Effect of Water Content on Glass Transition and Protein Aggregation of Whey Protein Powders During Short-Term Storage

The objectives of this study were to investigate the moisture-induced protein aggregation of whey protein powders and to elucidate the relationship of protein stability with respect to water content and glass transition. Three whey protein powder types were studied: whey protein isolate (WPI), whey protein hydrolysates (WPH), and beta-lactoglobulin (BLG). The water sorption isotherms were determined at 23 and 45°C, and they fit the Guggenheim–Andersson–DeBoer (GAB) model well. Glass transition was determined by differential scanning calorimeter (DSC). The heat capacity changes of WPI and BLG during glass transition were small (0.1 to 0.2 Jg⁻¹ °C⁻¹), and the glass transition temperature (T _g) could not be detected for all samples. An increase in water content in the range of 7 to 16% caused a decrease in T _g from 119 down to 75°C for WPI, and a decrease from 93 to 47°C for WPH. Protein aggregation after 2 weeks’ storage was measured by the increase in insoluble aggregates and change in soluble protein fractions. For WPI and BLG, no protein aggregation was observed over the range of 0 to 85% RH, whereas for WPH, ∼50% of proteins became insoluble after storage at 23°C and 85% RH or at 45°C and ≥73% RH, caused mainly by the formation of intermolecular disulfide bonds. This suggests that, at increased water content, a decrease in the T _g of whey protein powders results in a dramatic increase in the mobility of protein molecules, leading to protein aggregation in short-term storage.     

Plasma metabonomics as a novel diagnostic approach for major depressive disorder

Major depressive disorder (MDD) is a socially detrimental psychiatric disorder, contributing to increased healthcare expenditures and suicide rates. However, no empirical laboratory-based tests are available to support the diagnosis of MDD. In this study, a NMR-based plasma metabonomic method for the diagnosis of MDD was tested. Proton nuclear magnetic resonance ((1)H NMR) spectra of plasma sampled from first-episode drug-na??ve depressed patients (n = 58) and healthy controls (n = 42) were recorded and analyzed by orthogonal partial least-squares discriminant analysis (OPLS-DA). The OPLS-DA score plots of the spectra demonstrated that the depressed patient group was significantly distinguishable from the healthy control group. Moreover, the method accurately diagnosed blinded samples (n = 26) in an independent replication cohort with a sensitivity and specificity of 92.8% and 83.3%, respectively. Taken together, NMR-based plasma metabonomics may offer an accurate empirical laboratory-based method applicable to the diagnosis of MDD.     

Association of the CTLA4 Gene with Graves' Disease in the Chinese Han Population

To determine whether genetic heterogeneity exists in patients with Graves'' disease (GD), the cytotoxic T-lymphocyte associated 4 (CTLA-4) gene, which is implicated a susceptibility gene for GD by considerable genetic and immunological evidence, was used for association analysis in a Chinese Han cohort recruited from various geographic regions. Our association study for the SNPs in the CTLA4 gene in 2640 GD patients and 2204 control subjects confirmed that CTLA4 is the susceptibility gene for GD in the Chinese Han population. Moreover, the logistic regression analysis in the combined Chinese Han cohort revealed that SNP rs231779 (allele frequencies p = 2.81×10⁻⁹, OR = 1.35, and genotype distributions p = 2.75×10⁻⁹, OR = 1.42) is likely the susceptibility variant for GD. Interestingly, the logistic regression analysis revealed that SNP rs35219727 may be the susceptibility variant to GD in the Shandong population; however, SNP, rs231779 in the CTLA4 gene probably independently confers GD susceptibility in the Xuzhou and southern China populations. These data suggest that the susceptibility variants of the CTLA4 gene varied between the different geographic populations with GD.     

