Structure-function characterization and optimization of a plant-derived antibacterial peptide.

Crushed seeds of the Moringa oleifera tree have been used traditionally as natural flocculants to clarify drinking water. We previously showed that one of the seed peptides mediates both the sedimentation of suspended particles such as bacterial cells and a direct bactericidal activity, raising the possibility that the two activities might be related. In this study, the conformational modeling of the peptide was coupled to a functional analysis of synthetic derivatives. This indicated that partly overlapping structural determinants mediate the sedimentation and antibacterial activities. Sedimentation requires a positively charged, glutamine-rich portion of the peptide that aggregates bacterial cells. The bactericidal activity was localized to a sequence prone to form a helix-loop-helix structural motif. Amino acid substitution showed that the bactericidal activity requires hydrophobic proline residues within the protruding loop. Vital dye staining indicated that treatment with peptides containing this motif results in bacterial membrane damage. Assembly of multiple copies of this structural motif into a branched peptide enhanced antibacterial activity, since low concentrations effectively kill bacteria such as Pseudomonas aeruginosa and Streptococcus pyogenes without displaying a toxic effect on human red blood cells. This study thus identifies a synthetic peptide with potent antibacterial activity against specific human pathogens. It also suggests partly distinct molecular mechanisms for each activity. Sedimentation may result from coupled flocculation and coagulation effects, while the bactericidal activity would require bacterial membrane destabilization by a hydrophobic loop.

In vitro functionality of isolated embryonic hypothalamic vasopressinergic and oxytocinergic neurons: modulatory effects of brain-derived neurotrophic factor and angiotensin II.

There are only a few studies on the ontogeny and differentiation process of the hypothalamic supraoptic-paraventriculo-neurohypophysial neurosecretory system. In vitro neuron survival improves if cells are of embryonic origin; however, surviving hypothalamic neurons in culture were found to express small and minimal amounts of arginine-vasopressin (AVP) and oxytocin (OT), respectively. The aim of this study was to develop a primary neuronal culture design applicable to the study of magnocellular hypothalamic system functionality. For this purpose, a primary neuronal culture was set up after mechanical dissociation of sterile hypothalamic blocks from 17-day-old Sprague-Dawley rat embryos (E17) of both sexes. Isolated hypothalamic cells were cultured with supplemented (B27)-NeuroBasal medium containing an agent inhibiting non-neuron cell proliferation. The neurosecretory process was characterized by detecting AVP and OT secreted into the medium on different days of culture. Data indicate that spontaneous AVP and OT release occurred in a culture day-dependent fashion, being maximal on day 13 for AVP, and on day 10 for OT. Interestingly, brain-derived neurotrophic factor (BDNF) and Angiotensin II (A II) were able to positively modulate neuropeptide output. Furthermore, on day 17 of culture, non-specific (high-KCl) and specific (Angiotensin II) stimuli were able to significantly (P < 0.05) enhance the secretion of both neuropeptides over respective baselines. This study suggests that our experimental design is useful for the study of AVP- and OT-ergic neuron functionality and that BDNF and A II are positive modulators of embryonic hypothalamic cell development.

Fish oil for prevention of sudden death in hemodialysis patients?

Friedman et al. report that hemodialysis patients with the highest levels of n-3 fatty acids had impressively low odds of sudden cardiac death. The study is limited by a small sample size, and the analysis relies on only a single baseline measurement of blood levels. Recent randomized evidence indeed fails to support that n-3 fatty acids may prevent sudden death in nonrenal patients. More evidence is needed to advocate fish oil in this setting.

Effect of fructose overfeeding and fish oil administration on hepatic de novo lipogenesis and insulin sensitivity in healthy men.

