High pressure-low temperature processing of food proteins

High pressure-low temperature (HP-LT) processing is of interest in the food field in view of: (i) obtaining a "cold" pasteurisation effect, the level of microbial inactivation being higher after pressurisation at low or sub-zero than at ambient temperature; (ii) limiting the negative impact of atmospheric pressure freezing on food structures. The specific effects of freezing by fast pressure release on the formation of ice I crystals have been investigated on oil in water emulsions stabilized by proteins, and protein gels, showing the formation of a high number of small ice nuclei compared to the long needle-shaped crystals obtained by conventional freezing at 0.1 MPa. It was therefore of interest to study the effects of HP-LT processing on unfolding or dissociation/aggregation phenomena in food proteins, in view of minimizing or controlling structural changes and aggregation reactions, and/or of improving protein functional properties. In the present studies, the effects of HP-LT have been investigated on protein models such as (i) beta-lactoglobulin, i.e., a whey protein with a well known 3-D structure, and (ii) casein micelles, i.e., the main milk protein components, the supramolecular structure of which is not fully elucidated. The effects of HP-LT processing was studied up to 300 MPa at low or sub-zero temperatures and after pressure release, or up to 200 MPa by UV spectroscopy under pressure, allowing to follow reversible structural changes. Pressurisation of approximately 2% beta-lactoglobulin solutions up to 300 MPa at low/subzero temperatures minimizes aggregation reactions, as measured after pressure release. In parallel, such low temperature treatments enhanced the size reduction of casein micelles.     

Novel 4,5-diaryl-3-hydroxy-2(5H)-furanones as anti-oxidants and anti-inflammatory agents

In order to study the effect of phenol moieties on biological activities of ascorbic acid derivatives, we synthesized 13 novel 4,5-diaryl-3-hydroxy-2(5H)-furanones 5a-m with various substitution patterns. Compound 5 g bearing a 2,3-dihydroxy phenyl ring on the 5-position of the heterocycle appeared to be the most powerful anti-oxidant furanone with reducing activity against DPPH (IC(50)=10.3 microM), superoxide anion quenching capacity (IC(50)=0.187 mM) and lipid peroxidation inhibitory effect (IC(50)=0.129 mM). To ascertain determinant molecular features for anti-oxidant activities, structure-activity relationships were studied. Lipophilicity and molecular parameters related to electron distribution and structure (difference in heats of formation between the compound and its radical or its cation radical, energy of the highest occupied molecular orbital, HOMO) were found to correlate with the anti-oxidant action of compounds 5 in the different tests used. Oxygen-derived free radicals are known to contribute to inflammatory disorders; therefore we have investigated effects of compounds 5 in two models of inflammation: phorbol ester-induced ear edema in mice (TPA-test) and carrageenan-induced paw edema in rat. At 100 mg/kg ip in the TPA-test, the anti-inflammatory activity of compounds 5 was potent compared with that of indomethacin and ketorolac and all the results suggested a cyclooxygenase inhibition in the emergence of such properties. The combined pharmacological actions of compounds 5 associated with a favorable therapeutic index prompt with interesting perspectives for their use in heart and brain disorders as well as in inflammatory diseases.     

Cellular characterisation of Candida tropicalis presenting fluconazole-related trailing growth

We assessed fluconazole susceptibility in 52 Candida tropicalis clinical strains using seven antifungal susceptibility methods, including broth microdilution (BMD) [standard M27 A3 (with neutral and acid pH), ATB Fungus 3, Vitek 2 system and flow cytometric analysis] and agar-based methods (disk diffusion and E-test). Trailing growth, detection of cell-associated secreted aspartic proteases (Saps) and morphological and ultrastructural traits of these clinical strains were also examined. The ranges of fluconazole 24 h-minimum inhibitory concentration (MIC) values were similar among all methods. The essential agreement among the methods used for MIC determinations was excellent and all methods categorised all strains as susceptible, except for one strain that showed a minor error. The presence of the trailing effect was assessed by six methods. Trailing positivity was observed for 86.5-100% of the strains. The exception was the BMD-Ac method where trailing growth was not observed. Morphological and ultrastructural alterations were detected in C. tropicalis trailing cells, including mitochondrial swelling and cell walls with irregular shapes. We tested the production of Saps in 13 C. tropicalis strains expressing trailing growth through flow cytometry. Our results showed that all of the C. tropicalis strains up-regulated surface Sap expression after 24 h or 48 h of exposure to fluconazole, which was not observed in untreated yeast strains. We concluded that C. tropicalis strains expressing trailing growth presented some particular features on both biological and ultrastructural levels.     

