The effect of temperature on the retention and enantioseparation of dipeptides (DPs) with one and two chiral centers on surface-porous adsorbents Chiral-T and Chiral-V with grafted antibiotics teicoplanin and vancomycin by linear chromatography was studied. Multidirectional temperature dependences of the retention factor were revealed: decreasing — on Chiral-T and increasing — on Chiral-V. Adsorption of DP stereoisomers on Chiral-T is characterized by exothermic effect, and on Chiral-V — by endothermic thermal effect. It was found that the solvation effect and steric factors on Chiral-T have a greater influence on the retention and enantioseparation of the stereoisomers of the studied DPs than the energetic changes. The thermodynamic characteristics of adsorption were analyzed using extrathermodynamic relationships. Statistical analysis of the compensation effect revealed a different mechanism of retention and enantioseparation of DP stereoisomers on Chiral-T and Chiral-V. The mechanism of adsorption of DP stereoisomers on Chiral-T is more influenced by the spatial configuration of the DP molecule, and on Chiral-V — by the lipophilicity of DP.

Dipeptides (DPs), composed of two amino acids (AAs), hold significant therapeutic potential but remain underexplored. Given the crucial role of AAs in central nervous system (CNS) function, this study investigated the presence of DPs in cerebrospinal fluid (CSF) and their correlation with corresponding AAs, potentially indicating their role as AA donors. Plasma and CSF samples were collected from 43 children with neurological or metabolic conditions of unknown origin, including 23 with epilepsy. A panel of 33 DPs was quantified using UPLC-MS/MS. Out of 33 DPs, 18 were detectable in CSF and 20 in plasma, displaying high inter-individual variance. Gly-Asp, Gly-Pro, and Ala-Glu were consistently found in all CSF samples, while only Gly-Asp was universally detectable in plasma. Anserine and carnosine were prominent in CSF and plasma, respectively, with no other histidine-containing DPs observed. Generally, DP concentrations were higher in plasma than in CSF; however, anserine and Gly-Pro had similar concentrations in both fluids. Significant correlations were observed between specific DPs and their corresponding AAs in CSF (Gly-Glu, Gly-Pro and Ser-Gln) and plasma (Glu-Glu and Glu-Ser). Notably, patients with epilepsy had elevated medium anserine concentrations in CSF. This study is the first to demonstrate the presence of numerous DPs in CSF and plasma. Further research is needed to determine if DP patterns can support the diagnosis of neurological diseases and whether DP administration can modulate amino acid availability in the brain, potentially offering new therapeutic options, such as for defects in the amino acid transporter.

Retention mechanisms of dipeptides on superficially porous particle vancomycin- and teicoplanin-based chiral stationary phases

The investigation of collagen hydrolysates (CHs) is essential due to their widespread use in health, cosmetic, and therapeutic industries, attributing to the presence of bioactive dipeptides (DPs) and tripeptides (TPs). This study developed a novel targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with propyl chloroformate (PCF) derivatization to measure three bioactive peptides-Hydroxyprolyl-glycine (Hyp-Gly), Glycyl-prolyl-hydroxyproline (Gly-Pro-Hyp), and Prolyl-hydroxyproline (Pro-Hyp)-in CHs, with strong correlation coefficients (0.992, 1.000, and 0.995, respectively) and low limits of detection (LODs) of 1.40, 0.14, and 1.16 µM, respectively. Untargeted data-dependent acquisition (DDA) analyses measured peptide size distribution, while amino acid analysis assessed nutritional content. The analysis of ten commercial CHs revealed similar amino acid profiles but varied peptide lengths, indicating diverse hydrolysis conditions. Products with higher proportions of smaller peptides showed elevated levels of the targeted bioactive peptides, suggesting that a smaller peptide size may increase bioactivity. These findings can inform the optimization of CH supplements, providing consumers with detailed peptide content for more informed choices. Data are available via ProteomeXchange with the identifier PXD051699.