High-fructose diet stimulates hepatic de novo lipogenesis (DNL) and causes hypertriglyceridemia and insulin resistance in rodents. Fructose-induced insulin resistance may be secondary to alterations of lipid metabolism. In contrast, fish oil supplementation decreases triglycerides and may improve insulin resistance. Therefore, we studied the effect of high-fructose diet and fish oil on DNL and VLDL triglycerides and their impact on insulin resistance. Seven normal men were studied on four occasions: after fish oil (7.2 g/day) for 28 days; a 6-day high-fructose diet (corresponding to an extra 25% of total calories); fish oil plus high-fructose diet; and control conditions. Following each condition, fasting fractional DNL and endogenous glucose production (EGP) were evaluated using [1-13C]sodium acetate and 6,6-2H2 glucose and a two-step hyperinsulinemic-euglycemic clamp was performed to assess insulin sensitivity. High-fructose diet significantly increased fasting glycemia (7 +/- 2%), triglycerides (79 +/- 22%), fractional DNL (sixfold), and EGP (14 +/- 3%, all P < 0.05). It also impaired insulin-induced suppression of adipose tissue lipolysis and EGP (P < 0.05) but had no effect on whole- body insulin-mediated glucose disposal. Fish oil significantly decreased triglycerides (37%, P < 0.05) after high-fructose diet compared with high-fructose diet without fish oil and tended to reduce DNL but had no other significant effect. In conclusion, high-fructose diet induced dyslipidemia and hepatic and adipose tissue insulin resistance. Fish oil reversed dyslipidemia but not insulin resistance.

Numerical simulation of ambient seismic wavefield modification caused by pore-fluid effects in an oil reservoir

We have modeled numerically the seismic response of a poroelastic inclusion with properties applicable to an oil reservoir that interacts with an ambient wavefield. The model includes wave-induced fluid flow caused by pressure differences between mesoscopic-scale (i.e., in the order of centimeters to meters) heterogeneities. We used a viscoelastic approximation on the macroscopic scale to implement the attenuation and dispersion resulting from this mesoscopic-scale theory in numerical simulations of wave propagation on the kilometer scale. This upscaling method includes finite-element modeling of wave-induced fluid flow to determine effective seismic properties of the poroelastic media, such as attenuation of P- and S-waves. The fitted, equivalent, viscoelastic behavior is implemented in finite-difference wave propagation simulations. With this two-stage process, we model numerically the quasi-poroelastic wave-propagation on the kilometer scale and study the impact of fluid properties and fluid saturation on the modeled seismic amplitudes. In particular, we addressed the question of whether poroelastic effects within an oil reservoir may be a plausible explanation for low-frequency ambient wavefield modifications observed at oil fields in recent years. Our results indicate that ambient wavefield modification is expected to occur for oil reservoirs exhibiting high attenuation. Whether or not such modifications can be detected in surface recordings, however, will depend on acquisition design and noise mitigation processing as well as site-specific conditions, such as the geologic complexity of the subsurface, the nature of the ambient wavefield, and the amount of surface noise.

Development of bioreporter assays for the detection of bioavailability of long-chain alkanes based on the marine bacterium Alcanivorax borkumensis strain SK2.

Long-chain alkanes are a major component of crude oil and therefore potentially good indicators of hydrocarbon spills. Here we present a set of new bacterial bioreporters and assays that allow to detect long-chain alkanes. These reporters are based on the regulatory protein AlkS and the alkB1 promoter from Alcanivorax borkumensis SK2, a widespread alkane degrader in marine habitats. Escherichia coli cells with the reporter construct reacted strongly to octane in short-term (6 h) aqueous suspension assays but very slightly only to tetradecane, in line with what is expected from its low water solubility. In contrast, long-term assays (up to 5 days) with A. borkumensis bioreporters showed strong induction with tetradecane and crude oil. Gel-immobilized A. borkumensis reporter cells were used to demonstrate tetradecane and crude oil bioavailability at a distance from a source. Alcanivorax borkumensis bioreporters induced fivefold more rapid and more strongly when allowed physical contact with the oil phase in standing flask assays, suggesting a major contribution of adhered cells to the overall reporter signal. Using the flask assays we further demonstrated the effect of oleophilic nutrients and biosurfactants on oil availability and degradation by A. borkumensis. The fluorescence signal from flask assays could easily be captured with a normal digital camera, making such tests feasible to be carried out on, e.g. marine oil responder vessels in case of oil accidents.

Intracranial hypertension: what additional information can be derived from ICP waveform after head injury?

