Toward the development of a synthetic group A streptococcal vaccine of high purity and broad protective coverage

Using native chemical ligation, we synthesized a group A streptococcal. (GAS) vaccine that contained three different GAS M protein peptide epitopes in a chemically well-characterized construct in high purity. Two of the peptide epitopes represented variable amino terminal serotype determinants, and the third represented a carboxyl terminal conserved region determinant of the GAS M protein. We also synthesized a lipid core peptide (LCP) construct containing the same three peptides. Upon immunization of mice, the non-LCP construct only elicited antibody responses to all three epitopes with the use of adjuvant. The LCP construct, however, elicited excellent antibody responses to all three epitopes without the need for any additional adjuvant or carrier. We have synthesized the LCP synthetic vaccine system with good reproducibility.

Group A streptococcal vaccine delivery by immunization with a self-adjuvanting M protein-based lipid core peptide construct

Background & objectives: To develop a broad strain coverage GAS vaccine, several strategies have been investigated which included multi-epitope approaches as well as targeting the M protein conserved C-region. These approaches, however, have relied on the use of adjuvants that are toxic for human application. The development of safe and effective adjuvants for human use is a key issue in the development of effective vaccines. In this study, we investigated the lipid polylysine core peptide (LCP) system as a self-adjuvanting GAS vaccine delivery approach. Methods: An LCP-GAS construct was synthesised incorporating multiple copies of a protective peptide epitope (J8) from the conserved carboxy terminal C-repeat region of the M protein. B10.BR mice were immunized parenterally with the LCP-J8 construct, with or without conventional adjuvant, prior to the assessment of immunogenicity and the induction of serum opsonic antibodies. Results: Our data demonstrated immunogenicity of LCP-J8 when coadministered in complete Freund's adjuvant (CFA), or administered in the absence of conventional adjuvant. In both cases, immunization led to the induction of high-titre J8 peptide-specific serum IgG antibody responses, and the induction of heterologous opsonic antibodies that did not cross-react with human heart tissue proteins. Interpretation & conclusion: These data indicated the potential of a novel self-adjuvanting LCP vaccine delivery system incorporating a synthetic GAS M protein C-region peptide immunogen in the induction of broadly protective immune responses, and pointed to the potential application of this system in human vaccine development against infectious diseases.

Synthesis and immunological evaluation of M protein targeted tetra-valent and tri-valent group A streptococcal vaccine candidates based on the lipid-core peptide system

Group A streptococcus (GAS) is responsible for causing many clinical complications including the relatively benign streptococcal pharyngitis and impetigo. However. if left untreated. these conditions may lead to more severe diseases such as rheumatic fever (RF) and rheumatic heart disease (RHD). These diseases exhibit high morbidity and mortality, Particularly in developing countries and in indigenous populations of affluent countries. Only ever occur following GAS infection, a vaccine offers Promise for their Prevention. As stich, we have investigated the Use of the lipid-core peptide (LCP) system for the development of multi-valent Prophylactic GAS vaccines. The current study has investigated the capacity of this system to adjuvant LIP to four different GAS peptide epitopes. Presented are the synthesis and immunological assessment of tetra-valent and tri-valent GAS LCP systems. We demonstrated their capacity to elicit systemic IgG antibody responses in B10.BR mice to all GAS peptide epitopes. The data also showed that the LCP systems Were self-adjuvanting. These findings are particularly encouraging for the development of multi-valent LCP-based GAS vaccines.

