Generation of Broad Protection against Influenza with Di-Tyrosine-Cross-Linked M2e Nanoclusters.

Tyrosine cross-linking has recently been used to produce nanoclusters (NCs) from peptides to enhance their immunogenicity. In this study, NCs were generated using the ectodomain of the ion channel Matrix 2 (M2e) protein, a conserved influenza surface antigen. The NCs were administered via intranasal (IN) or intramuscular (IM) routes in a mouse model in a prime-boost regimen in the presence of the adjuvant CpG. After boost, a significant increase in anti-M2e IgG and its subtypes was observed in the serum and lungs of mice vaccinated through the IM and IN routes; however, significant enhancement in anti-M2e IgA in lungs was observed only in the IN group. Analysis of cytokine concentrations in stimulated splenocyte cultures indicated a Th1/Th17-biased response. Mice were challenged with a lethal dose of A/California/07/2009 (H1N1pdm), A/Puerto Rico/08/1934 (H1N1), or A/Hong Kong/08/1968 (H3N2) strains. Mice that received M2e NCs + CpG were significantly protected against these strains and showed decreased lung viral titers compared with the naive mice and M2e NC-alone groups. The IN-vaccinated group showed superior protection against the H3N2 strain as compared to the IM group. This research extends our earlier efforts involving the tyrosine-based cross-linking method and highlights the potential of this technology in enhancing the immunogenicity of short peptide immunogens.

Self-Assembled Materials Based on Fully Aromatic Peptides: The Impact of Tryptophan, Tyrosine, and Dopa Residues.

Peptides are able to self-organize in structural elements including cross-ß structures. Taking advantage of this tendency, in the last decades, peptides have been scrutinized as molecular elements for the development of multivalent supramolecular architectures. In this context, different classes of peptides, also with completely aromatic sequences, were proposed. Our previous studies highlighted that the (FY)3 peptide, which alternates hydrophobic phenylalanine and more hydrophilic tyrosine residues, is able to self-assemble, thanks to the formation of both polar and apolar interfaces. It was observed that the replacement of Phe and Tyr residues with other noncoded aromatic amino acids like 2-naphthylalanine (Nal) and Dopa affects the interactions among peptides with consequences on the supramolecular organization. Herein, we have investigated the self-assembling behavior of two novel (FY)3 analogues with Trp and Dopa residues in place of the Phe and Tyr ones, respectively. Additionally, PEGylation of the N-terminus was analyzed too. The supramolecular organization, morphology, and capability to gel were evaluated using complementary techniques, including fluorescence, Fourier transform infrared spectroscopy, and scanning electron microscopy. Structural periodicities along and perpendicular to the fiber axis were detected by grazing incidence wide-angle X-ray scattering. Finally, molecular dynamics studies provided interesting insights into the atomic structure of the cross-ß that constitutes the basic motif of the assemblies formed by these novel peptide systems.

Molecular Recognition of Tyrosine-Containing Polypeptides with Pseudopeptidic Cages Unraveled by Fluorescence and NMR Spectroscopies.

The molecular recognition of Tyr-containing peptide copolymers with pseudopeptidic cages has been studied using a combination of fluorescence and NMR spectroscopies. Fluorescence titrations rendered a reasonable estimation of the affinities, despite the presence of dynamic quenching masking the unambiguous detection of the supramolecular complexes. Regarding NMR, the effect of polypeptide (PP) binding on relaxation and diffusion parameters of the cages is much more reliable than the corresponding chemical shift perturbations. To that, purification of the commercial PPs is mandatory to obtain biopolymers with lower polydispersity. Thus, the relaxation/diffusion-filtered 1H spectra of the cages in the absence vs presence of the PPs represent a suitable setup for the fast detection of the noncovalent interactions. Additional key intermolecular NOE cross-peaks supported by molecular models allow the proposal of a structure of the supramolecular species, stabilized by the Tyr encapsulation within the cage cavity and additional attractive polar interactions between the side chains of cage and PP, thus defining a binding epitope with a potential for implementing sequence selectivity. Accordingly, the cages bearing positive/negative residues prefer to bind the peptides having complementary negative/positive side chains close to the target Tyr, suggesting an electrostatic contribution to the interaction. Overall, our results show that both techniques represent a powerful and complementary combination for studying cage-to-PP molecular recognition processes.

Free-Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitors Using Metadynamics.