UV-crosslinking of protein and RNA in direct contacts has been widely used to study protein-RNA complexes while our understanding of the photo-crosslinking mechanisms remains poor. This knowledge gap is due to the challenge of precisely mapping the crosslink sites in protein and RNA simultaneously in their native sequence and structural contexts. Here we systematically analyze protein-RNA interactions and photo-crosslinking by bridging crosslinked nucleotides and amino acids mapped using different assays with protein-RNA complex structures. We developed a computational method PxR3D-map which reliably predicts crosslink sites using structural information characterizing protein-RNA interaction interfaces. Analysis of the informative features revealed that photo-crosslinking is facilitated by base stacking with not only aromatic residues, but also dipeptide bonds that involve glycine, and distinct mechanisms are utilized by different RNA-binding domains. Our work suggests protein-RNA photo-crosslinking is highly selective in the cellular environment, which can guide data interpretation and further technology development for UV-crosslinking-based assays.

A chemical derivatization-based pseudotargeted LC-MS/MS method for high coverage determination of dipeptides

A series of new ursolic acid (UA) derivatives substituted with various amino acids (AAs) or dipeptides (DP) at the C-3 position of the steroid skeleton was designed and synthesized. The compounds were obtained by the esterification of UA with the corresponding AAs. The cytotoxic activity of the synthesized conjugates was determined using the hormone-dependent breast cancer cell line MCF-7 and the triple-negative breast cancer cell line MDA. Three derivatives (l-seryloxy-, l-prolyloxy- and l-alanyl-l-isoleucyloxy-) showed micromolar IC50 values and reduced the concentrations of matrix metalloproteinases 2 and 9. Further studies revealed that for two compounds (l-seryloxy- and l-alanyl-l-isoleucyloxy-), a possible mechanism of their antiproliferative action is the activation of caspase-7 and the proapoptotic Bax protein in the apoptotic pathway. The third compound (l-prolyloxy- derivative) showed a different mechanism of action as it induced autophagy as measured by an increase in the concentrations of three autophagy markers: LC3A, LC3B, and beclin-1. This derivative also showed statistically significant inhibition of the proinflammatory cytokines TNF-α and IL-6. Finally, for all synthesized compounds, we computationally predicted their ADME properties as well as performed molecular docking to the estrogen receptor to assess their potential for further development as anticancer agents.

A simple geometrical model of the electrostatic environment around the catalytic center of the ribosome and its significance for the elongation cycle kinetics

Assisted dipeptide bond formation: glycine as a case study

We describe a fast (5 min) liquid chromatography tandem mass spectrometry method (LC-MS/MS) based on a 46 Da neutral loss of formic acid (H2 O and CO) to identify tri- and dipeptides (DIPEP) in whey protein and porcine liver protein hydrolysates and confirmed by further de novo sequencing. Sample solutions were acidified to favor [dipep + H]+ ions, and a m/z range of 50-300 was used to improve sensitivity. All dipeptide candidates were selected based on all possibilities of the 20 amino acid combinations, and their collision-induced dissociation fragments were screened via de novo sequencing. To determine their biological activities, sequenced dipeptides were compared with the Biopep database and other data from literature. Altogether, 18 dipeptides and 7 tripeptides were identified from the whey protein hydrolysate; they seemed to be broadly active, and peptides were identified as active dipeptidyl peptidase IV inhibitors and active angiotensin-converting enzyme (ACE), according to available information. Porcine liver hydrolysate showed 14 dipeptides which exhibit similar biological activities to whey protein hydrolysate.

DFT study of cyclic glycine-alanine dipeptide binding to gold nanoclusters

The antioxidant dipeptide L-carnosine has potential neuroprotective and beneficial metabolic effects, and studies have indicated cognitive benefits.

Metallogelation was observed selectively for CuSO4, ZnSO4 and Pb(OAc)2 and a dipeptide containing N-phenylglycine and l-Phe, whereas other metals and analogue dipeptides failed to form gel.

A novel “turn on” fluorescent probe enabled analysis of the relative rates of intracellular disulfide and dipeptide bond cleavage.

