Inflammatory pain resolution by mouse serum-derived small extracellular vesicles.

Chronic pain is a significant public health issue. Current treatments have limited efficacy and significant side effects, warranting research on alternative strategies for pain management. One approach involves using small extracellular vesicles (sEVs) to transport beneficial biomolecular cargo to aid pain resolution. Exosomes are 30-150 nm sEVs that can carry RNAs, proteins, and lipid mediators to recipient cells via circulation. Exosomes can be beneficial or harmful depending on their source and contents. To investigate the short and long-term effects of mouse serum-derived sEVs in pain modulation, sEVs from naïve control or spared nerve injury (SNI) model donor mice were injected intrathecally into naïve recipient mice. Basal mechanical thresholds transiently increased in recipient mice. This effect was mediated by opioid signaling as this outcome was blocked by naltrexone. Mass Spectrometry of sEVs detected endogenous opioid peptide leu-enkephalin. A single prophylactic intrathecal injection of sEVs two weeks prior to induction of the pain model in recipient mice delayed mechanical allodynia in SNI model mice and accelerated recovery from inflammatory pain after complete Freund's adjuvant (CFA) injection. ChipCytometry of spinal cord and dorsal root ganglion (DRG) from sEV treated mice showed that prophylactic sEV treatment reduced the number of natural killer (NK) and NKT cells in spinal cord and increased CD206+ anti-inflammatory macrophages in (DRG) after CFA injection. Further characterization of sEVs showed the presence of immune markers suggesting that sEVs can exert immunomodulatory effects in recipient mice to promote the resolution of inflammatory pain. Collectively, these studies demonstrate multiple mechanisms by which sEVs can attenuate pain.

Bioactive Compounds and Antioxidant Activity in Seeds of Bred Lines of Common Bean Developed from Interspecific Crosses.

Knowledge is limited about the level of bioactive compounds and antioxidant activity of seeds from bred lines of common beans developed from interspecific crosses using four different Phaseolus species (P. vulgaris L., P. coccineus L., P. acutifolius A. Gray. Gray., and P. dumosus). In this study, differences in the nutritional quality of seeds among 112 bean genotypes were evaluated by measuring the levels of phenolic compounds, pigments, antioxidant activity, and sugars. The bean genotypes were grown under high temperatures and acid soil conditions in the Amazon region of Colombia. Five typology groups of bean genotypes were identified based on the level of bioactive compounds and their functional capacity: (1) highly bioactive and functional (HBF); (2) moderately bioactive and functional (MBF); (3) moderate antioxidant content with pigment influence (MACP); (4) moderately antinutritional with limited antioxidant potential (MALAP); and (5) antinutritional, low bioactive, and functional (ALBF). We developed a nutritional quality index (NQI) with values ranging from 0 to 1 based on the nutritional and anti-nutritional balance of each genotype and the higher values of the NQI of a genotype indicating greater nutritional quality. We found three interspecific bred lines (SER 212, SER 213, and RRA 81), with NQI values higher than 0.8. These three lines belong to the typology group of HBF. The superior nutritional quality of these three interspecific bred lines is attributed to a greater level of bioactive compounds and antioxidant capacity. These three bred lines may serve as useful parents to develop nutritionally superior and stress-resilient beans from bean breeding programs. Further research is needed to explore the role of testa color in improving the nutritional quality of seeds of common bean genotypes grown under different climatic conditions.

Antibody titrations are critical for microflow cytometric analysis of extracellular vesicles.

Optimization of flow cytometry assays for extracellular vesicles (EVs) often fail to include appropriate reagent titrations - the most critically antibody titration is either not performed or is incomplete. Using nonoptimal antibody concentration is one of the main sources of error leading to a lack of reproducible data. Antibody titration for the analysis of antigens on the surface of EVs is challenging for a variety of technical reasons. Using platelets as surrogates for cells and platelet-derived particles as surrogates for EV populations, we demonstrate our process for antibody titration, highlighting some of the key analysis parameters that may confound and surprise new researchers moving into the field of EV research. Additional care must be exercised to ensure instrument and reagent controls are utilized appropriately. Complete graphical analysis of positive and negative signal intensities, concentration, and separation or stain index data is highly beneficial when paired with visual analysis of the cytometry data. Using analytical flow cytometry procedures optimized for cells for EV analysis can lead to misleading and nonreproducible results.