Aromatic side chains (phenylalanine and tyrosine) of a protein flip by 180° around the Cß-Cγ axis (χ2 dihedral of the side chain), producing two symmetry-equivalent states. The study of ring flip dynamics with nuclear magnetic resonance (NMR) experiments helps to understand local conformational fluctuations. Ring flips are categorized as slow (milliseconds and onward) or fast (nanoseconds to near milliseconds) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to estimate the flip rate and discriminate between slow and fast ring flips for eight individual aromatic side chains (F4, Y10, Y21, F22, Y23, F33, Y35, and F45) of the basic pancreatic trypsin inhibitor. Well-tempered metadynamics simulations were performed to estimate the ring-flipping free-energy surfaces for all eight aromatic residues. The results indicate that χ2 as a standalone collective variable (CV) is not sufficient to obtain computationally consistent results. Inclusion of a complementary CV, such as χ1(Cα-Cß), solved the problem for most residues and enabled us to classify fast and slow ring flips. This indicates the importance of librational motions in ring flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events were observed for residues F22 and F33, indicating a possible role of friction effects in ring flipping. The results demonstrate the successful application of infrequent metadynamics to estimate ring flip rates and identify certain limitations of the approach.

Ultraviolet Resonance Raman Spectra of Serum Albumins.

The ultraviolet resonance Raman (UVRR) spectra of the two proteins bovine serum albumin (BSA) and human serum albumin (HSA) in an aqueous solution are compared with the aim to distinguish between them based on their very similar amino acid composition and structure and to obtain signals from tryptophan that has only very few residues. Comparison of the protein spectra with solutions of tryptophan, tyrosine, and phenylalanine in comparative ratios as in the two proteins shows that at an excitation wavelength of 220 nm, the spectra are dominated by the strong resonant contribution from these three amino acids. While the strong enhancement of two and one single tryptophan residue in BSA and HSA, respectively, results in pronounced bands assigned to fundamental vibrations of tryptophan, its weaker overtones and combination bands do not play a major role in the spectral range above 1800 cm-1. There, the protein spectra clearly reveal the signals of overtones and combination bands of phenylalanine and tyrosine. Assignments of spectral features in the range of Raman shifts from 3800 to 5100 cm-1 to combinations comprising fundamentals and overtones of tyrosine were supported by spectra of amino acid mixtures that contain deuterated tyrosine. The information in the high-frequency region of the UVRR spectra could provide information that is complementary to near-infrared absorption spectroscopy of the proteins.

MicroCrystalline Tyrosine-adsorbed immunotherapy.

PURPOSE OF REVIEW: The purpose of this article is to provide an overview of the literature pertaining to the use of MicroCrystalline Tyrosine (MCT) in the immunotherapy with an emphasis on recent developments. RECENT FINDINGS: In addition to significant effectiveness and safety profiles, additional aspects of interest such as booster immunotherapy concepts, sustained clinical effects, long-term efficacy and disease-modifying effects are being focused on in the recently published studies. The depot adjuvant MCT also shows potential in promising disease-challenge models such as for malaria and melanoma. SUMMARY: MCT-adsorbed immunotherapy products have been shown to provide convincing overall safety, tolerability and efficacy outcomes, as well in vulnerable groups such as children and asthmatic patients.

Protein nitration induced by Hemin/NO: A complementary mechanism through the catalytic functions of hemin and NO-scavenging.

Hemin and heme-peroxidases have been considered essential catalysts for the nitrite/hydrogen peroxide (H2O2)-mediated protein nitration in vitro, understood as one of the main pathways for protein modification in biological systems. However, the role of nitric oxide (●NO) in the heme/hemin-induced protein nitration has not been studied in-depth. This is despite its reductive nitrosylating effects following binding to hemin and the possible involvement of the reactive nitrogen species in the nitration of various functional proteins. Here, the ●NO-binding affinity of hemin has been studied along with the influence of ●NO on the internalization of hemin into MDA-MB-231 cells and the accompanying changes in the profile of intracellular nitrated proteins. Moreover, to further understand the mechanism involved, bovine serum albumin (BSA) nitration was studied after treatment with hemin and ●NO, with an investigation of the effects of pH of the reaction medium, generation of H2O2, and the oxidation of the tyrosine residues as the primary sites for the nitration. We demonstrated that hemin nitrosylation enhanced its cellular uptake and induced the one-electron oxidation and nitration of different intracellular proteins along with its ●NO-scavenging efficiency. Moreover, the hemin/NO-mediated BSA nitration was proved to be dependent on the concentration of ●NO and the pH of the reaction medium, with a vital role being played by the scavenging effects of protein for the free hemin molecules. Collectively, our results reaffirm the involvement of hemin and ●NO in the nitration mechanism, where the nitrosylation products can induce protein nitration while promoting the effects of the components of the nitrite/H2O2-mediated pathway.

Effectiveness and safety of a microcrystalline tyrosine-adjuvanted Dermatophagoides pteronyssinus allergoid immunotherapy in adult patients with allergic asthma and rhinitis: A real-life prospective observational study.

INTRODUCTION: Although clinical trials have shown the efficacy and safety of allergen-specific immunotherapy (AIT) in the treatment of allergic asthma, there is a need for real-life studies. We aimed to assess the effectiveness and safety of a microcrystalline tyrosine-adjuvanted Dermatophagoides pteronyssinus allergoid (Acarovac Plus®) in patients with house dust mite (HDM)-induced allergic asthma in a real-life study. METHODS: A subanalysis of a multicenter, prospective, observational, real-life study. Patients with rhinitis and allergic asthma caused by HDMs were assessed before AIT with Acarovac Plus® and at 6 and 12 months after this treatment. Assessment parameters were percentage of days with asthma symptoms, percentage of days on asthma medication, classification of asthma according to Spanish guidelines for the management of asthma, asthma-related quality of life (quality of life in adults with asthma questionnaire [QLAAQ]), perception of symptoms (visual analog scale [VAS]), and treatment satisfaction (treatment satisfaction questionnaire for medication [TSQM]). Safety was assessed by the number and severity of adverse reactions. RESULTS: This subanalysis included 55 patients. Treatment with Acarovac Plus® showed significant differences in the analyzed variables when the baseline visit was compared with the 12-month visit: reduction of the mean (SD) percentage of days with asthma symptoms (23.9 [9.2] vs. 5.1 [12.8]; p = .002), of the mean [SD] percentage of days on asthma medication (67.6 [42.9] vs. 45.1 [46.8]; p = .002), and of the percentage of patients with persistent asthma (78.2% vs. 38.9%; p = .009). Acarovac Plus® significantly improved asthma-related quality of life, as shown by a decrease of 1.39 points in QLAAQ score at 12 months (p < .001), and in the subjective perception of symptoms on the VAS (-3.50, p < .0001). Patients showed high treatment satisfaction according to the TSQM, and it was well tolerated. No serious adverse events were reported. CONCLUSIONS: Acarovac Plus® was effective and safe for the treatment of patients with HDM-induced allergic asthma in a real-life study.

A Tyrosine Phosphoproteome Analysis Approach Enabled by Selective Dephosphorylation with Protein Tyrosine Phosphatase.

Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 µg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.

Effect of catechin and commercial preparation of green tea essence on the pharmacokinetics of l-dopa in rabbits.

The aim of this study was to investigate drug interactions of L-dopa/carbidopa with catechin and green tea essence in rabbits following the simultaneous administration via an intramuscular injection of catechin or via an intragastric route for green tea essence with L-dopa/carbidopa. The results indicated that catechin at doses of 10, 20 and 50 mg/kg increased the area under the plasma concentration-time curve from time zero to the time of the last quantifiable concentration (AUC0-t ) of L-dopa by about 69, 78 and 42%, respectively. The metabolic ratios of the AUC0-t for 3-O-methyldopa (3-OMD)/L-dopa significantly decreased by about 56, 68 and 76% (P < 0.05), respectively. In addition, a single dose of 5/1.25 mg/kg L-dopa/carbidopa was co-administrated with 150 mg/kg green tea essence via an intragastric route with an oral-gastric tube. Comparing the related pharmacokinetic parameters of L-dopa, the clearance and metabolic ratio of L-dopa decreased by 20 and 19% (P < 0.05), respectively. In conclusion, catechin and green tea essence can significantly affect the metabolism of L-dopa by the catechol-O-methyltransferase (COMT) metabolic pathway. Catechin can enhance L-dopa bioavailability, and both catechin and green tea essence decreased 3-OMD formation. Therefore, catechin and green tea essence may increase L-dopa efficacy for Parkinson's disease treatment.

Mass spectrometry-based urinary metabolomics for exploring the treatment effects of Radix ginseng-Schisandra chinensis herb pair on Alzheimer's disease in rats.