Delivery of tumor-associated antigens for cancer immunotherapy is generally limited due to poor stability and low cellular uptake. Use of nanoparticles as delivery vehicles has been an exciting development but toxicity associated with nanoparticles has remained a concern. Here, we have used conformationally constraint self-assembling dipeptide nanotubes for the delivery of Cancer-Testis antigens (CTAs). A dipeptide Phenylalanine-α, β-dehydrophenylalanine (F-ΔF) was synthesized and was found to self-assemble into nanotubes. CTAs (NY-ESO-1 and MAGE-3) were also synthesized and loaded onto F-ΔF nanotubes using physical adsorption method for delivery purposes. CTA loaded F-ΔF nanotubes showed higher cellular uptake in macrophages. In animal studies, F-ΔF-CTA nanocomplex elicited much higher immune response compared to CTAs alone and showed an inhibition of ~65-70% tumor growth in mouse melanoma model. Our results indicated that F-ΔF nanotubes are biocompatible and are efficient delivery vehicles for CTAs in vivo and therefore are excellent candidates for further development as delivery agents.

Objective To study the effect of a new immunomodulator composed of muramyl dipeptide(MDP) and anti-CD10 monoclonal antibody (MDP-Ab) on the dendritic cells(DC) of children with acute leukemia. Methods DC was adopted to divide the children with acute lymphoblastic leukemia into 6 groups, including the control group, unconjugated anti-CD10 alone, unconjugated MDP alone, MDP-Ab alone, lipopolysaccharide (LPS) alone and MDP-Ab + LPS.The immunophenotypes, the endocytosis interleukin-12 (IL-12) were detected.The stimulation index of autologous lymphocytes was assayed by adopting 5-(and 6)-carboxyfluorescein diacetate, succinimidyl ester(CFSE)-staining method.The supernatants of DC and autologous lymphocytes were used to detect the level of interferon-γ(IFN-γ) by using enzyme-linked immunosorbent assay. Results (1) DC immunophenotype: The expressions of human leukocyte antigen-DR(HLA-DR), mature molecule (CD83) and co-stimulatory molecules (CD80 and CD86) were increased significantly upon DC triggered with MDP-Ab, compared with the control group, unconjugated anti-CD10 group, and unconjugated MDP group, but lower than those in LPS and combination of MDP-Ab with LPS(F=629.62, P=0.000). (2) The level of IL-12: a significant increase in IL-12 level was detected in MDP-Ab group, LPS group, and combination of MDP-Ab with LPS group, compared with the control group, unconjugated anti-CD10 group, and unconjugated MDP group (F=857.87, P=0.000). There were significant differences among the first three groups. (3) Endocytosis assay: The uptake of DCs stimulated by unconjugated anti-CD10, unconjugated MDP, MDP-Ab immunoconjugate, LPS or combination was lower than that of immature DC in the control group which was (81.3±10.1)%. (4)Mixed lymphocyte reaction and IFN-γ level: DC, treated with MDP-Ab, LPS and combination, stimulated more CFSE positive cells and higher level of IFN-γ secretion than the control group and unconjugated anti-CD10 group, unconjugated MDP group. The most significance was observed in combination of MDP-Ab with LPS(F=393.36, P=0.000; F=2 497.18, P=0.000). Conclusion It is concluded that MDP-Ab could promote the proliferation and maturation of DC derived from blood of children with acute leukemia. Key words: Cancer vaccine; Anti-CD10 monoclonal antibody; Muramyl dipeptide; Dendritic cells; Leukemia; Child

Recent evidence suggests that increased brain serotonin synthesis impairs performance in high-intensity intermittent exercise and specific amino acids may modulate this condition, delaying fatigue. This study investigated the effects of glutamine and alanine supplementation on central fatigue markers in rats submitted to resistance training (RT). Wistar rats were distributed in: sedentary (SED), trained (CON), trained and supplemented with alanine (ALA), glutamine and alanine in their free form (G + A), or as dipeptide (DIP). Trained groups underwent a ladder-climbing exercise for eight weeks, with progressive loads. In the last 21 days, supplementations were offered in water with a 4% concentration. Albeit without statistically significance difference, RT decreased liver glycogen, and enhanced the concentrations of plasma glucose, free fatty acids (FFA), hypothalamic serotonin, and ammonia in muscle and the liver. Amino acids affected fatigue parameters depending on the supplementation form. G + A prevented the muscle ammonia increase by RT, whereas ALA and DIP augmented ammonia and glycogen concentrations in muscle. DIP also increased liver ammonia. ALA and G + A reduced plasma FFA, whereas DIP increased this parameter, free tryptophan/total tryptophan ratio, hypothalamic serotonin, and the serotonin/dopamine ratio. The supplementations did not affect physical performance. In conclusion, glutamine and alanine may improve or impair central fatigue markers depending on their supplementation form.