Novel high water content hydrogels

Hydrogels may be described as cross-linked hydrophilic polymers that swell but do not dissolve in water. The production of high water content hydrogels was the subject of investigation. Based upon copolymer compositions that had already achieved commercial success as biomaterials, new monomers were added or substituted in and the effects observed. The addition of N-isopropyl acrylamide to an acrylamide-based composition that had previously been designed to become a contact lens, produced materials that showed smart effects in that the water content showed dependence on the temperature of the hydrating solution. Such thermo-responsive materials have potential uses in drug delivery, ultrafiltration and cell culture surfaces. Proteoglycans in nature have an important role to play in structural support where a highly hydrophilic structure maintains lubricious surfaces. Certain functional groups that impart this hydrophilicity are present in certain sulphonate monomers, Bis(3-sulphopropyl ester) itaconate, dipotassium salt (SPI), 3-Sulphopropyl ester acrylate, potassium salt (SPA) and Sodium 2-(acrylamido)-2-methyl propane sulphonate (NaAMPS). These monomers were incorporated into a HEMA-based copolymer that had been designed initially as a contact lens and the resulting effects examined. Highly hydrophilic materials resulted that showed reduced protein deposition over the neutral core material. It is postulated that a sulphonate group would have a larger number of hydration shells around it than for example methacrylic acid, leading to more dynamic exchange and so reducing the adsorption of biological solutes. A cationic monomer was added to bring back the net anionic nature of the sulphonate hydrogels and the effects studied. Ionic interactions were found to cause a reduction in the water content of the resulting materials as the mobility of the network decreased, leading to stiffer but less extensible materials. The presence of a net dominant charge, whether negative or positive, appeared to act to reduce protein deposition, but increasing equivalence in the amount of both charges served to present a more 'neutral' surface and deposition subsequently increased. The grafting of hydrophilic hydrogel layers onto silicone elastomer was attempted and the results evaluated using dynamic contact angle measurements. Following plasma oxidation to reduce the surface energy barrier to aqueous grafting chemistry, it was found that the wettability of the modified elastomers could be significantly enhanced by such treatment. The SPA-grafted material in particular hinted at an osmotic drive for rehydration that may be exploited in biomaterials.

Effect of maternal protein restriction during pregnancy and postweaning high-fat feeding on diet-induced thermogenesis in adult mouse offspring

Purpose: Prenatal undernutrition followed by postweaning feeding of a high-fat diet results in obesity in the adult offspring. In this study, we investigated whether diet-induced thermogenesis is altered as a result of such nutritional mismatch. Methods: Female MF-1 mice were fed a normal protein (NP, 18 % casein) or a protein-restricted (PR, 9 % casein) diet throughout pregnancy and lactation. After weaning, male offspring of both groups were fed either a high-fat diet (HF; 45 % kcal fat) or standard chow (C, 7 % kcal fat) to generate the NP/C, NP/HF, PR/C and PR/HF adult offspring groups (n = 7-11 per group). Results: PR/C and NP/C offspring have similar body weights at 30 weeks of age. Postweaning HF feeding resulted in significantly heavier NP/HF offspring (P <0.01), but not in PR/HF offspring, compared with their chow-fed counterparts. However, the PR/HF offspring exhibited greater adiposity (P <0.01) v the NP/HF group. The NP/HF offspring had increased energy expenditure and increased mRNA expression of uncoupling protein-1 and β-3 adrenergic receptor in the interscapular brown adipose tissue (iBAT) compared with the NP/C mice (both at P <0.01). No such differences in energy expenditure and iBAT gene expression were observed between the PR/HF and PR/C offspring. Conclusions: These data suggest that a mismatch between maternal diet during pregnancy and lactation, and the postweaning diet of the offspring, can attenuate diet-induced thermogenesis in the iBAT, resulting in the development of obesity in adulthood. © 2014 Springer-Verlag Berlin Heidelberg.

High protein flexibility and reduced hydration water dynamics are key pressure adaptive strategies in prokaryotes

Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

Association of the TNF-alpha -308 G/A polymorphisms with metabolic responses secondary to a high protein/low carbohydrate versus a standard hypocaloric diet

