Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterize the major angiotensin converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilization of β-lactoglobulin and casein derived peptides (CDP) from whey. The adsorbed proteins were hydrolyzed in-situ which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC50 =287μg/mL) and (ii) from β-lactoglobulin (IC50=128μg/mL). IC50 is the concentration of inhibitor needed to inhibit ACE by half. The well known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterized in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC50).

Staphylococcal extracellular adherence protein induces platelet activation by stimulation of thiol isomerases

OBJECTIVE: Staphylococcus aureus can induce platelet aggregation. The rapidity and degree of this correlates with the severity of disseminated intravascular coagulation, and depends on platelet peptidoglycans. Surface-located thiol isomerases play an important role in platelet activation. The staphylococcal extracellular adherence protein (Eap) functions as an adhesin for host plasma proteins. Therefore we tested the effect of Eap on platelets. METHODS AND RESULTS: We found a strong stimulation of the platelet-surface thiol isomerases protein disulfide isomerase, endoplasmic reticulum stress proteins 57 and 72 by Eap. Eap induced thiol isomerase-dependent glycoprotein IIb/IIIa activation, granule secretion, and platelet aggregation. Treatment of platelets with thiol blockers, bacitracin, and anti-protein disulfide isomerase antibody inhibited Eap-induced platelet activation. The effect of Eap on platelets and protein disulfide isomerase activity was completely blocked by glycosaminoglycans. Inhibition by the hydrophobic probe bis(1-anilinonaphthalene 8-sulfonate) suggested the involvement of hydrophobic sites in protein disulfide isomerase and platelet activation by Eap. CONCLUSIONS: In the present study, we found an additional and yet unknown mechanism of platelet activation by a bacterial adhesin, involving stimulation of thiol isomerases. The thiol isomerase stimulatory and prothrombotic features of a microbial secreted protein are probably not restricted to S aureus and Eap. Because many microorganisms are coated with amyloidogenic proteins, it is likely that the observed mechanism is a more general one.

Purified galactooligosaccharide, derived from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium adhesion and invasion in vitro and in vivo

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 411 71 using lactose as the substrate Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno (R) in reducing Salmonella enterica serovar Typhimurium (S Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS Here we wished to test the hypothesis that GOS, derived from Bimuno (R) may confer the direct anti-invasive and protective effects of Bimuno (R) In this study the efficacy of Bimuno (R), a basal solution of Bimuno (R) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno (R)] and purified GOS to reduce S Typhimurium adhesion and invasion was assessed using a series of in vitro and in vivo models The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics in vitro demonstrated that the presence of similar to 5 mg Bimuno (R) ml(-1) or similar to 2 5 mg GOS ml(-1) significantly reduced the invasion of S Typhimurium (SL1344nal(r)) (P<0 0001) Furthermore, similar to 2 5 mg GOS ml(-1) significantly reduced the adherence of S Typhimurium (SU 344nal(r)) (P<0 0001) It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions In the murine ligated deal gut loops, the presence of Bimuno (R) or GOS prevented the adherence or invasion of S Typhimurium to enterocytes, and thus reduced its associated pathology This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen In all assays, Bimuno (R) without GOS conferred no such protection, indicating that the basal solution confers no protective effects against S Typhimurium Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno (R) can be attributed to GOS

A mixture containing galactoollgosaccharide, produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium infection in mice

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharide, produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 41 .vertical bar 71 in the presence of lactose. Previous studies have implicated prebiotics in reducing infections by enteric pathogens, thus it was hypothesized that Bimuno (R) may confer some protection in the murine host from Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. In this study, infection caused by S. Typhimurium SL1344nal(r) in the presence or absence of Bimuno (R) was assessed using tissue culture assays, a murine ligated ileal gut loop model and a murine oral challenge model. In tissue culture adherence and invasion assays with HT-29-1 6E cells, the presence of similar to 2 mM Bimuno) significantly reduced the invasion of S. Typhimuriurn SL1 344nal(r) (p < 0.0001). In the murine ligated ileal gut loops, the presence of Bimuno (R) prevented colonization and the associated pathology of S. Typhimurium. In the BALB/c mouse mocel, the oral delivery of Bimuno prior to challenge with S. Typhimurium resulted in significant reductions in colonization in the five organs sampled, with highly significant reductions being observed in the spleen at 72 and 96 h post-challenge (P=0.0002, < 0.0001, respectively). Collectively, the results indicate that Bimuno (R) significantly reduced the colonization and pathology associated with S. Typhimurium infection in a murine model system, possibly by reducing the invasion of the pathogen into host cells.