Paclitaxel (PTX) is among the most commonly used first-line drugs for cancer chemotherapy. However, its poor water solubility and indiscriminate distribution in normal tissues remain clinical challenges. Here we design and synthesize a highly water-soluble nucleolin aptamer-paclitaxel conjugate (NucA-PTX) that selectively delivers PTX to the tumor site. By connecting a tumor-targeting nucleolin aptamer (NucA) to the active hydroxyl group at 2′ position of PTX via a cathepsin B sensitive dipeptide bond, NucA-PTX remains stable and inactive in the circulation. NucA facilitates the uptake of the conjugated PTX specifically in tumor cells. Once inside cells, the dipeptide bond linker of NucA-PTX is cleaved by cathepsin B and then the conjugated PTX is released for action. The NucA modification assists the selective accumulation of the conjugated PTX in ovarian tumor tissue rather than normal tissues, and subsequently resulting in notably improved antitumor activity and reduced toxicity.

Influence of an ω3-fatty acid-enriched enteral diet with and without added glutamine on the metabolic response to injury in a rat model of prolonged acute catabolism.

Objective To assess the short-time economics of various glutamine dipeptide-enriched parenteral nutrition (PN) for patients undergoing elective surgery for gastrointestinal tumors, with an attempt to provide evidence for decision makers on clinical nutrition support. Methods A prospective cohort study was designed. From payer/disburser's perspective, a cost-effectiveness decision-tree model was developed to assess the clinical outcomes and short-time economic effects of glutamine dipeptide-enriched PN that used in different time points (early postoperative or perioperative). Cost-effectiveness analysis, cost-utility analysis, and incremental cost-effectiveness analysis were adopted in the decision-tree model. One-way sensitivity analysis was performed to determine the robustness of the results. Results Totally 107 patients were included. There was no significant difference between the perioperative alanine(Ala) glutamine(Gln) nutrition support (group A) and early postoperative Ala-Gln nutrition support (group B) in the ratio of 5% weight declines on the 8th day after surgery and infection-related postoperative complications (72.1% vs. 78.1%, χ2=0.509, P=0.498 and 2.32% vs. 4.69%, χ2=0.060, P=0.806). The levels of prealbumin (PA) and albumin(Alb) and the level of total lymphocyte count(TLC) also the time of recovering gastrointestinal function, length of stay nutritional discharge index(LOSNDI), and direct costs were significantly different [PA: (208.19±56.92)mg/L vs. (187.97±62.05)mg/L, t=2.283, P=0.039; Alb: (33.82±3.91)×109vs. (31.96±4.57)×109,t=2.184, P=0.036; TLC: (1.19±0.55)×109vs. (0.89±0.66)×109,t=2.461, P=0.015; the time of recovering gastrointestinal function(3.06±0.28)d vs. (3.39 ± 0.34)d, t=-3.675, P=0.000; LOSNDI: (16.84±2.92)d vs. (18.52 ±3.47)d, t=-2.613, P=0.011; direct costs: ￥(17 029.05±317.28) vs. ￥(15 610.64±292.56), t=23.764, P=0.000]. When LOSNDI and quality-adjusted life years (QALYs) were estimated as indicators of effectiveness, the incremental cost-effectiveness ratios and incremental cost-utility ratios of group A were ￥844.3 and ￥70 920.5, respectively. Net monetary benefit of group B was more than that of group A. One-way sensitivity analysis showed that parameters had no significant effect on the model. Conclusion When using local per capita gross domestic product as threshold, early postoperative Ala-Gln PN was more economical than perioperative Ala-Gln PN strategy evaluation. Key words: Parenteral nutrition; Decision trees; Glutamine dipeptide; Gastro-intestinal tumors; Health economic evaluation