Herb pairs are the unique combinations of two relatively fixed herbs, intrinsically convey the basic idea of traditional Chinese medicine prescriptions. The compatibility of Radix ginseng and Schisandra chinensis has been used in traditional Chinese medicine for treating Alzheimer's disease for many years. However, there are few studies on Radix ginseng-Schisandra chinensis herb pair, and the underlying action mechanism is still unclear. In this study, the mechanism of Radix ginseng-Schisandra chinensis herb pair on Alzheimer's disease was investigated by using the mass spectrometry-based urinary metabolomics method. Sixteen urinary endogenous metabolites were identified as potential biomarkers. Meanwhile, 10 biomarkers were quantified with tandem mass spectrometry. The study result showed that the brain pathologic symptoms of model rats were improved and the potential biomarkers were adjusted backward significantly after the herb pair administration. The metabolic pathways linked to the herb pair-regulated endogenous biomarkers included phenylalanine and tyrosine metabolism, tryptophan metabolism, purine metabolism, and so on. The above metabolic pathways reflected that Radix ginseng-Schisandra chinensis herb pair mainly regulates abnormal energy metabolism, reduces inflammation, and regulates gut microbiota and neurotransmitters in the treatment of Alzheimer's disease.

Identification of the vibrational marker of tyrosine cation radical using ultrafast transient infrared spectroscopy of flavoprotein systems.

Tryptophan and tyrosine radical intermediates play crucial roles in many biological charge transfer processes. Particularly in flavoprotein photochemistry, short-lived reaction intermediates can be studied by the complementary techniques of ultrafast visible and infrared spectroscopy. The spectral properties of tryptophan radical are well established, and the formation of neutral tyrosine radicals has been observed in many biological processes. However, only recently, the formation of a cation tyrosine radical was observed by transient visible spectroscopy in a few systems. Here, we assigned the infrared vibrational markers of the cationic and neutral tyrosine radical at 1483 and 1502 cm-1 (in deuterated buffer), respectively, in a variant of the bacterial methyl transferase TrmFO, and in the native glucose oxidase. In addition, we studied a mutant of AppABLUF blue-light sensor domain from Rhodobacter sphaeroides in which only a direct formation of the neutral radical was observed. Our studies highlight the exquisite sensitivity of transient infrared spectroscopy to low concentrations of specific radicals.

Accessing p-Hydroxycinnamic Acids: Chemical Synthesis, Biomass Recovery, or Engineered Microbial Production?

p-Hydroxycinnamic acids (i. e., p-coumaric, ferulic, sinapic, and caffeic acids) are phenolic compounds involved in the biosynthesis pathway of lignin. These naturally occurring molecules not only exhibit numerous attractive properties, such as antioxidant, anti-UV, and anticancer activities, but they also have been used as building blocks for the synthesis of tailored monomers and functional additives for the food/feed, cosmetic, and plastics sectors. Despite their numerous high value-added applications, the sourcing of p-hydroxycinnamic acids is not ensured at the industrial scale except for ferulic acid, and their production cost remains too high for commodity applications. These compounds can be either chemically synthesized or extracted from lignocellulosic biomass, and recently their production through bioconversion emerged. Herein the different strategies described in the literature to produce these valuable molecules are discussed.

Shaping Modern Vaccines: Adjuvant Systems Using MicroCrystalline Tyrosine (MCT®).

The concept of adjuvants or adjuvant systems, used in vaccines, exploit evolutionary relationships associated with how the immune system may initially respond to a foreign antigen or pathogen, thus mimicking natural exposure. This is particularly relevant during the non-specific innate stage of the immune response; as such, the quality of this response may dictate specific adaptive responses and conferred memory/protection to that specific antigen or pathogen. Therefore, adjuvants may optimise this response in the most appropriate way for a specific disease. The most commonly used traditional adjuvants are aluminium salts; however, a biodegradable adjuvant, MCT®, was developed for application in the niche area of allergy immunotherapy (AIT), also in combination with a TLR-4 adjuvant-Monophosphoryl Lipid A (MPL®)-producing the first adjuvant system approach for AIT in the clinic. In the last decade, the use and effectiveness of MCT® across a variety of disease models in the preclinical setting highlight it as a promising platform for adjuvant systems, to help overcome the challenges of modern vaccines. A consequence of bringing together, for the first time, a unified view of MCT® mode-of-action from multiple experiments and adjuvant systems will help facilitate future rational design of vaccines while shaping their success.

