No abstract

Linaridins are members of the ribosomally synthesized and post-translationally modified peptide (RiPP) family of natural products. Five linaridins have been reported, which are defined by the presence of dehydrobutyrine, a dehydrated threonine residue. This work describes the development of a linaridinspecific scoring module for Rapid ORF Description and Evaluation Online (RODEO), a genome-mining tool tailored towards RiPP discovery. Upon mining publicly accessible genomes available in the NCBI database, RODEO identified 561 (382 non-redundant) linaridin biosynthetic gene clusters (BGCs). Linaridin BGCs with unique gene architectures and precursor sequences markedly different from previous predictions were uncovered during these efforts. To aid in dataset validation, two new linaridins, pegvadin A and B, were detected through reactivity-based screening (RBS) and isolated from Streptomyces noursei and Streptomyces auratus, respectively. RBS involves the use of a reactive chemical probe that chemoselectively modifies a functional group present in the natural product. The dehydrated amino acids present in linaridins as a/b-unsaturated carbonyls were appropriate electrophiles for nucleophilic 1,4addition using a thiol-functionalized probe. The data presented within significantly expands the number of predicted linaridin BGCs and serves as a road map for future work in the area. The combination of bioinformatics and RBS is a powerful approach to accelerate natural product discovery.

D-Amino acid-containing peptides (DAACPs) make up a class of post-translationally modified peptides in animals that play important roles as cell-to-cell signaling molecules. Despite the functional importance of L-to D-residue isomerization, little is known about its prevalence, mostly due to difficulties associated with detecting differences in peptide stereochemistry. Prior efforts to discover DAACPs have been largely focused on pursuing peptides based on homology to known DAACPs or DAACP-encoding precursors. Here, we used a combination of enzymatic screening, mass spectrometry, and chromatographic analysis to identify novel DAACPs in the central nervous system (CNS) of Aplysia californica. We identified five new DAACPs from the pleurin precursor and three DAACPs from previously uncharacterized proteins. In addition, two peptides from the pleurin precursor, Plrn2 and Plrn3, exist as DAACPs with the D-residue found at position 2 or 3. These differentially modified forms of Plrn2 and Plrn3 are located in specific regions of the animal's CNS. Plrn2 and Plrn3 appear to be the first animal DAACPs in which the D-residue is found at more than one position, and this suggests that L-to D-residue isomerization may be a more variable/dynamic modification than previously thought. Overall, this study demonstrates the utility of nontargeted DAACP discovery approaches for identifying new DAACPs and demonstrates that isomerization is prevalent throughout the CNS of A. californica.

Single cell analysis strives to probe molecular heterogeneity in morphologically similar cell populations through quantitative or qualitative measurements of genetic, proteomic, or metabolic products. Here, we applied mass analysis of single neurons to investigate cell–cell signaling peptides. The multiplicity of endogenous cell–cell signaling peptides is a common source of chemical diversity among cell populations. Certain peptides can undergo post-translational isomerization of select residues, which has important physiological consequences. The limited number of single cell analysis techniques that are sensitive to peptide stereochemistry make it challenging to study isomerization at the individual cell level. We performed capillary electrophoresis (CE) with mass spectrometry (MS) detection to characterize the peptide content of single cells. Using complementary trapped ion mobility spectrometry (TIMS) separations, we measured the stereochemical configurations of three neuropeptide gene products derived from the pleurin precursor in individual neurons (N = 3) isolated from the central nervous system of Aplysia californica. An analysis of the resultant mobility profiles indicated >98% of the detectable pleurin-derived peptides exist as the nonisomerized, all-l forms in individual neuron cell bodies. However, we observed 44% of the Plrn2 peptide from the pleurin precursor was present as the isomerized, d-residue-containing form in the nerve tissue. These findings demonstrate an unusual distribution of isomerized peptides in A. californica and establish CE–TIMS MS as a powerful analytical tool for investigating peptide stereochemistry at the single cell level.

In a clinical study we tested the following parameters: free fatty acids, beta-hydroxybutyrate, acid-base-balance, lactate, bilirubin, uric-acid, fructose, xylitole, glucose in blood and urine. The tests were executed in 9 patients who were undergoing stomac operations. The cardio pulmonary system of all patients was normal, and there was a homeostasis in water and electrolytes preoperatively. In combination with the amino-acids we received a ratio of 1:1:1 for glucose, levulose and xylitole. Totally, the patients received 0.36 g per kg body weight and per hour of carbohydrates. Beta-hydroxybutyrate, aceto-acetat, and free fatty acids show normal values under conditions of parenteral nutrition as well as lactate, uric acid, and acid-base-balance. The ratio of the different carbohydrates in serum and urine prove that the infusion time and volume were extremely favourable. The loss of carbohydrates in urine was very low.