Dynamics in Polysaccharide Glasses and Their Impact on the Stability of Encapsulated Flavors

The aim of this work is to examine the correlation between measured instability of model flavor compounds in glassy matrices with the calorimetric relaxation times of the matrices. Spray-dried carbohydrate matrices were chosen as the model compounds for this study. Enthalpy relaxation times were determined for spray-dried carbohydrate matrices using differential and isothermal calorimetric methods. The losses of the volatile methyl acetate, ethyl acetate and limonene, as well as formation of limonene oxidation products, were measured by gas chromatography. Storage conditions were 30 and 40 °C, with samples equilibrated with 11, 23, 33 and 43 % RH at each temperature. A comparison of the relaxation times for temperatures below Tg was made using Modulated DSC (MDSC) and a Thermal Activity Monitor (TAM). TAM yields significantly lower values for relaxation times implying that it is capturing some of the faster dynamics as well as dynamics that are activated near Tg. However, plots of relaxation times as determined by both techniques versus temperature appear to converge at Tg. An increase in the relative humidity results in moderately higher loss of volatiles (methyl acetate, ethyl acetate and limonene) and greater oxidation rates. In general, there is a good correlation between relaxation time and stability, with greater enthalpy relaxation time associated with better stability. Enthalpy relaxation time appears to be a useful predictor of stability for both loss of volatiles and oxidation of limonene.     

Identifying Patient-Specific Epstein-Barr Nuclear Antigen-1 Genetic Variation and Potential Autoreactive Targets Relevant to Multiple Sclerosis Pathogenesis

Background

Epstein-Barr virus (EBV) infection represents a major environmental risk factor for multiple sclerosis (MS), with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1)-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1*15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome.

Methods

Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1*1501 epitopes (NetMHCII, NetMHCIIpan) and candidates were evaluated for cross recognition with human brain proteins.

Results

EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cut-off). In silico epitope mapping revealed two known HLA-DRB1*1501-restricted epitopes (‘AEG’: aa 481–496 and ‘MVF’: aa 562–577), and two putative epitopes between positions 502–543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1*15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis.

Conclusions

This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains. This approach has identified a number of immunogenic regions of EBNA-1 as well as known and novel targets for autoreactive HLA-DRB1*15-restricted T cells within the central nervous system that could arise as a result of cross-reactivity with EBNA-1-specific immune responses.     

Semisynthetic hemoglobin A: reconstitution of functional tetramer from semisynthetic alpha-globin

The optimal conditions for the semisynthesis of alpha-globin through Staphylococcus aureus V8 protease condensation of a synthetic fragment (alpha 1-30) with the complementary apo fragment (alpha 31-141) in the presence of structure-inducing organic cosolvents and the reconstitution of the functional tetramer from semisynthetic alpha-globin have been investigated. The protease-catalyzed ligation of the complementary apo fragments alpha 1-30 and alpha 31-141 proceeds with very high selectivity at pH 6.0 and 4 degrees C in the presence of 1-propanol as the organic cosolvent. A 30% 1-propanol solution was optimal for the semisynthetic reaction, and the synthetic reaction attained an equilibrium (approximately 50%) in 72 h. The synthetic reaction proceeds smoothly over a wide pH range (pH 5-8). Besides, the semisynthetic system is flexible, and it also proceeded well if trifluoroethanol or 2-propanol was used instead of 1-propanol. However, glycerol, a versatile organic cosolvent used in all other proteosynthetic reactions reported in the literature, was not very efficient as an organic cosolvent in the present synthetic reaction. The semisynthetic alpha-globin prepared with 1-propanol as the organic cosolvent has been reconstituted into HbA. The semisynthetic HbA was then purified by CM-cellulose chromatography. The semisynthetic HbA is indistinguishable from native HbA, in terms of its structural and functional properties. The semisynthetic approach provides the flexibility in protein engineering studies for the incorporation of spectroscopic labels (13C- and/or 15N-labeled amino acids), noncoded amino acids, or unnatural bond functionalities, which at present is not possible with genetic approaches.     

