Natural blues: structure meets function in plant natural products

Colour plays a crucial role in food manufacturing, providing product consistency, addressing off-colouring issues, and enhancing consumer appeal. However, the availability of blue food colorants is severely limited, with only a few options in the US (Brilliant blue FCF and indigo carmine) and an additional choice in Europe. Given the rising demand for naturally derived additives, confectionery companies have struggled to find a suitable naturally derived substitute. Anthocyanins, plant-based pigments that exhibit a range of colours from red to blue, have been explored as potential food colorants. However, their inherent instability at the pH required for generating blue colours has presented a significant challenge. The complex mixture of anthocyanins derived from butterfly pea consists of 15 delphinidin-3(6”-malonyl), 3',5'-O-triglucoside derivatives acylated with p-coumarate which are highly stable and produce a blue colour at mildly acid pH. In this study, a new flash chromatography method for the rapid fractionation of ternatins was developed, allowing for efficient and food safe separation based on the terminal sidechain residues. This method offers a high capacity and quick separation technique for food applications. Through this research, it was demonstrated empirically that acyl residues stabilised the anthocyanin against degradation by stacking over the delphinidin C-ring. Glycosylation weakens these sidechain interactions, thereby decreasing stability. In both cases, acylation and glycosylation resulted in a bathochromic shift. The mechanisms by which acylation and glycosylation cause bathochromic shifts were explored using a combination of molecular dynamics and quantum mechanical simulations. Quantum mechanics simulations of delphinidin-3-O-glucoside rotamers have demonstrated that UV-VIS absorbance λmax increases with an increasing dihedral angle. Molecular dynamics simulations of ternatins showed decreased interaction between the acyl residue and chromophore upon glycosylation, with an associated increase in dihedral angle. This thesis establishes ternatins as potential ‘natural’ replacements for synthetic blue food colourants

In Vitro Study of Histatins: Studies of the Free and Copper Complexed Bioligand's Structure and Catalytic Activity.

The ability of the histidine-rich peptides, histatin-5 (Hst-5) and -8 (Hst-8), to support the generation of reactive oxygen species (ROS) during the copper-catalyzed oxidation of ascorbate and cysteine has been evaluated. High levels of hydrogen peroxide (70-580 mol/mol Cu/hr) are produced by aqueous solutions containing Cu(II), Hst-8 or Hst-5, and a reductant, either ascorbate or cysteine, as determined by the post-reaction Amplex Red (AR) assay. When the reactions are conducted in the presence of superoxide dismutase (SOD), the total hydrogen peroxide produced is decreased, more so in the presence of the peptides (up to 50%), suggesting the intermediacy of superoxide in these reactions. On the other hand, the presence of sodium azide or sodium formate, traps for hydroxyl radicals, has no appreciable effect on the total hydrogen peroxide production for the Cu-Hst systems. Electron paramagnetic resonance (EPR) spectroscopy spin-trapping studies using CYPMPO (5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide) in the cysteine/Cu(II) reactions reveal the formation of the CYPMO-hydroperoxyl and -hydroxyl radical adducts in the presence of Hst-8, whereas only the latter was observed with Cu alone.As in vitro ROS production involved copper ions, the complex formation of the peptide-metal species was also of interest. The specific residues involved in coordinating the copper were investigated using nuclear magnetic resonance spectroscopy (NMR) experiments, specifically by 1H-15N heteronuclear multiple bond correlation (HMBC), heteronuclear single quantum correlation (HSQC), and 1H-13C HSQCAD experiments. Protonation of Hst-8 and model poly-imidazole ligands resulted in distinct downfield shifts of proton, carbon and nitrogen resonances of the imidazolyl units. Tight binding metal complexes of model compounds were also observed to have downfield resonance shifts upon binding Cu(I). Unfortunately, Hst-8 does not bind copper tightly enough to permit observations of downfield shifted peaks. Peaks were instead observed to be broadened and in some cases absent. However, because these were localized to only certain residues, assignments were made on the basis of which resonances exhibited such behavior. This evidence in addition to computational results led to the conclusion that residues histidine 3, histidine 6, and histidine 7 are involved in the binding of Cu(I) in Hst-8 at a 1:1 Cu(I):Hst-8 ratio of concentration in DMSO.The conformational structure of Hst-8 in DMSO, both in the presence and absence of a copper (I) ion, has also been investigated using NMR experiments. The Nuclear Overhauser Effect (NOE), which is a nuclear spin transfer through space, was used in experimental series to identify protons in nearby space. This was useful in determining the larger three dimensional picture of the peptide in solution when the identities of the proton resonances were identified via total correlation spectroscopy (TOCSY) and 1H-13C HSQC experiments. After the spatial contacts were identified, boundary restriction data was generated by MARDIGRAS and the restriction data was input into a molecular dynamics (MD) simulator CNS, which output the structure. We found that Hst-8 did not have either beta strand or alpha helical secondary structural elements, in DMSO either as a free ligand or in the presence of Cu(I)

Investigating Habituation to Premonitory Urges in Behavior Therapy for Tic Disorders