Immobilization of 6-O-α-maltosyl-ß-cyclodextrin on the surface of black phosphorus nanosheets for selective chiral recognition of tyrosine enantiomers.

A novel chiral sensing platform, 6-O-α-maltosyl-ß-cyclodextrin (Mal-ßCD)-based film, is proposed for selective electrochemical recognition of tyrosine (Tyr) enantiomers. Black phosphorus nanosheets (BP NSs) and Mal-ßCD modified glassy carbon electrode (Mal-ßCD/BP NSs/GCE) were prepared by a layer-to-layer drop-casting method, and the platform was easy to fabricate and facile to operate. It is proposed that the amino and hydroxyl groups of the Tyr enantiomers and the chiral hydroxyl groups of Mal-ßCD selectively form intermolecular hydrogen bonds to dominate effective chiral recognition. Two linear equations of Ip (µA) = 11.40 CL-Tyr (mM) + 0.28 (R2 = 0.99147) and Ip (µA) = 7.96 CD-Tyr (mM) + 0.22 (R2 = 0.99583) in the concentration range 0.01-1.00 mM have been obtained. The limits of detection (S/N=3) for L-Tyr and D-Tyr were 4.81 and 6.89 µM, respectively. An interesting phenomenon was that the value of IL-Tyr/ID-Tyr (1.51) in this work was slightly higher than the value of IL-Trp/ID-Trp (1.49) reported in our previous study, where tryptophan (Trp) enantiomers were electrochemically recognized by Nafion (NF)-stabilized BPNSs-G2-ß-CD composite. The two similar sensors fabricated by different methods showed different recognition ability toward either Tyr or Trp enantiomers, and the underlying mechanism was discussed in detail. More importantly, the proposed chiral sensor enables prediction of the percentages of D-Tyr in racemic Tyr mixtures. The chiral sensor may provide a novel approach for the fabrication of novel chiral platforms in the practical detection of L- or D-enantiomer in racemic Tyr mixtures.Graphical abstract.

Honey as Source of Nitrogen Compounds: Aromatic Amino Acids, Free Nucleosides and Their Derivatives.

The content of selected major nitrogen compounds including nucleosides and their derivatives was evaluated in 75 samples of seven varieties of honey (heather, buckwheat, black locust, goldenrod, canola, fir, linden) by targeted ultra-high performance liquid chromatography-diode array detector - high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QqTOF-MS) and determined by UHPLC-DAD. The honey samples contained nucleosides, nucleobases and their derivatives (adenine: 8.9 to 18.4 mg/kg, xanthine: 1.2 to 3.3 mg/kg, uridine: 17.5 to 51.2 mg/kg, guanosine: 2.0 to 4.1 mg/kg; mean amounts), aromatic amino acids (tyrosine: 7.8 to 263.9 mg/kg, phenylalanine: 9.5 to 64.1 mg/kg; mean amounts). The amounts of compounds significantly differed between some honey types. For example, canola honey contained a much lower amount of uridine (17.5 ± 3.9 mg/kg) than black locust where it was most abundant (51.2 ± 7.8 mg/kg). The presence of free nucleosides and nucleobases in different honey varieties is reported first time and supports previous findings on medicinal activities of honey reported in the literature as well as traditional therapy and may contribute for their explanation. This applies, e.g., to the topical application of honey in herpes infections, as well as its beneficial activity on cognitive functions as nootropic and neuroprotective, in neuralgia and is also important for the understanding of nutritional values of honey.

Real-life effect of a microcrystalline tyrosine adjuvanted mite immunotherapy in patients with allergic rhinitis.

Aim: Evaluate the effectiveness and safety of immunotherapy with Acarovac Plus® in a 1-year prospective multicentered real-life study. Methods: A total of 118 adults with allergic rhinitis sensitized to Dermatophagoides received subcutaneous immunotherapy with Acarovac Plus. Treatment outcomes were evaluated at baseline, 6 months and 1 year after treatment initiation. Primary end point was the evolution of the combined symptom and medication score. Secondary end points included other effectiveness outcomes and measurement of product tolerability. Results: Acarovac Plus induced significant improvements in primary and secondary end points after 6 months compared with baseline. These differences persisted after 1 year of treatment (p < 0.001; baseline vs 1 year): combined symptom and medication score (1.60 vs 0.79). No serious adverse events were recorded. Conclusion: Acarovac Plus for 1 year was effective and well tolerated in a real-life setting.