Background: Mutation analysis has identified a G-> A transition in the promoter region of TNF-alpha gene at position -308 (rs1800629). Objective: The aim of our study was to investigate the influence of polymorphism in -308 GA promoter variant of the TNF alpha gene on metabolic response and weight loss secondary to two hypocaloric diets. Method: A sample of 283 obese subjects was enrolled in a consecutive prospective way. In the basal visit, patients were randomly allocated during 9 months to diet HP (high protein/low carbohydrate hypocaloric diet) and diet S (standard hypocaloric diet). Results: There were no significant differences between the positive effects on weight loss in either genotype group with both diets. With both diets and only in wild genotype (diet HP vs. diet S), total cholesterol (-9.1 ± 3.4 mg/dL vs. -6.9 ± 2.0 mg/dL; p > 0.05), LDL cholesterol (-9.0 ± 2.9 mg/dL vs. -6.5 ± 2.1 mg/dL; p > 0.05) and triglycerides (-23.1 ± 5.1 mg/dL vs. -12.3 ± 4.8 mg/dL; p < 0.05) decreased. The improvement in triglycerides was higher in subjects without A allele. With diet HP and only in wild genotype, insulin levels (-3.1 ± 1.8 UI/L; p < 0.05) and HOMA-R (-0.8 ± 0.1 units; p < 0.05) decreased. Conclusion: Carriers of -308 GG promoter variant of TNF-alpha gene have a better metabolic response than -308 GA obese with a high protein hypocaloric diet.

Magnesium-deficient High-fat Diet: Effects On Adiposity, Lipid Profile And Insulin Sensitivity In Growing Rats.

To determine if magnesium deficiency aggravates the effects of a high-fat diet in growing rats in terms of obesity, lipid profile and insulin resistance. The study population comprised 48 newly weaned male Wistar Hannover rats distributed into four groups according to diet, namely, control group (CT; n = 8), control diet provided ad libitum; pair-feeding control group (PF; n = 16), control diet but in the same controlled amount as animals that received high-fat diets; high-fat diet group (HF; n = 12), and magnesium-deficient high-fat diet group (HFMg(-); n = 12). The parameters investigated were adiposity index, lipid profile, magnesium status, insulin sensitivity and the phosphorylation of proteins involved in the insulin-signaling pathway, i.e. insulin receptor β-subunit, insulin receptor substrate 1 and protein kinase B. The HF and HFMg(-) groups were similar regarding gain in body mass, adiposity index and lipid profile, but were significantly different from the PF group. The HFMg(-) group exhibited alterations in magnesium homeostasis as revealed by the reduction in urinary and bone concentrations of the mineral. No inter-group differences were observed regarding glucose homeostasis. Protein phosphorylation in the insulin-signaling pathway was significantly reduced in the high-fat groups compared with the control groups, demonstrating that the intake of fat-rich diets increased insulin resistance, a syndrome that was aggravated by magnesium deficiency. Under the experimental conditions tested, the intake of a magnesium-deficient high-fat diet led to alterations in the insulin-signaling pathway and, consequently, increased insulin resistance.

Taurine Supplementation Reduces Blood Pressure And Prevents Endothelial Dysfunction And Oxidative Stress In Post-weaning Protein-restricted Rats.

Taurine is a sulfur-containing amino acid that exerts protective effects on vascular function and structure in several models of cardiovascular diseases through its antioxidant and anti-inflammatory properties. Early protein malnutrition reprograms the cardiovascular system and is linked to hypertension in adulthood. This study assessed the effects of taurine supplementation in vascular alterations induced by protein restriction in post-weaning rats. Weaned male Wistar rats were fed normal- (12%, NP) or low-protein (6%, LP) diets for 90 days. Half of the NP and LP rats concomitantly received 2.5% taurine supplementation in the drinking water (NPT and LPT, respectively). LP rats showed elevated systolic, diastolic and mean arterial blood pressure versus NP rats; taurine supplementation partially prevented this increase. There was a reduced relaxation response to acetylcholine in isolated thoracic aortic rings from the LP group that was reversed by superoxide dismutase (SOD) or apocynin incubation. Protein expression of p47phox NADPH oxidase subunit was enhanced, whereas extracellular (EC)-SOD and endothelial nitric oxide synthase phosphorylation at Ser 1177 (p-eNOS) were reduced in aortas from LP rats. Furthermore, ROS production was enhanced while acetylcholine-induced NO release was reduced in aortas from the LP group. Taurine supplementation improved the relaxation response to acetylcholine and eNOS-derived NO production, increased EC-SOD and p-eNOS protein expression, as well as reduced ROS generation and p47phox expression in the aortas from LPT rats. LP rats showed an increased aortic wall/lumen ratio and taurine prevented this remodeling through a reduction in wall media thickness. Our data indicate a protective role of taurine supplementation on the high blood pressure, endothelial dysfunction and vascular remodeling induced by post-weaning protein restriction. The beneficial vascular effect of taurine was associated with restoration of vascular redox homeostasis and improvement of NO bioavailability.

