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.

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.

Stem cell populations derived from heart and pancreas show a with a unique phenotype and give rise to functional cardiomyocytes and insulin-producing cells, respectively

Introduction: Recently, mesenchymal stem cells (MSC) of perivascular origin have been identified in several organs not including the heart. Using a novel cell isolation protocol, we have isolated cells sharing common characteristics from mouse hearts and pancreas. The aim of the present study was to characterize these cells in vitro.Methods: Cells were isolated from neonatal and adult mouse hearts and pancreas and cultured for more than 6 months. Surface marker expression was analyzed by flow cytometry and immunocytochemistry. Cell differentiation was tested using multiple differentiation media. Insulin production by pancreas-derived cells was tested by dithizone staining.Results: Cells showing a similar, distinctive morphology were obtained from the heart and pancreas after 4-8 weeks of culture. Cells from the two organs also showed a very similar immunophenotype, characterized by expression of c-kit (stem cell factor receptor), CD44, the common leukocyte marker CD45, and the monocytic markers CD11b and CD14. A significant proportion of cardiac and pancreatic cells expressed NG2, a marker for pericytes and other vascular cells. A significant proportion of cardiac, but not of pancreatic cells expressed stem cell antigen-1 (Sca-1). However, cells did not express T, B or dendritic cell markers. Cells of both cardiac and pancreatic origin spontaneously formed "spheres" (spherical cell aggregates similar to "neurospheres" formed by neural stem cells) in vitro. Cardiosphere formation was enhanced by TNF-alpha. Several cardiospheres (but no "pancreatospheres") derived from neonatal (but not adult) cells showed spontaneous rhythmic contractions, thus demonstrating cardiac differentiation (this was confirmed by immunostaining for alpha-sarcomeric actinin). Beating activity was enhanced by low serum conditions. Cells from both organs formed adipocytes, osteocytes and osteocytes under appropriate conditions, the typical differentiation pattern of MSCs. Pancreas-derived cells also formed dithizonepositive insulin-producing cells.Conclusions: We have defined cardiac and pancreatic cell populations that share a common morphology, growth characteristics, and a unique immunophenotype. Expression of perivascular and monocytic markers, along with stem/priogenitor cell markers by these cells suggests a relationship with pericytes-mesoangioblasts and so-called multipotent monocytes. Cells show MSC-typical growth and differentiation patterns, together with tissue-specific differentiation potential: cardiomyocytes for cardiac-derived cells and insulinproducing cells for pancreas-derived cells.

Nanosized Nb-TiO2 gas sensors derived from alkoxides hydrolization

Nanocrystalline TiO2 modified with Nb has been produced through the sol-gel technique. Nanopowders have been obtained by means of the hydrolysis of pure alkoxides with deionized water and peptization of the resulting hydrolysate with diluted acid nitric at 100 C. The addition of Nb stabilizes the anatase phase to higher temperatures. XRD spectra of the undoped and the Nb-doped samples show that the undoped sample has been almost totally converted to rutile at 600 C, meanwhile the doped samples present still a low percentage of rutile phase. Nanocrystalline powders stabilized at 600 C with grain sizes of about 17 nm have successfully been synthesized by the addition of Nb with a concentration of 2% at., which appears to be an adequate additive concentration to improve the gas sensor performances, such as it is suggested by the catalytic conversion efficiency experiments performed from FTIR measurements. FTIR absorbance spectra show that catalytic conversion of CO occurs at lower temperatures when niobium is introduced. The electrical response of the films to different concentrations of CO and ethanol has been monitored in dry and wet environments in order to test the influence of humidity in the sensor response. The addition of Nb decreases the working temperature and increases the stability of the layers. Also, large enhancement of the response time is obtained even with lower working temperatures. Moreover, humidity effects on the gas sensor response toward CO and ethanol are less important in Nb-doped samples than in the undoped ones.

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. ©2010 AACR.

Measuring the efficiency in Spanish municipal refuse collection services

The objective of this study is to analyse the technical or productive efficiency ofthe refuse collection services in 75 municipalities located in the Spanish regionof Catalonia. The analysis has been carried out using various techniques. Firstly we have calculated a deterministic parametric frontier, then a stochastic parametric frontier, and finally, various non-parametric approaches (DEA and FDH). Concerning the results, these naturally differ according to the technique used to approach the frontier. Nevertheless, they have an appearance of solidity, at least with regard to the ordinal concordance among the indices of efficiency obtained by the different approaches, as is demonstrated by the statistical tests used. Finally, we have attempted to search for any relation existing between efficiency and the method (public or private) of managing the services. No significant relation was found between the type of management and efficiencyindices

Nanosized Nb-TiO2 gas sensors derived from alkoxides hydrolization

Nanocrystalline TiO2 modified with Nb has been produced through the sol-gel technique. Nanopowders have been obtained by means of the hydrolysis of pure alkoxides with deionized water and peptization of the resulting hydrolysate with diluted acid nitric at 100 C. The addition of Nb stabilizes the anatase phase to higher temperatures. XRD spectra of the undoped and the Nb-doped samples show that the undoped sample has been almost totally converted to rutile at 600 C, meanwhile the doped samples present still a low percentage of rutile phase. Nanocrystalline powders stabilized at 600 C with grain sizes of about 17 nm have successfully been synthesized by the addition of Nb with a concentration of 2% at., which appears to be an adequate additive concentration to improve the gas sensor performances, such as it is suggested by the catalytic conversion efficiency experiments performed from FTIR measurements. FTIR absorbance spectra show that catalytic conversion of CO occurs at lower temperatures when niobium is introduced. The electrical response of the films to different concentrations of CO and ethanol has been monitored in dry and wet environments in order to test the influence of humidity in the sensor response. The addition of Nb decreases the working temperature and increases the stability of the layers. Also, large enhancement of the response time is obtained even with lower working temperatures. Moreover, humidity effects on the gas sensor response toward CO and ethanol are less important in Nb-doped samples than in the undoped ones.

Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production.

The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase.

Utilization of Boron (10B) derived from fertilizer by sugar cane

The response to B in agricultural systems of sugar cane is still an unexplored issue; B application has however recently been widely publicized and used with a certain degree of frequency. The use of 10B-labeled fertilizers may further contribute to clarify this practice. With the objective of evaluating sugar cane use of B (10B) derived from fertilizer (boric acid), an experiment was conducted under field conditions in the 2005/2006 growing season. The experiment consisted of the installation of microplots (2 x 1.5 m) where 4 kg ha-1 B (boric acid with 85.95 % in 10B atoms) dissolved in water was applied 90 days after planting (May 2005). The solution was applied to the soil on both sides of the plant row at a distance of 20 cm. After harvest (June 2006) the B content and 10B abundance in % atoms in all parts of the sugar cane plants (stalks, dry leaves, tips and roots) were determined. Results showed that the total B accumulated was 471 g ha-1 in the entire plant (35 % in the stalks, 22 % in the dry leaves, 9 % in the tips and 34 % in the roots). The sugar cane plants used on average 14 % of the total accumulated B in the above-ground part (44 g ha-1) and 11 % in the roots (19 g ha-1), totaling 13 % in the entire plant (63 g ha-1). The recovery of 10B-fertilizer by sugar cane plants was low, around 2 % of the total applied amount.