vitro biological test results were used together with the molecular structures of the various intermediates in a concise SAR analysis. Several of the intermediates revealed cytotoxicity (IC₅₀<10⁻⁴ M), although the final natural product carbazomycin G did not reveal cytotoxicity versus the two said human cell lines.