Whey And Soy Protein Supplements Changes Body Composition In Patients With Crohn's Disease Undergoing Azathioprine And Anti-tnf-alpha Therapy.

Crohn´s disease (CD) is a chronic transmural inflammation of the gastrointestinal tract of unknown cause. Malnutrition associated with active CD has been reduced although obesity has increased. Dietary strategies such as those with high-protein have been proposed to reduce body fat. This study compares the effects of two supplements on the nutritional status of CD patients. 68 CD patients were randomized in two groups: whey protein group (WP) and soy protein group (SP). Using bioimpedance analysis, anthropometry and albumin and pre-albumin dosages the nutritional status was measured before starting the intervention and after 8 and 16 weeks. The disease activity was determined by Crohn's Disease Activity Index and serum C-reactive protein dosage and dietary intake by 24h dietary recalls. Forty-one patients concluded the study and both supplements changed body composition similarly. Triceps skin fold thickness (p< 0.001) and body fat percentage (p=0.001) decreased, whereas mid-arm muscle circumference (p=0.004), corrected arm muscle area (p=0.005) and body lean percentage (p=0.001) increased. For Crohn's disease patients undergoing anti TNF-alpha and azatioprine therapies, supplementation with whey and soy proteins changes body composition through reduction of body fat and thus contributes to control inflammation.

Increased Expression Of Hes5 Protein In Notch Signaling Pathway In The Hippocampus Of Mice Offspring Of Dams Fed A High-fat Diet During Pregnancy And Suckling.

Maternal high-fat diet (HFD) impairs hippocampal development of offspring promoting decreased proliferation of neural progenitors, in neuronal differentiation, in dendritic spine density and synaptic plasticity reducing neurogenic capacity. Notch signaling pathway participates in molecular mechanisms of the neurogenesis. The activation of Notch signaling leads to the upregulation of Hes5, which inhibits the proliferation and differentiation of neural progenitors. This study aimed to investigate the Notch/Hes pathway activation in the hippocampus of the offspring of dams fed an HFD. Female Swiss mice were fed a control diet (CD) and an HFD from pre-mating until suckling. The bodyweight and mass of adipose tissue in the mothers and pups were also measured. The mRNA and protein expression of Notch1, Hes5, Mash1, and Delta1 in the hippocampus was assessed by RT-PCR and western blotting, respectively. Dams fed the HFD and their pups had an increased bodyweight and amount of adipose tissue. Furthermore, the offspring of mothers fed the HFD exhibited an increased Hes5 expression in the hippocampus compared with CD offspring. In addition, HFD offspring also expressed increased amounts of Notch1 and Hes5 mRNA, whereas Mash1 expression was decreased. However, the expression of Delta1 did not change significantly. We propose that the overexpression of Hes5, a Notch effector, downregulates the expression of the proneural gene Mash1 in the offspring of obese mothers, delaying cellular differentiation. These results provide further evidence that an offspring's hippocampus is molecularly susceptible to maternal HFD and suggest that Notch1 signaling in this brain region is important for neuronal differentiation.