OBJECTIVE: Although intracranial hypertension is one of the important prognostic factors after head injury, increased intracranial pressure (ICP) may also be observed in patients with favourable outcome. We have studied whether the value of ICP monitoring can be augmented by indices describing cerebrovascular pressure-reactivity and pressure-volume compensatory reserve derived from ICP and arterial blood pressure (ABP) waveforms. METHOD: 96 patients with intracranial hypertension were studied retrospectively: 57 with fatal outcome and 39 with favourable outcome. ABP and ICP waveforms were recorded. Indices of cerebrovascular reactivity (PRx) and cerebrospinal compensatory reserve (RAP) were calculated as moving correlation coefficients between slow waves of ABP and ICP, and between slow waves of ICP pulse amplitude and mean ICP, respectively. The magnitude of 'slow waves' was derived using ICP low-pass spectral filtration. RESULTS: The most significant difference was found in the magnitude of slow waves that was persistently higher in patients with a favourable outcome (p<0.00004). In patients who died ICP was significantly higher (p<0.0001) and cerebrovascular pressure-reactivity (described by PRx) was compromised (p<0.024). In the same patients, pressure-volume compensatory reserve showed a gradual deterioration over time with a sudden drop of RAP when ICP started to rise, suggesting an overlapping disruption of the vasomotor response. CONCLUSION: Indices derived from ICP waveform analysis can be helpful for the interpretation of progressive intracranial hypertension in patients after brain trauma.

Extent and patterns of MGMT promoter methylation in glioblastoma- and respective glioblastoma-derived spheres.

Purpose: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. Experimental Design: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. Results: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity. Conclusions: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy. Clin Cancer Res; 17(2); 255-66. (C)2010 AACR.

Immunotherapy with Bet v 1 derived contiguous overlapping peptides leads to long-term immunoregulatory responses

Background: Allergen-specific immunotherapy with whole pollen extract may induce anaphylaxis, is poorly standardized and of long duration.We thus designed a randomized, placebo-controlled phase I/II clinical trial in volunteers with birch pollen allergic rhinitis and asthma to evaluate the safety and immunogenicity of a novel immunotherapy based on contiguous overlapping peptides (COPs) derived from Bet v 1, the major birch pollen allergen. Methods: A mixture of three COPs (AllerT™, Anergis SA, Switzerland) spanning the whole Bet v 1 molecule was selected for its inability to bind IgE. Prior to the pollen season, AllerT (in Alum) was injected subcutaneously to 15 adult volunteers at D0 (57 g), D7, D14, D21 and D51 (95 g each). Control volunteers (n = 5) only received the adjuvant. Results: Overall AllerT was safe. No serious adverse events and no immediate allergic reactions were reported. AllerT induced a vigorous early Bet v 1 specific immune response marked by vaccine associated INF- and IL- 10 secretion. This contributed to a strong anti-Bet v 1-specific IgG4 enhancement. Moreover, 2 months after the second season post treatment (July 2010), serum Bet v 1 specific IgG4 response was still markedly increased as compared to pre-treatment values and to placebo whereas post seasonal Bet v 1 specific IgE titers were similar to baseline values. Conclusion: Our data indicate that immunotherapy with a mixture of three COPs derived from Bet v 1 (AllerT) was safe and immunogenic, and led to long-term immunological memory.

Brain-derived neurotrophic factor-like immunoreactivity is localized mainly in small sensory neurons of rat dorsal root ganglia.

Brain-derived neurotrophic factor (BDNF) is a protein capable of supporting the survival and fiber outgrowth of peripheral sensory neurons. It has been argued that histological detection of BDNF has proven difficult because of its low molecular weight and relatively low expression. In the present study we report that rapid removal of dorsal root ganglia (DRG) from the rat, followed by rapid freezing and appropriate fixation with cold acetone, preserves BDNF in situ without altering protein antigenicity. Under these conditions, specific BDNF-like immunoreactivity was detected in DRG both in vivo and in vitro. During DRG development in vivo, BDNF-like immunoreactivity (BDNF-LI) was observed only in a subset of sensory neurons. BDNF-LI was confined to small neurons, after neurons became morphologically distinct on the basis of size. BDNF-L immunoprecipitate was detected only in neuronal cells, and not in satellite or Schwann cells. While in vivo BDNF localization was restricted to small neurons, practically all neurons in DRG cell culture displayed BDNF-LI. Small or large primary afferent neurons exhibited a faint but clear BDNF-LI during the whole life span of cultures. Again, non-neuronal cells were devoid of BDNF-LI. In conclusion, in DRG in vivo, specific BDNF-LI was confined to small B sensory neurons. In contrast, all DRG sensory neurons displayed BDNF-LI in vitro. The finding that BDNF expressed in all DRG neurons in vitro but not in vivo suggests that BDNF expression may be modulated by environmental factors.