Antidepressant-Like Effect of ß-Lactolin, a Glycine-Threonine-Tryptophan-Tyrosine Peptide.

The number of patients with mental illnesses, including depression, is rapidly increasing, and daily lifestyle is closely associated with the development of symptoms. Consequently, corrective measures, such as diet-based treatment for diseases, are receiving great attention. We previously showed that ß-lactolin, a ß-lactopeptide of glycine-threonine-tryptophan-tyrosine peptide, inhibits monoamine oxidase and improves memory impairment in mice, but the effects on depression have not been investigated. Here we showed that ß-lactolin improved depression-like behavior via dopamine-D1-like receptor. Orally administered ß-lactolin reduced immobility time in tail suspension test (TST). Pretreatment with SCH23390, dopamine D1-like receptor antagonist, attenuated the reduction in TST by ß-lactolin. These effects were observed by the treatment with whey digest rich in ß-lactolin. In addition, ß-lactolin increased the levels of dopamine in the frontal cortex associated with the depression-like behavior. The present study suggests that supplements or nutraceutical compounds in whey digests (such as ß-lactolin) show antidepressant-like effect.

Characterization and prediction of positional 4-hydroxyproline and sulfotyrosine, two post-translational modifications that can occur at substantial levels in CHO cells-expressed biotherapeutics.

Biotherapeutics may contain a multitude of different post-translational modifications (PTMs) that need to be assessed and possibly monitored and controlled to ensure reproducible product quality. During early development of biotherapeutics, unexpected PTMs might be prevented by in silico identification and characterization together with further molecular engineering. Mass determinations of a human IgG1 (mAb1) and a bispecific IgG-ligand fusion protein (BsAbA) demonstrated the presence of unusual PTMs resulting in major +80 Da, and +16/+32 Da chain variants, respectively. For mAb1, analytical cation exchange chromatography demonstrated the presence of an acidic peak accounting for 20%. A + 79.957 Da modification was localized within the light chain complementarity-determining region-2 and identified as a sulfation based on accurate mass, isotopic distribution, and a complete neutral loss reaction upon collision-induced dissociation. Top-down ultrahigh resolution MALDI-ISD FT-ICR MS of modified and unmodified Fabs allowed the allocation of the sulfation to a specific Tyr residue. An aspartate in amino-terminal position-3 relative to the affected Tyr was found to play a key role in determining the sulfation. For BsAbA, a + 15.995 Da modification was observed and localized to three specific Pro residues explaining the +16 Da chain A, and +16 Da and +32 Da chain B variants. The BsAbA modifications were verified as 4-hydroxyproline and not 3-hydroxyproline in a tryptic peptide map via co-chromatography with synthetic peptides containing the two isomeric forms. Finally, our approach for an alert system based on in-house in silico predictors is presented. This system is designed to prevent these PTMs by molecular design and engineering during early biotherapeutic development.

Antidepressant-like effects of dietary gardenia blue pigment derived from genipin and tyrosine.

Gardenia blue pigments derived from genipin reacting with amino acids have been used as natural food colorants for nearly 30 years in East Asia. However, their pharmacological effects, especially antidepressant-like effects, have not been reported so far. In this study, one of the gardenia blue pigments, was obtained from the reaction of genipin with tyrosine (genipin-tyrosine derivant (GTD)), and its antidepressant-like effects were investigated in lipopolysaccharide (LPS) or chronic unpredictable mild stress (CUMS) models. The results showed that GTD could attenuate depressive-like behaviors in both animal models. GTD reversed the LPS-induced cytokine increase of TNF-α, IL-6, and corticosterone (CORT) in mice plasma and hippocampus. In CUMS rats, GTD treatment significantly reduced hypothalamic-pituitary-adrenal (HPA) axis-related stress hormone levels in plasma including those of CORT, adrenocorticotropic hormone (ACTH), and corticotropin-releasing hormone (CRH). Besides, GTD increased plasma testosterone and hippocampal brain-derived neurotrophic factor (BDNF) levels in CUMS rats. GTD increased serotonin (5-HT), dopamine (DA), and norepinephrine (NE) in rat hippocampus and corpus striatum. Consistently, hippocampal metabolomic analysis demonstrated that GTD restored monoamine neurotransmitter metabolism, mitochondrial oxidative function, and membrane structural integrity. Our data suggested that GTD produced antidepressant-like activity through the restoration of the HPA axis hormone balance and the regulation of neurotransmitter release.