Fracionamento dos carboidratos pelas equações do Cornell Net Carbohydrate and Protein System de três cultivares de girassol na presença ou não de irrigação

Objetivou-se quantificar as frações de carboidratos pelas equações do Cornell Net Carbohydrate and Protein System (CNCPS) de três cultivares de girassol (Helianthus annuus L.) cultivados na presença ou não de irrigação. A utilização de uma preparação fibrosa, denominada parede celular (PC), nas equações da CNCPS, em substituição à fibra em detergente neutro (FDN) não promoveu diferenças nas frações de carboidratos B1 e C, mas influenciou as frações A e B2. Como os valores da fração B1, obtidos pelo modelo CNCPS foram menores que os teores de amido e pectina determinados em laboratório, supõe-se que a pectina e outros oligossacarídeos da parede celular, solubilizados pela solução de detergente neutro (fibra solúvel), nunca fizeram parte da fração B1, e sim da fração A. Apesar de os carboidratos da fibra solúvel apresentarem elevadas taxas de degradação, não parece adequada a caracterização da fibra solúvel na fração A. Parece mais adequado que a fibra solúvel (que inclui a pectina) seja alocada a uma fração exclusivamente sua, que pode ser a fração B2, e que seja criada uma nova fração, a B3, para os carboidratos digeríveis da parede celular. Assim, a fração B1 seria composta apenas de amido. A equação da fração C, que estima os carboidratos indigeríveis da parede celular, pode ser simplificada, relacionando a fração indigerível ao teor de lignina na matéria seca, e não à FDN isenta de cinzas e proteína, como atualmente utilizado. Esta proposta tem implicações práticas, uma vez que a fração indigerível da parede celular tem sido expressa em relação à FDN, e não na MS, com base no fato de que os efeitos inibitórios da lignina ocorrem sobre os componentes fibrosos da parede celular vegetal, e não sobre o conteúdo celular.

Leucine supplementation improves adiponectin and total cholesterol concentrations despite the lack of changes in adiposity or glucose homeostasis in rats previously exposed to a high-fat diet

Background: Studies suggest that leucine supplementation (LS) has a therapeutic potential to prevent obesity and to promote glucose homeostasis. Furthermore, regular physical exercise is a widely accepted strategy for body weight maintenance and also for the prevention of obesity. The aim of this study was to determine the effect of chronic LS alone or combined with endurance training (ET) as potential approaches for reversing the insulin resistance and obesity induced by a high-fat diet (HFD) in rats. Methods: Forty-seven rats were randomly divided into two groups. Animals were fed a control diet-low fat (n = 10) or HFD (n = 37). After 15 weeks on HFD, all rats received the control diet-low fat and were randomly divided according to treatment: reference (REF), LS, ET, and LS+ET (n = 7-8 rats per group). After 6 weeks of treatment, the animals were sacrificed and body composition, fat cell volume, and serum concentrations of total cholesterol, HDL-cholesterol, triacylglycerol, glucose, adiponectin, leptin and tumor necrosis factor-alpha (TNF-alpha) were analyzed. Results: At the end of the sixth week of treatment, there was no significant difference in body weight between the REF, LS, ET and LS+ET groups. However, ET increased lean body mass in rats (P = 0.019). In addition, ET was more effective than LS in reducing adiposity (P = 0.019), serum insulin (P = 0.022) and TNF-alpha (P = 0.044). Conversely, LS increased serum adiponectin (P = 0.021) levels and reduced serum total cholesterol concentration (P = 0.042). Conclusions: The results showed that LS had no beneficial effects on insulin sensitivity or adiposity in previously obese rats. On the other hand, LS was effective in increasing adiponectin levels and in reducing total cholesterol concentration.

The Function and Three-Dimensional Structure of a Thromboxane A(2)/Cysteinyl Leukotriene-Binding Protein from the Saliva of a Mosquito Vector of the Malaria Parasite

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD(7). In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the omega-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

Identification of protein-coding and non-coding RNA expression profiles in CD34(+) and in stromal cells in refractory anemia with ringed sideroblasts

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods: In this study, gene expression profiles of CD34(+) cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results: In CD34(+) cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value <= 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value <= 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions: These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34(+) cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.