Emotional States Modulate the Recognition Potential during Word Processing

This study examined emotional modulation of word processing, showing that the recognition potential (RP), an ERP index of word recognition, could be modulated by different emotional states. In the experiment, participants were instructed to compete with pseudo-competitors, and via manipulation of the outcome of this competition, they were situated in neutral, highly positive, slightly positive, highly negative or slightly negative emotional states. They were subsequently asked to judge whether the referent of a word following a series of meaningless character segmentations was an animal or not. The emotional induction task and the word recognition task were alternated. Results showed that 1) compared with the neutral emotion condition, the peak latency of the RP under different emotional states was earlier and its mean amplitude was smaller, 2) there was no significant difference between RPs elicited under positive and negative emotional states in either the mean amplitude or latency, and 3) the RP was not affected by different degrees of positive emotional states. However, compared to slightly negative emotional states, the mean amplitude of the RP was smaller and its latency was shorter in highly negative emotional states over the left hemisphere but not over the right hemisphere. The results suggest that emotional states influence word processing.     

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer¹. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells^2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/α-ketoglutarate-dependent dioxygenases^{2, 3}. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development^{2, 5-10}. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC¹¹. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically¹².Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by β-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.     

Characterisation of Ilomastat for Prolonged Ocular Drug Release

We are developing tablet dosage forms for implantation directly into the subconjunctival space of the eye. The matrix metalloproteinase inhibitor, ilomastat, has previously been shown to be efficacious at suppressing scarring following glaucoma filtration surgery (GFS). We report on the physical characterisation of ilomastat which is being developed for ocular implantation. Since ilomastat is being considered for implantation it is necessary to examine its polymorphs and their influence on aspects of the in vitro drug release profile. X-ray powder diffraction identified two polymorphs of ilomastat from different commercial batches of the compound. Tablets were prepared from the two different polymorphs. Isothermal perfusion calorimetry was used to show that amorphous content is not increased during tablet formulation. The melting points of the two polymorphs are 188 and 208°C as determined by differential scanning calorimetry. Utilising single crystal X-ray diffraction, the structural conformations and packing arrangements of the different polymorphs were determined. The orthorhombic crystal crystallised as a monohydrate while the second monoclinic crystal form is non-solvated. Ilomastat tablets prepared from the two different solid forms exhibited similar drug release profiles in vitro under conditions mimicking the aqueous composition, volume and flow of the subconjunctival space after GFS. This suggests that a reproducible dose at each time point during release after implantation should be achievable in vivo with ilomastat tablets prepared from the two polymorphs identified.     

Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies

A major challenge in combating the human immunodeficiency virus (HIV) epidemic is the development of vaccines capable of inducing potent, persistent cellular immunity and broadly reactive neutralizing antibody responses to HIV type 1 (HIV-1). We report here the results of a preclinical trial using the chimpanzee model to investigate a combination vaccine strategy involving sequential priming immunizations with different serotypes of adenovirus (Ad)/HIV-1(MN)env/rev recombinants and boosting with an HIV envelope subunit protein, oligomeric HIV(SF162) gp140deltaV2. The immunogenicities of replicating and nonreplicating Ad/HIV-1(MN)env/rev recombinants were compared. Replicating Ad/HIV recombinants were better at eliciting HIV-specific cellular immune responses and better at priming humoral immunity against HIV than nonreplicating Ad-HIV recombinants carrying the same gene insert. Enhanced cellular immunity was manifested by a greater frequency of HIV envelope-specific gamma interferon-secreting peripheral blood lymphocytes and better priming of T-cell proliferative responses. Enhanced humoral immunity was seen in higher anti-envelope binding and neutralizing antibody titers and better induction of antibody-dependent cellular cytotoxicity. More animals primed with replicating Ad recombinants mounted neutralizing antibodies against heterologous R5 viruses after one or two booster immunizations with the mismatched oligomeric HIV-1(SF162) gp140deltaV2 protein. These results support continued development of the replicating Ad-HIV recombinant vaccine approach and suggest that the use of replicating vectors for other vaccines may prove fruitful.