Behavior therapy is effective for Persistent Tic Disorders (PTDs), but behavioral processes facilitating tic reduction are not well understood. One process, habituation, is thought to create tic reduction through decreases in premonitory urge severity. The current study tested whether premonitory urges decreased in youth with PTDs (N = 126) and adults with PTDs (N = 122) who participated in parallel randomized clinical trials comparing behavior therapy to psychoeducation and supportive therapy (PST). Trends in premonitory urges, tic severity, and treatment outcome were analyzed according to the predictions of a habituation model, whereby urge severity would be expected to decrease in those who responded to behavior therapy. Although adults who responded to behavior therapy showed a significant trend of declining premonitory urge severity across treatment, results failed to demonstrate that behavior therapy specifically caused changes in premonitory urge severity. In addition, reductions in premonitory urge severity in those who responded to behavior therapy were significant greater than those who did not respond to behavior therapy but no different than those who responded or did not respond to PST. Children with PTDs failed to show any significant changes in premonitory urges. Reductions in premonitory urge severity did not mediate the relationship between treatment and outcome in either adults or children. These results cast doubt on the notion that habituation is the therapeutic process underlying the effectiveness of behavior therapy, which has immediate implications for the psychoeducation and therapeutic rationale presented in clinical practice. Moreover, there may be important developmental changes in premonitory urges in PTDs, and alternative models of therapeutic change warrant investigation

Antiviral T cell responses: phalanx or multipronged attack?

Around 700 BCE, a new military formation called the phalanx was established in ancient Greece: a tight column of heavy infantry carrying long spears, or pikes, used in a single prong of attack. Later, in the battle of Marathon described by Herodotus, the Greeks learned the advantages of multipronged attacks, a strategy still used in modern warfare. Is the immune system similar in its approach to combating pathogens or tumors

Assignment of the Human TYRP (brown) Locus to Chromosome Region 9p23 by Nonradioactive in Situ Hybridization

The TYRP (brown) locus determines pigmentation and coat color in the mouse. The human homolog of the TYRP locus has been recently identified and shown to encode a 75-KDA transmembrane melanosomal glycoprotein called gp75. The gp 75 glycoprotein is homologous to tyrosinase, an enzyme in evolved in the synthesis of melanin, forming a family of tyrosinase-related proteins. A genomic clone of human gp75 was used to map the human TYRP locus to chromosome 9, region 9p23, by nonradioactive fluorescent in situ hybridization. Specificity of hybridization was tested with a genomic fragment of hu- man tyrosinase that mapped to a distinct site on 1lq2 1. The 9p region has been reported to be nonrandomly altered in human melanoma, suggesting a role for the region near the TYRP locus in melanocyte transformation

Blockade of Treg derived TGF-β abrogates suppression of effector T cell function within the tumor microenvironment

Regulatory T cells (Treg) play a role in suppression of anti-melanoma immunity; however, the exact mechanism is poorly understood. Through intravital two photon microscopy, we found that Pmel-1 effectors engage in cell-cell interactions with tumor resident Tregs. To determine if contact between Tregs and T effectors (Teff) hinders killing of tumor cells in vivo, we utilized ex-vivo three-dimensional collagen-fibrin gel cultures of B16 melanoma cells. Collagen-fibrin gel cultures recapitulated the in vivo suppression, rendering the dissociated tumor resistant to killing by in vitro activated antigen specific Teff. In vivo depletion of Tregs in foxp3-DTR mice prior to tumor excision reversed the suppression. Additionally, In vivo modulation of intra-tumor Tregs suppressive function by GITR ligation had a similar effect, leading to ex-vivo tumor killing. Using neutralizing antibodies, we found that blocking TGF-β reversed the suppression. In addition, soluble factors from collagen-fibrin gel tumors do not inhibit killing suggesting that suppression is contact or proximity dependent. The CD8 Teff recovered from these gels exhibit a decrease in Granzyme B expression and an increase in expression of T cell exhaustion marker PD-1. These findings support the conclusion that intra-tumor contact with Tregs during the effector phase of the immune response is responsible for inhibiting anti-melanoma immunity in a TGF-β dependent manner, elucidating a novel way to target intratumoral Tregs

Concomitant Tumor Immunity to a Poorly Immunogenic Melanoma Is Prevented by Regulatory T Cells

Concomitant tumor immunity describes immune responses in a host with a progressive tumor that rejects the same tumor at a remote site. In this work, concomitant tumor immunity was investigated in mice bearing poorly immunogenic B16 melanoma. Progression of B16 tumors did not spontaneously elicit concomitant immunity. However, depletion of CD4+ T cells in tumor-bearing mice resulted in CD8+ T cell–mediated rejection of challenge tumors given on day 6. Concomitant immunity was also elicited by treatment with cyclophosphamide or DTA-1 monoclonal antibody against the glucocorticoid-induced tumor necrosis factor receptor. Immunity elicited by B16 melanoma cross-reacted with a distinct syngeneic melanoma, but not with nonmelanoma tumors. Furthermore, CD8+ T cells from mice with concomitant immunity specifically responded to major histocompatibility complex class I–restricted epitopes of two melanocyte differentiation antigens. RAG1−/− mice adoptively transferred with CD8+ and CD4+ T cells lacking the CD4+CD25+ compartment mounted robust concomitant immunity, which was suppressed by readdition of CD4+CD25+ cells. Naturally occurring CD4+CD25+ T cells efficiently suppressed concomitant immunity mediated by previously activated CD8+ T cells, demonstrating that precursor regulatory T cells in naive hosts give rise to effective suppressors. These results show that regulatory T cells are the major regulators of concomitant tumor immunity against this weakly immunogenic tumor