Some effects of organic acid supplementation on utilization of ammonium compounds by three pathogenic fungi

Utilization of ammonium nitrogen byFusarium moniliforme Sheld.,Curvularia verruciformis Agarwal &Sahni andSclerotium rolfsii Sacc., with and without organic acid supplementation was studied. Ammonium utilization by these organisms generally improved with small amounts of organic acid supplementation except in the case ofC. verruciformis on ammonium chloride where fumaric acid was observed to suppress utilization. Effects of supplementation were seen to be dependent on the nature of the organic acid, the ammonium source used and the organism employed. Further evidence obtained by varying the quantities of succinic acid in ammonium chloride media showed that higher levels of supplementation gave increased growth of the fungi and prevented the pH from falling. It seems that these acids play a dual role as pH buffers as well as nutritional compounds.     

Effect of phenolic acids on anthocyanin content in maize roots

Supply of 0.01 to 5.0 mM salicylic, caffeic and gallic acids, either during imbibition of seeds for 24 to 48 h or during seedling growth increased anthocyanin production in maize (Zea mays L. cv. Ganga safed-2) roots. While tyrosine had no effect, phenylalanine either in the presence or absence of the phenolic acids increased anthocyanin content. Glucose in a concentration range of 1 to 20 mM and shikimic acid in 0.01 to 5.0 mM range also increased pigment level, which was higher in the presence of salicylic acid than in it.s absence. The experiments demonstrate the possibility of some indirect effects of salicylic acid and other phenolic acids on anthocyanin synthesis.     

Synthesis and application of β-carbolines as novel multi-functional anti-Alzheimer’s disease agents

The design, synthesis and assessment of β-carboline core-based compounds as potential multifunctional agents against several processes that are believed to play a significant role in Alzheimer’s disease (AD) pathology, are described. The activity of the compounds was determined in Aβ self-assembly (fibril and oligomer formation) and cholinesterase (AChE, BuChE) activity inhibition, and their antioxidant properties were also assessed. To obtain insight into the mode of action of the compounds, HR-MS studies were carried out on the inhibitor-Aβ complex formation and molecular docking was performed on inhibitor-BuChE interactions. While several compounds exhibited strong activities in individual assays, compound 14 emerged as a promising multi-target lead for the further structure-activity relationship studies.     

Molecular insights into development of Trichoderma interfusants for multistress tolerance enhancing antagonism against Sclerotium rolfsii Sacc

The current study aimed at developing diverse Trichoderma fusants for fungicides, drought, and salt tolerance with enhanced antagonistic activity against Sclerotium rolfsii Sacc. Trichoderma virens NBAII Tvs12 (mycoparasitic) and Trichoderma koningii MTCC796 (multistress tolerant) were used as parental strains for development of interspecific protoplast fusants. A total of 36 stable fusants were used for mycoparasitism, fungicides, and abiotic stresses (drought and salt) tolerance. The results revealed 20 homozygous progenies showing characteristics of either one parental strain and 14 heterozygous mutants depicting traits of both parental strains. A novel concept of inhibition coefficient was established using growth-related key parameters that represent the pathogen biology and the biocontrol-related biophysics of Trichoderma fusants. The results indicated a differential inhibition coefficient of the test pathogen and the highest (92.88%) inhibition coefficient of S. rolfsii was observed by interstable fusant Fu21. It also grew better under fungicides and abiotic stress (drought and salt) conditions. The molecular characterization and heterozygosity analysis evidenced the highest observed heterozygosity (0.5441) and gene flow (0.3872) in stable heterozygous Fu21. Principal coordinates analysis exhibited 62.7% of total variability. The ecofriendly heterozygous Trichoderma fusant (Fu21) might be useful for biocontrol of stem rot disease under adverse conditions or as a part of integrated disease management.