Interacting epidemics? Sleep curtailment, insulin resistance, and obesity

In the last 50 years, the average self-reported sleep duration in the United States has decreased by 1.5-2 hours in parallel with an increasing prevalence of obesity and diabetes. Epidemiological studies and meta-analyses report a strong relationship between short or disturbed sleep, obesity, and abnormalities in glucose metabolism. This relationship is likely to be bidirectional and causal in nature, but many aspects remain to be elucidated. Sleep and the internal circadian clock influence a host of endocrine parameters. Sleep curtailment in humans alters multiple metabolic pathways, leading to more insulin resistance, possibly decreased energy expenditure, increased appetite, and immunological changes. On the other hand, psychological, endocrine, and anatomical abnormalities in individuals with obesity and/or diabetes can interfere with sleep duration and quality, thus creating a vicious cycle. In this review, we address mechanisms linking sleep with metabolism, highlight the need for studies conducted in real-life settings, and explore therapeutic interventions to improve sleep, with a potential beneficial effect on obesity and its comorbidities.     

Influence of temperature, light and nutrients on the growth rates of the macroalga Gracilaria domingensis in synthetic seawater using experimental design

In the present study, the daily relative growth rates (DRGR, in percent per day) of the red macroalga Gracilaria domingensis in synthetic seawater was investigated for the combined influence of five factors, i.e., light (L), temperature (T), nitrate (N), phosphate (P), and molybdate (M), using a statistical design method. The ranges of the experimental cultivation conditions were T, 18–26°C; L, 74–162?μmol photons m^?2?s^?1; N, 40–80?μmol?L^?1; P, 8–16?μmol?L^?1; and M, 1–5?nmol?L^?1. The optimal conditions, which resulted in a maximum growth rate of ≥6.4% d^?1 from 7 to 10?days of cultivation, were determined by analysis of variance (ANOVA) multivariate factorial analysis (with a 2⁵ full factorial design) to be L, 74?μmol photons m^?2?s^?1; T, 26°C; N, 80?μmol?L^?1; P, 8?μmol?L^?1; and M, 1?nmol?L^?1. In additional, these growth rate values are close to the growth rate values in natural medium (von Stosch medium), i.e., 6.5–7.0% d^?1. The results analyzed by the ANOVA indicate that the factors N and T are highly significant linear terms, X _L, (α?=?0.05). On the other hand, the only significant quadratic term (X _Q) was that for L. Statistically significant interactions between two different factors were found between T vs. L and N vs. T. Finally, a two-way (linear/quadratic interaction) model provided a quite reasonable correlation between the experimental and predicted DRGR values (R _adjusted ² ?=?0.9540).     

High-fat diets rich in soy or fish oil distinctly alter hypothalamic insulin signaling in rats

Hypothalamic insulin inhibits food intake, preventing obesity. High-fat feeding with polyunsaturated fats may be obesogenic, but their effect on insulin action has not been elucidated. The present study evaluated insulin hypophagia and hypothalamic signaling after central injection in rats fed either control diet (15% energy from fat) or high-fat diets (50% energy from fat) enriched with either soy or fish oil. Soy rats had increased fat pad weight and serum leptin with normal body weight, serum lipid profile and peripheral insulin sensitivity. Fish rats had decreased body and fat pad weight, low leptin and corticosterone levels, and improved serum lipid profile. A 20-mU dose of intracerebroventricular (ICV) insulin inhibited food intake in control and fish groups, but failed to do so in the soy group. Hypothalamic protein levels of IR, IRS-1, IRS-2, Akt, mTOR, p70S6K and AMPK were similar among groups. ICV insulin stimulated IR tyrosine phosphorylation in control (68%), soy (36%) and fish (34%) groups. Tyrosine phosphorylation of the pp185 band was significantly stimulated in control (78%) and soy (53%) rats, but not in fish rats. IRS-1 phosphorylation was stimulated only in control rats (94%). Akt serine phosphorylation was significantly stimulated only in control (90%) and fish (78%) rats. The results showed that, rather than the energy density, the fat type was a relevant aspect of high-fat feeding, since blockade of hypothalamic insulin signal transmission and insulin hypophagia was promoted only by the high-fat soy diet, while they were preserved in the rats fed with the high-fat fish diet.     