Learning the structure-activity relationship (SAR) of the Wittig reaction from genetically-encoded substrates.

The Wittig reaction can be used for late stage functionalization of proteins and peptides to ligate glycans, pharmacophores, and many other functionalities. In this manuscript, we modified 160 000 N-terminal glyoxaldehyde peptides displayed on phage with the Wittig reaction by using a biotin labeled ylide under conditions that functionalize only 1% of the library population. Deep-sequencing of the biotinylated and input populations estimated the rate of conversion for each sequence. This "deep conversion" (DC) from deep sequencing correlates with rate constants measured by HPLC. Peptide sequences with fast and slow reactivity highlighted the critical role of primary backbone amides (N-H) in accelerating the rate of the aqueous Wittig reaction. Experimental measurement of reaction rates and density functional theory (DFT) computation of the transition state geometries corroborated this relationship. We also collected deep-sequencing data to build structure-activity relationship (SAR) models that can predict the DC value of the Wittig reaction. By using these data, we trained two classifier models based on gradient boosted trees. These classifiers achieved area under the ROC (receiver operating characteristic) curve (ROC AUC) of 81.2 ± 0.4 and 73.7 ± 0.8 (90-92% accuracy) in determining whether a sequence belonged to the top 5% or the bottom 5% in terms of its reactivity. This model can suggest new peptides never observed experimentally with 'HIGH' or 'LOW' reactivity. Experimental measurement of reaction rates for 11 new sequences corroborated the predictions for 8 of them. We anticipate that phage-displayed peptides and related mRNA or DNA-displayed substrates can be employed in a similar fashion to study the substrate scope and mechanisms of many other chemical reactions.

Genetically-encoded discovery of proteolytically stable bicyclic inhibitors for morphogen NODAL.

In this manuscript, we developed a two-fold symmetric linchpin (TSL) that converts readily available phage-displayed peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. TSL combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX1CX2X3X4X5X6X7C sequences, where X is any amino acid but Cys, were converted to a library of bicyclic TSL-[S]X1[C]X2X3X4X5X6X7[C] peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 µM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma cells. The TSL-[S]Y[C]KRAHKN[C] bicycle inhibited NODAL-induced proliferation of NODAL-TYK-nu ovarian carcinoma cells with apparent IC50 of 1 µM. The same bicycle at 10 µM concentration did not affect the growth of the control TYK-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (Pronase™) for 21 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides to expand the previously reported repertoire of phage-displayed bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets.

Tandem Wittig/Diels-Alder diversification of genetically encoded peptide libraries.

In this paper, we developed a tandem of two carbon-carbon bond-forming reactions to chemically diversify the libraries of peptides displayed on a bacteriophage. The Wittig reaction of a biotin-ester from a stabilized phosphorane ylide with model peptides containing N-terminal glyoxal exhibits reaction rates of 0.07 to 5 M-1 s-1 in water at pH 6.5-8. The log(k) scaled linearly with pH from pH 6 to 8; above pH 9 the reaction is accompanied by the hydrolysis of the ester functionality. Capture of the phage displaying the biotinylated product by streptavidin beads confirmed the rate of this reaction in a library of 108 peptides (k = 0.23 M-1 s-1 at pH = 6.5) and also confirmed the regioselectivity of this modification. Olefins introduced into the Wittig reaction can act as Michael acceptors: addition of glutathione, cysteamine, and DYKDDDDKC ("FLAG-Cys") peptide occurs with k = 0.12-4.1 M-1 s-1 at pH 7.8. Analogous reactions with the DYKDDDDKC peptide take place on phage-displayed peptides modified via the Wittig reaction. This reaction is manifested as a progressive emergence of a FLAG-epitope on the phage and detected by the capture of this phage using an anti-FLAG antibody. Olefins introduced into the Wittig reaction also act as dienophiles in the Diels-Alder reaction with cyclopentadiene. The conversion of the dienophile to norbornene-like adducts on the phage was observed by monitoring the disappearance of the thiol-reactive olefin on the phage. This report broadens the reaction scope of genetically-encoded peptide libraries displayed on the phage, expanding the structural diversity of these platforms and increasing their potential to be used in screening against important protein targets. The possibility of monitoring tandem reactions by the use of different labels illustrates the feasibility of obtaining highly functionalized peptides with chemical motifs impossible to achieve using conventional translational machinery.