Does high-frequency repetitive transcranial magnetic stimulation produce residual and/or cumulative effects within an experimental session?

A common procedure for studying the effects on cognition of repetitive transcranial magnetic stimulation (rTMS) is to deliver rTMS concurrent with task performance, and to compare task performance on these trials versus on trials without rTMS. Recent evidence that TMS can have effects on neural activity that persist longer than the experimental session itself, however, raise questions about the assumption of the transient nature of rTMS that underlies many concurrent (or "online") rTMS designs. To our knowledge, there have been no studies in the cognitive domain examining whether the application of brief trains of rTMS during specific epochs of a complex task may have effects that spill over into subsequent task epochs, and perhaps into subsequent trials. We looked for possible immediate spill-over and longer-term cumulative effects of rTMS in data from two studies of visual short-term delayed recognition. In 54 subjects, 10-Hz rTMS trains were applied to five different brain regions during the 3-s delay period of a spatial task, and in a second group of 15 subjects, electroencephalography (EEG) was recorded while 10-Hz rTMS was applied to two brain areas during the 3-s delay period of both spatial and object tasks. No evidence for immediate effects was found in the comparison of the memory probe-evoked response on trials that were vs. were not preceded by delay-period rTMS. No evidence for cumulative effects was found in analyses of behavioral performance, and of EEG signal, as a function of task block. The implications of these findings, and their relation to the broader literature on acute vs. long-lasting effects of rTMS, are considered.

Prefrontal control of familiarity and recollection in working memory

Left inferior frontal gyrus (IFG) is a critical neural substrate for the resolution of proactive interference (PI) in working memory. We hypothesized that left IFG achieves this by controlling the influence of familiarity- versus recollection-based information about memory probes. Consistent with this idea, we observed evidence for an early (200 msec)-peaking signal corresponding to memory probe familiarity and a late (500 msec)-resolving signal corresponding to full accrual of trial-related contextual ("recollection-based") information. Next, we applied brief trains of repetitive transcranial magnetic stimulation (rTMS) time locked to these mnemonic signals, to left IFG and to a control region. Only early rTMS of left IFG produced a modulation of the false alarm rate for high-PI probes. Additionally, the magnitude of this effect was predicted by individual differences in susceptibility to PI. These results suggest that left IFG-based control may bias the influence of familiarity- and recollection-based signals on recognition decisions.

Emulsifying, rheological and physicochemical properties of exopolysaccharide produced by Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205

The rheological, emulsification and certain physicochemical properties of purified exopolysaccharides (EPS) of Bifidobacterium longum subsp. infantis CCUG 52486 and Bifidobacterium infantis NCIMB 702205 were studied and compared with those of guar gum and xanthan gum. The two strains were grown in skim milk supplemented with 1.5% (w/v) casein hydrolysate at 37 ◦C for 24 h; they both produced heteropolysaccharides with different molecular mass and composition. The carbohydrate content of both polymers was more than 92% and no protein was detected. The EPS of B. longum subsp. infantis CCUG 52486 showed highly branched entangled porous structure under scanning electron microscopy. Higher intrinsic viscosity was observed for the EPS of B. longum subsp. infantis CCUG 52486 compared to the EPS of B. infantis NCIMB 702205 and guar gum. Both polymers showed pseudoplastic non-Newtonian fluid behaviour in an aqueous solution. The EPS of B. infantis NCIMB 702205 and B. longum subsp. infantis CCUG 52486 produced more stable emulsions with orange oil, sunflower seed oil, coconut oil and xylene compared to guar and xanthan gum. The EPS of B. longum subsp. infantis CCUG 52486 is the most promising one for applications in the food industry, as it had higher intrinsic viscosity, higher apparent viscosity in aqueous solution, porous dense entangled structure and good emulsification activity.

The effect of wind shear and curvature on the gravity wave drag produced by a ridge

The analytical model proposed by Teixeira, Miranda, and Valente is modified to calculate the gravity wave drag exerted by a stratified flow over a 2D mountain ridge. The drag is found to be more strongly affected by the vertical variation of the background velocity than for an axisymmetric mountain. In the hydrostatic approximation, the corrections to the drag due to this effect do not depend on the detailed shape of the ridge as long as this is exactly 2D. Besides the drag, all the perturbed quantities of the flow at the surface, including the pressure, may be calculated analytically.

Drag produced by trapped lee waves and propagating mountain waves in a two-layer atmosphere

The surface drag force produced by trapped lee waves and upward propagating waves in non-hydrostatic stratified flow over a mountain ridge is explicitly calculated using linear theory for a two-layer atmosphere with piecewise-constant static stability and wind speed profiles. The behaviour of the drag normalized by its hydrostatic single-layer reference value is investigated as a function of the ratio of the Scorer parameters in the two layers l_2/l_1 and of the corresponding dimensionless interface height l_1 H, for selected values of the dimensionless ridge width l_1 a and ratio of wind speeds in the two layers. When l_2/l_1 → 1, the propagating wave drag approaches 1 in approximately hydrostatic conditions, and the trapped lee wave drag vanishes. As l_2/l_1 decreases, the propagating wave drag progressively displays an oscillatory behaviour with l_1 H, with maxima of increasing magnitude due to constructive interference of reflected waves in the lower layer. The trapped lee wave drag shows localized maxima associated with each resonant trapped lee wave mode, occurring for small l_2/l_1 and slightly higher values of l_1 H than the propagating wave drag maxima. As l1a decreases, i.e. the flow becomes more non-hydrostatic, the propagating wave drag decreases and the regions of non-zero trapped lee wave drag extend to higher l_2/l_1. These results are confirmed by numerical simulations for l_2/l_1 = 0.2. In parameter ranges of meteorological relevance, the trapped lee wave drag may have a magnitude comparable to that of propagating wave drag, and be larger than the reference single-layer drag. This may have implications for drag parametrization in global climate and weather-prediction models.

Spatial coding of touch at the fingers: Insights from double simultaneous stimulation within and between hands

We studied the effect of tactile double simultaneous stimulation (DSS) within and between hands to examine spatial coding of touch at the fingers. Participants performed a go/no-go task to detect a tactile stimulus delivered to one target finger (e.g., right index), stimulated alone or with a concurrent non-target finger, either on the same hand (e.g., right middle finger) or on the other hand (e.g., left index finger=homologous; left middle finger=non-homologous). Across blocks we also changed the unseen hands posture (both hands palm down, or one hand rotated palm-up). When both hands were palm-down DSS interference effects emerged both within and between hands, but only when the non-homologous finger served as non-target. This suggests a clear segregation between the fingers of each hand, regardless of finger side. By contrast, when one hand was palm-up interference effects emerged only within hand, whereas between hands DSS interference was considerably reduced or absent. Thus, between hands interference was clearly affected by changes in hands posture. Taken together, these findings provide behavioral evidence in humans for multiple spatial coding of touch during tactile DSS at the fingers. In particular, they confirm the existence of representational stages of touch that distinguish between body-regions more than body-sides. Moreover, they show that the availability of tactile stimulation side becomes prominent when postural update is required.

Vaccine glycoproteins tagged with the human Fc domain as second generation vaccine candidates

Traditional vaccines such as inactivated or live attenuated vaccines, are gradually giving way to more biochemically defined vaccines that are most often based on a recombinant antigen known to possess neutralizing epitopes. Such vaccines can offer improvements in speed, safety and manufacturing process but an inevitable consequence of their high degree of purification is that immunogenicity is reduced through the lack of the innate triggering molecules present in more complex preparations. Targeting recombinant vaccines to antigen presenting cells (APCs) such as dendritic cells however can improve immunogenicity by ensuring that antigen processing is as efficient as possible. Immune complexes, one of a number of routes of APC targeting, are mimicked by a recombinant approach, crystallizable fragment (Fc) fusion proteins, in which the target immunogen is linked directly to an antibody effector domain capable of interaction with receptors, FcR, on the APC cell surface. A number of virus Fc fusion proteins have been expressed in insect cells using the baculovirus expression system and shown to be efficiently produced and purified. Their use for immunization next to non-Fc tagged equivalents shows that they are powerfully immunogenic in the absence of added adjuvant and that immune stimulation is the result of the Fc-FcR interaction.

The neurochemically diverse intermedius nucleus of the medulla as a source of excitatory and inhibitory synaptic input to the nucleus tractus solitarii

Sensory afferent signals from neck muscles have been postulated to influence central cardiorespiratory control as components of postural reflexes, but neuronal pathways for this action have not been identified. The intermedius nucleus of the medulla (InM) is a target of neck muscle spindle afferents and is ideally located to influence such reflexes but is poorly investigated. To aid identification of the nucleus, we initially produced three-dimensional reconstructions of the InM in both mouse and rat. Neurochemical analysis including transgenic reporter mice expressing green fluorescent protein in GABA-synthesizing neurons, immunohistochemistry, and in situ hybridization revealed that the InM is neurochemically diverse, containing GABAegric and glutamatergic neurons with some degree of colocalization with parvalbumin, neuronal nitric oxide synthase, and calretinin. Projections from the InM to the nucleus tractus solitarius (NTS) were studied electrophysiologically in rat brainstem slices. Electrical stimulation of the NTS resulted in antidromically activated action potentials within InM neurons. In addition, electrical stimulation of the InM resulted in EPSPs that were mediated by excitatory amino acids and IPSPs mediated solely by GABA(A) receptors or by GABA(A) and glycine receptors. Chemical stimulation of the InM resulted in (1) a depolarization of NTS neurons that were blocked by NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonoamide) or kynurenic acid and (2) a hyperpolarization of NTS neurons that were blocked by bicuculline. Thus, the InM contains neurochemically diverse neurons and sends both excitatory and inhibitory projections to the NTS. These data provide a novel pathway that may underlie possible reflex changes in autonomic variables after neck muscle spindle afferent activation.

Microbiological, chemical and rheological properties of low fat set yoghurt produced with exopolysaccharide (EPS) producing Bifidobacterium strains.

In this work, the microbiological and physicochemical differences of three types of low fat set yoghurts were studied, as well as the changes taking place during storage at 4 °C for 28 days. The first yoghurt was produced with yoghurt starters and exopolysaccharide (EPS) producing Bifidobacterium longum subsp. infantis CCUG 52486 (CCUGY), the second with yoghurt starters and Bifidobacterium infantis NCIMB 702205 (NCIMBY) and the third with just yoghurt starters (control yoghurt). No significant differences were observed in terms of cell concentrations; for all three yoghurts, similar final cell concentrations were obtained for the yoghurt starter cultures (~7.5 log cfu g−1) and the Bifidobacterium strains (~7.8 log cfu g−1). Both Bifidobacterium survived well during storage, as in both cases the cell viability decreased by less than 0.5 log cfu g−1after 28 days of storage. A decrease in pH followed by an increase in lactic acid was observed during storage for all three yoghurts, which was mostly attributed to the activity of the yoghurt starter cultures. The two yoghurts with the EPS producing Bifidobacterium strains exhibited lower syneresis than the control yoghurt. The lowest was shown by CCUGY, which also exhibited the highest storage modulus and firmness, and a well defined porous web-like structure in cryo-SEM. Examination of the micro-structure of the yoghurts using cryo-scanning electron microscopy (cryo-SEM) indicated that the above observations were due to the interaction between the EPS and the milk proteins. Overall, the results indicated that the EPS producing Bifidobacterium longum subsp. infantis CCUG 52486 is the most promising strain, and can be used with yoghurt starter cultures to manufacture low fat set yoghurt with probiotic activities and at the same time enhanced physicochemical and rheological properties.

Electrical stimulation for trunk control in paraplegia:A feasibility study

Paraplegic subjects lack trunk stability due to the loss of voluntary muscle control.This leads to a restriction of the volume of bi-manual workspace available,and hence has a detrimental impact on activities of daily living. Electrical Stimulation of paralysed muscles can be used to stabilize the trunk, but has never been applied in closed loop for this purpose. This paper describes the development of two closed loop controllers(PID and LQR),and their experimental evaluation on a human subject. Advantages and disadvantages of the two are discussed,considering a potential use of this technology during daily activities.

Micelle size characterization of lipopeptides produced by B. subtilis and their recovery by the two-step ultrafiltration process

The aim of this work was to investigate the lipopeptides aggregation behavior in single and mixed solutions in a wide range of concentrations, in order to optimize their separation and purification following the two-step ultrafiltration process and using large pore size membranes (up to MWCO = 300 kDa). Micelle size was determined by dynamic light scattering. In single solutions of lipopeptide both surfactin and mycosubtilin formed micelles of different size depending on their concentration, micelles of average diameter = 5–105 nm for surfactin and 8–18 nm for mycosubtilin. However when the lipopeptides were in the same solution they formed mixed micelles of different size (d = 8 nm) and probably conformation to that formed by the individual lipopeptide, this prevents their separation according to size. These lipopeptides were purified from fermentation culture by the two-step ultrafiltration process using different MWCO membranes ranging from 10 to 300 kDa. This led to their effective rejection in the first ultrafiltration step by membranes with MCWO = 10–100 kDa but poor rejection by the 300 KDa membrane. The lipopeptides were recovered at 90% purity (in relation to protein) and with 2.34 enrichment in the permeate of the second ultrafiltration step with the 100 KDa membrane upon addition of 75% ethanol.