Genetic structure, nestmate recognition and behaviour of two cryptic species of the invasive big-headed ant Pheidole megacephala

Background

Biological invasions are recognized as a major cause of biodiversity decline and have considerable impact on the economy and human health. The African big-headed ant Pheidole megacephala is considered one of the world''s most harmful invasive species.

Methodology/Principal Findings

To better understand its ecological and demographic features, we combined behavioural (aggression tests), chemical (quantitative and qualitative analyses of cuticular lipids) and genetic (mitochondrial divergence and polymorphism of DNA microsatellite markers) data obtained for eight populations in Cameroon. Molecular data revealed two cryptic species of P. megacephala, one inhabiting urban areas and the other rainforests. Urban populations belong to the same phylogenetic group than those introduced in Australia and in other parts of the world. Behavioural analyses show that the eight populations sampled make up four mutually aggressive supercolonies. The maximum distance between nests from the same supercolony was 49 km and the closest distance between two nests belonging to two different supercolonies was 46 m. The genetic data and chemical analyses confirmed the behavioural tests as all of the nests were correctly assigned to their supercolony. Genetic diversity appears significantly greater in Africa than in introduced populations in Australia; by contrast, urban and Australian populations are characterized by a higher chemical diversity than rainforest ones.

Conclusions/Significance

Overall, our study shows that populations of P. megacephala in Cameroon adopt a unicolonial social structure, like invasive populations in Australia. However, the size of the supercolonies appears several orders of magnitude smaller in Africa. This implies competition between African supercolonies and explains why they persist over evolutionary time scales.     

Nitroxyl (HNO) stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation

Background

New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated.

Methods

Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli''s salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined.

Results

We now demonstrate that Angeli''s salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli''s salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli''s salt were mimicked by BNP. We also demonstrate that the effects of Angeli''s salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP).

Conclusions

Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.     

Proteome turnover in the green alga Ostreococcus tauri by time course 15N metabolic labeling mass spectrometry

Protein synthesis and degradation determine the cellular levels of proteins, and their control hence enables organisms to respond to environmental change. Experimentally, these are little known proteome parameters; however, recently, SILAC-based mass spectrometry studies have begun to quantify turnover in the proteomes of cell lines, yeast, and animals. Here, we present a proteome-scale method to quantify turnover and calculate synthesis and degradation rate constants of individual proteins in autotrophic organisms such as algae and plants. The workflow is based on the automated analysis of partial stable isotope incorporation with (15)N. We applied it in a study of the unicellular pico-alga Ostreococcus tauri and observed high relative turnover in chloroplast-encoded ATPases (0.42-0.58% h(-1)), core photosystem II proteins (0.34-0.51% h(-1)), and RbcL (0.47% h(-1)), while nuclear-encoded RbcS2 is more stable (0.23% h(-1)). Mitochondrial targeted ATPases (0.14-0.16% h(-1)), photosystem antennae (0.09-0.14% h(-1)), and histones (0.07-0.1% h(-1)) were comparatively stable. The calculation of degradation and synthesis rate constants k(deg) and k(syn) confirms RbcL as the bulk contributor to overall protein turnover. This study performed over 144 h of incorporation reveals dynamics of protein complex subunits as well as isoforms targeted to different organelles.     

Myocardial Chemokine Expression and Intensity of Myocarditis in Chagas Cardiomyopathy Are Controlled by Polymorphisms in CXCL9 and CXCL10

Background

Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium.

Methods and Results

Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2–6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes.

Conclusions

Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC.