Electroencephalographic (EEG) neurofeedback (NF) is considered a nonpharmacological alternative for medication in attention-deficit/hyperactivity disorder (ADHD). Comparisons of the behavioral efficacy of NF and medication have produced inconsistent results. EEG measures can provide insight into treatment mechanisms, but have received little consideration. In this randomized controlled trial (RCT), effects of NF were compared with methylphenidate (MPH), and physical activity (PA) in children with ADHD on event-related potential (ERP) indices of response inhibition, which are involved in ADHD psychopathology.

Using a multicenter three way parallel group RCT design, 112 children with a Diagnostic and Statistical Manual of Mental Disorders, 4th ed. (DSM-IV) (American Psychiatric Association 1994 ) diagnosis of ADHD, between 7 and 13 years of age, were initially included. NF training consisted of 30 sessions of theta/beta training at Cz over a 10 week period. PA training was a semiactive control group, matched in frequency and duration. MPH was titrated using a double-blind placebo controlled procedure in 6 weeks, followed by a stable dose for 4 weeks. ERP measures of response inhibition, N2 and P3, were available for 81 children at pre- and postintervention (n = 32 NF, n = 25 MPH, n = 24 PA).

Only the medication group showed a specific increase in P3 amplitude compared with NF (partial eta-squared [ηp(2) ] = 0.121) and PA (ηp(2) = 0.283), which was related to improved response inhibition. Source localization of medication effects on P3 amplitude indicated increased activation primarily in thalamic and striatal nuclei.

This is the first study that simultaneously compared NF with stimulant treatment and a semiactive control group. Only stimulant treatment demonstrated specific improvements in brain function related to response inhibition. These results are in line with recent doubts on the efficacy and specificity of NF as treatment for ADHD.

Train Your Brain? Exercise and Neurofeedback Intervention for ADHD, https://clinicaltrials.gov/show/NCT01363544 , Ref. No. NCT01363544.

The effect of hormonal interactions on human erythroid colony growth has been studied using the beta adrenergic agonist, isoproterenol. The growth of colonies derived from both erythroid burst-forming cells (BFU-E) and erythroid colony-forming cells (CFU-E) was enhanced in the presence of isoproterenol. While isoproterenol was effective at all concentrations of erythropoietin (Ep) in cultures of marrow cells, an increase in BFU-E-derived colonies in peripheral blood cultures could be detected only at suboptimal levels of Ep concentrations. The isoproterenol effect was blocked by an agent with beta 2 receptor specificity (butoxamine), and L-isomeric configuration (L-propranolol). The stimulatory effect appeared to be mediated by a receptor different from that for Ep or for phytohaemagglutinin-stimulated leucocyte conditioned medium. Circulating BFU-E from eight patients with polycythaemia vera were also studied. Those colonies which grew in the absence of added Ep increased with isoproterenol; in cultures from normal subjects and in patients with secondary erythrocytosis, agonist stimulation was seen only in the presence of Ep. These studies provide evidence that the growth of both normal and neoplastic erythroid progenitors may be modulated by hormonal interactions.

Stem cell-based therapy (SC-BT) is emerging as an alternative for endodontic therapies. The interaction between stem cells and scaffolds plays a crucial role in the generation of a 'friendly cell' microenvironment. The aim of this systematic review was to explore techniques applied to regenerate the pulp-dentine complex tissue using SC-BT. An electronic search into the SciVerse Scopus (SS), ISI Web Science (IWS) and Entrez PubMed (EP) using specific keywords was performed. Specific inclusion and exclusion criteria were predetermined. The search yielded papers, out of which full-text papers were included in the final analyses. Data extraction pooled the results in four main topics: (a) influence of the chemical properties of the scaffolds over cell behaviour; (b) influence of the physical characteristics of scaffolds over cell behaviour; (c) strategies applied to improve the stem cell/scaffold interface; and (d) influence of cue microenvironment on stem cell differentiation towards odontoblast-like cells and pulp-like tissue formation. The relationship between the scaffolds, the environment and the growth factors released from dentine are critical for de novo pulp tissue regeneration. The preconditioning of dentine walls with ethylenediaminetetraacetic acid (EDTA) was imperative for successful pulp-dentine complex regeneration. An analyses of the grouped results revealed that pulp regeneration was an attainable goal.

Bacillus amyloliquefaciens C06, a potential agent in biological preservation of post-harvest fruit, was found to secrete extra-cellular γ-polyglutamic acid (γ-PGA) in liquid culture. In this work, M306, a transposon mutant of B. amyloliquefaciens C06, defective in forming structured colony and displaying enhanced ability of producing γ-PGA, was obtained. Inverse PCR and quantitative reverse transcription PCR (qRT-PCR) analysis demonstrated that the defective phenotype in M306 was associated with an ORF showing high similarity to RBAM_034550 from B. amyloliquefaciens FZBB, the gene directing γ-PGA synthesis. Evaluations in γ-PGA productivity of wild-type C06 and its mutants C06ΔepsA and C06ΔtasA, respectively, deficient in producing exopolysaccharides (EPS) and TasA, revealed that γ-PGA overproduction in M306 was probably due to the redistributed metabolic flux caused by defective production of EPS.

There is compelling evidence that endogenous opioid peptides are regulated by exogenous opiates. Our previous studies have shown that the mu-opioid receptor protein and mRNA are down-regulated in the mediobasal hypothalamus of the female guinea pig following chronic morphine treatment. In addition, electrophysiological studies have shown that hypothalamic beta-endorphin (beta-EP) neurons express mu-opioid receptors that are uncoupled and down-regulated following chronic morphine treatment. Currently, we tested the hypothesis that chronic morphine, which produces down-regulation of mu-opioid receptors, causes a down-regulation of pro-opiomelanocortin (POMC, the precursor of beta-EP) mRNA expression in female guinea pig hypothalamus. Female guinea pigs were ovariectomized and implanted subcutaneously (s.c.) with 4 x 75 mg pellets for 2 days plus six more pellets of either morphine (n = 6) or placebo (n = 6) for another 5 days. Animals were sacrificed between 1000 and 1100 h on day 7. The expression of POMC mRNA were investigated using in situ hybridization histochemistry with a guinea pig specific 35S-labeled cRNA probe in hypothalamic tissue sections. POMC mRNA was localized to the arcuate nucleus (Arc) and median eminence (ME) of the medial basal hypothalamus. The distribution pattern was the same in both morphine and placebo control animals. However, the density of silver grains was less in morphine treated animals versus placebo control animals. Overall, the level of POMC mRNA was decreased by 22% in the Arc of morphine-treated guinea pigs as compared with the placebo controls (p < 0.05). This decrease in POMC mRNA expression was even greater in the caudal Arc (28%, p < 0.01) in morphine-treated animals. These results suggested that the biosynthetic activity of POMC neurons is down-regulated with chronic exposure to morphine.

Photolysis of alpha-diazo-N-methoxy-N-methyl (Weinreb) beta-ketoamides derived from enantiomerically pure (EP) alpha-amino acids affords the corresponding EP beta-lactams via an intramolecular Wolff rearrangement. The photochemistry is promoted with either standard UV irradiation or through the use of a 100 W compact fluorescent light; the latter affords a safe and environmentally friendly alternative to standard photolysis conditions. A continuous-flow photochemical reactor made from inexpensive laboratory equipment reduced reaction times and was amenable to scale-up. The diastereoselectivity (cis or trans) of the product beta-lactams has been shown to vary from modest to nearly complete. An extremely facile, atom-economical method for the epimerization of the product mixture to the trans isomer, which is generally highly crystalline, has been developed. Evidence for C3 epimerization of Weinreb amide structures via a nonbasic, purely thermal route is presented. Subsequent transformations of both the Weinreb amide at C3 (beta-lactam numbering) and the amino acid side chain at C4 are well-tolerated, allowing for a versatile approach to diverse beta-lactam structures. The technology is showcased in the synthesis of a common intermediate used toward several carbapenem-derived structures starting from unfunctionalized aspartic acid.

Lines of T lymphocytes reactive against the basic protein of myelin (BP) were found in previous studies to mediate experimental autoimmune encephalomyelitis (EAE) in rats. Moreover, inoculation of rats with attenuated anti-BP line cells vaccinated them against subsequent attempts to induce active EAE by injection of BP in adjuvant. In the present study, we investigated the effects of T lymphocyte lines reactive to different antigenic determinants on the BP molecule, they are the major encephalitogenic peptide (EP) determinant present on guinea pig BP (G-BP), and minor, non-EP determinants present on bovine BP (B-BP). We found that both lines of T lymphocytes could mediate EAE. Resistance to active EAE acquired by spontaneous recovery from line mediated EAE or by vaccination with attenuated cells, however, was found to be specific for the particular BP determinant. Thus, EAE may be mediated by lines of T lymphocytes reactive to different determinants on the BP molecule, but the resistance to EAE acquired by exposure to line cells is determinant specific. This suggests that acquired resistance to EAE is directed by the receptor specificity of the autoimmune anti-BP T cells.

The bceA gene is part of the Burkholderia cepacia IST408 exopolysaccharide (EPS) biosynthetic cluster. It encodes a 55.3-kDa bifunctional protein (type II PMI family) with phosphomannose isomerase (PMI) and GDP-mannose pyrophosphorylase (GMP) activities. GMP activity is strongly dependent on the presence of Ca(2+) or Mn(2+), while PMI activity can use a broader variety of divalent cations (Ca(2+>>Mn(2+>>Mg(2+>>Co(2+>>Ni(2+)). The lack of a functional bceA gene does not affect EPS production yield in a non-polar insertion bceA mutant. The in silico search for putative bceA homologues revealed the presence of 2-5 bceA orthologues in the Burkholderia genomes available. This suggests that in B. cepacia IST408 putative bceA functional homologues may compensate the bceA mutation. However, the viscosity of aqueous solutions prepared with the EPS produced by the bceA mutant was significantly reduced compared with wild-type biopolymer and the mutant forms biofilms with a size reduced by 6-fold.

We investigated whether prostaglandins regulate endothelial nitric oxide synthase (eNOS) in the pig cerebral vasculature during the neonatal period. Prostaglandins, eNOS mRNA, eNOS protein, and NO production were higher in cerebral microvessels of newborn (1 day old) than in those of adult (6- to 8-month-old) pigs. The treatment of isolated cerebral microvessels of newborn animals with ibuprofen for 24 h reduced eNOS mRNA and nitrite production to levels in the adult; this effect of ibuprofen was prevented by concurrent treatment with prostaglandin (PG)E(2) analog 16,16-dimethyl-PGE(2), nonselective PGE(2) receptor analog 11-deoxy PGE(1), and prostaglandin EP(3) receptor agonists sulprostone and M&B 28,767 but was not modified by PGI(2) analog carbaprostacyclin, PGD(2), and EP(1) receptor agonist 17-phenyl trinor PGE(2). Correspondingly, 16, 16-dimethyl-PGE(2) and M&B 28,767 increased eNOS mRNA expression of adult microvessels to values in the newborn. Data similar to those with isolated cerebral vessels were obtained through histochemical analysis (NADPH-diaphorase positivity) of brain from newborn animals treated in vivo with ibuprofen in combination or not with sulprostone. Furthermore, substance P-induced NO-mediated cerebral vasorelaxation was decreased to adult values through the treatment of newborn pigs with ibuprofen; this effect was prevented by concomitant treatment with sulprostone. It is concluded that PGE(2) regulates eNOS in newborn pig cerebral microvessels via EP(3) receptors; this may be physiologically required during normal neurovascular development.

Prostaglandins act through specific receptors to stimulate cyclic AMP formation which inhibits platelet activation and relaxes vascular smooth muscle. We have used RT-PCR combined with Southern blot analysis to determine the subtypes of prostaglandin receptor on platelets. Platelets expressed the EPEPEP receptor subtype, whereas vascular smooth muscle cells predominantly expressed the EPEPEPB cell contamination in the platelet RNA, PCR was performed using primers specific for the cell specific cluster determinants CD2 (a T-cell marker) and CD20 (a B-cell marker). The finding that platelets express both stimulatory and inhibitory prostaglandin receptors provides confirmation of a homeostatic model of regulation of platelet adenylyl cyclase previously proposed.

Like many other prokaryotes, rhizobacteria of the genus Azospirillum produce high levels of poly(beta-hydroxybutyrate) (PHB) under suboptimal growth conditions. Utilization of PHB by bacteria under stress has been proposed as a mechanism that favors their compatible establishment in competitive environments, thus showing great potential for the improvement of bacterial inoculants for plants and soils. The three genes that are considered to be essential in the PHB biosynthetic pathway, phbA (beta-ketothiolase), phbB (acetoacetyl coenzyme A reductase), and phbC (PHB synthase), were identified in Azospirillum brasilense strain Sp7, cloned, and sequenced. The phbA, -B, and -C genes were found to be linked together and located on the chromosome. An A. brasilense phbC mutant was obtained by insertion of a kanamycin resistance cassette within the phbC gene. No PHB production was detected in this mutant. The capability of the wild-type strain to endure starvation conditions was higher than that of the mutant strain. However, motility, cell aggregation, root adhesion, and exopolysaccharide (EPS) and capsular polysaccharide (CPS) production were higher in the phbC mutant strain than in the wild type.

The release of vasopressin and oxytocin is regulated by the electrical activity of magnocellular neurosecretory cells in the supraoptic and paraventricular nuclei, which is under the control of a great variety of neurotransmitters and neuromodulators. The major neural signals to the supraoptic nucleus are from excitatory glutamate inputs and inhibitory GABA inputs. In recent studies, the voltage-clamp mode of the whole-cell patch-clamp technique has been applied to slice preparations from rat hypothalamus to monitor synaptic inputs to supraoptic neurones. Spontaneous excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) are abolished by CNQX and picrotoxin, respectively, but are insensitive to tetrodotoxin, indicating that they represent quantal release of glutamate and GABA, respectively, from nerve terminals of presynaptic neurones. GABA and glutamate show remarkable suppressive effects on both EPSCs and IPSCs via presynaptic GABA(B) and mGlu receptors, respectively. Noradrenaline, which excites supraoptic neurones via postsynaptic alpha1-receptors, also suppresses IPSCs and potentiates EPSCs. On the other hand, prostaglandin E2, which excites supraoptic neurones via postsynaptic prostaglandin E2 (EP) receptors of the EPEPEPSCs. Thus pre- and postsynaptic mechanisms may act cooperatively to excite supraoptic neurones. Nitric oxide, which inhibits supraoptic neurones, potentiates IPSCs without affecting EPSCs. This provides another example for the preferential modulation of IPSCs of supraoptic neurones. On the other hand, PACAP, which causes a long-lasting increase in the firing frequency via the postsynaptic receptors, has no effect on EPSCs and IPSCs, suggesting that some ligands act only at postsynaptic receptors. Thus multiple patterns for pre- and postsynaptic modulation are present in the supraoptic nucleus, and the electrical activity of supraoptic neurones is regulated via complex mechanisms at both pre- and postsynaptic sites.

In order to determine how brain beta-endorphin (beta-EP) and its precursor proopiomelanocortin (POMC) adapt to chronic opioid blockade we have examined the effects of treatment with the opioid receptor antagonist naltrexone (NTX) on POMC gene expression and peptide levels in the hypothalamus. Male rats were treated with NTX by daily injection or constant minipump infusion. RNA was isolated from the medial basal hypothalamus (MBH) after an aliquot was removed for peptide RIA and the amount of POMC mRNA was measured by solution hybridization SI nuclease protection assay. beta-EP and several other POMC-derived peptides including alpha-melanocyte-stimulating hormone (alpha-MSH) and corticotropin-like intermediate lobe peptide (CLIP) or gamma(3)-MSH were measured in the MBH and anterior hypothalamus (AH) by RIA. In an initial experiment POMC peptide levels were measured after 7 days of NTX (4.8 mg/day) infusion. There was a marked fall in the concentrations of beta-EP, alpha-MSH, and CLIP; levels in the MBH declined by more than 60% (P < 0.001). In the next experiment NTX (1 mg) was injected daily and POMC peptides and mRNA were measured after 2 and 5 days of treatment. (beta-EP) and alpha-MSH levels fell progressively in the MBH and AH and were significantly less than those of the controls by 5 days of treatment (P < 0.02). POMC mRNA levels, however, did not change after 2 or 5 days. When NTX was infused for 3 weeks there was a decrease in the concentrations of beta-EP, alpha-MSH, and gamma(3)-MSH in the MBH (P < 0.001). The concentration of POMC mRNA in the MBH, however, was significantly higher in the NTX-treated animals, 0.99 +/- 0.06 pg/mug RNA vs 0.81 +/- 0.05 pg/mug RNA (P < 0.05). Since NTX can affect LH and testosterone release, the study was repeated in castrated rats. POMC peptide levels again fell after 3 weeks of NTX. POMC mRNA levels were higher in the castrated rats than in the intact rats, 1.14 +/- 0.06 pg/mug RNA vs 0.85 +/- 0.09 pg/mug RNA (P < 0.05), consistent with our previous findings in longterm castrated rats. However POMC mRNA increased further to 1.40 +/- 0.09 pg/mug RNA in the castrated animals treated with NTX (P < 0.05). We conclude that opioid receptor blockade has significant effects on POMC peptide levels and gene expression in the MBH. The increase in POMC gene expression associated with a fall in peptide levels is consistent with a compensatory increase in brain beta-EP synthesis and release in the setting of chronic opioid receptor blockade.

A new type of IgG serum inhibitor in adult pure red cell aplasia (PRCA) has been investigated. This inhibitor is directed against circulating erythropoietin (Ep) (PRCA type B), rather than the erythroid marrow (PRCA type A). Thus, the IgG inhibitor, after interaction with Ep in solution, is precipitated together with Ep by addition of goat anti-human gamma-globulins. Pre-therapy PRCA serum, although apparently devoid of Ep, shows considerable Ep activity following acidification and boiling. The inhibitor is absent from post-therapy serum, while Ep levels are elevated. An experimental model for PRCA type B has been established in normal mice after prolonged administration of pre-remission serum IgG.

In the present work we investigated the toxic activities of two Bothrops snake venoms using in vivo and in vitro experimental protocols in mice and tested the protective effect of dexamethasone (DEXA) in different conditions, comparing it with the polyvalent antivenom. We also expanded the investigations on the antiophidic effect of the Eclipta prostrata (EP) crude extract. The administration of Bothrops jararaca and Bothrops jararacussu snake venoms induced muscle damage demonstrated in vivo by the elevation on plasma creatine kinase (CK) activity in mice and by the decrease in CK content in the extensor digitorum longus (EDL) muscle of these animals, and in vitro by the increase in the rate of CK release from the isolated EDL muscle. We also observed inflammatory response following perimuscular injection of B. jararacussu venom (1.0 mg/kg). Treatment with DEXA (1.0 mg/kg) preserved over 50% of the EDL muscle CK content in vivo when evaluated 24 and 72 h after the injection of B. jararacussu venom in mice, and likewise reduced about 20% of the edema induced by this venom. DEXA reduced in 50% the presence of inflammatory cells and their activity in EDL muscle. The EP extract (50 mg/kg) showed similar ability in preventing the induction of edema and the decrease in muscle CK content, and its association with DEXA showed additive effect. EP reduced over 77% of the plasma CK activity induced by the B. jararacussu venom. In the in vitro experiments, DEXA was not able to change the rate of CK release from EDL muscles exposed to 25 μg/mL of B. jararacussu venom, neither to prevent the fall in the amplitude of the indirectly evoked twitch at the phrenic-diaphragm preparation. EP extract showed otherwise a protective effect on these protocols, reaching up to 100% of protection when concentrations of 50.0 and 100.0 μg/mL were used. Altogether our results show that inflammation is at least in part responsible for the tissue damage induced by Bothrops snake venoms, once the steroidal anti-inflammatory drug dexamethasone was able to decrease the myotoxic effects of these venoms, by reducing the inflammatory response to the venom injection.

The influence of carbohydrate source on growth, exopolysaccharide (EPS) production and on the activity of the enzymes implicated in energy generation and UDP-glucose synthesis in Pediococcus parvulus 2.6 was evaluated. The highest EPS production was obtained on glucose, while fructose was a poor substrate for EPS synthesis. HPLC and NMR analysis on monomer composition and structure of the EPS showed that this strain produced the same beta-glucan, regardless of the carbohydrate source. The alpha-phosphoglucomutase specific activities were dependent on the carbohydrate source and a high correlation between the activity of this enzyme and the amount of EPS was found in glucose- and maltose-grown cultures. alpha-UDP-glucose pyrophosphorylase activity, necessary for the activation of glucose, was very low, but significantly higher on glucose as sugar source. In vitro phosphorylation assays and transport activities showed that glucose is taken up by a proton motive force-dependent permease, while fructose is internalized by an inducible phosphotransferase system, which renders fructose-6-phosphate. The levels of 6-phosphofructokinase activity and alpha-phosphoglucomutase activities determined on fructose were higher and lower, than those found on glucose or maltose, respectively. This suggests that fructose-6-phosphate is mainly diverted to glycolysis and explains the low EPS synthesis on fructose. Results indicate that alpha-phosphoglucomutase and/or alpha-UDP-glucose pyrophosphorylase might be the bottlenecks for EPS biosynthesis, opening the field for metabolic-engineering strategies aimed to improve EPS production.

Certain strains of Bradyrhizobium japonicum and B. elkanii produce a polysaccharide within the root nodules of their legume host, soybean. These nodule polysaccharides (NPSs) were isolated and characterized. The NPS produced by B. elkanii strains proved to be identical in glycosyl composition and linkages to the extracellular polysaccharide (EPS) of this species indicating that the NPS and EPS for B. elkanii have identical structures (W.F. Dudman, Carbohydr. Res., 66 (1978) 9-23), [formula: see text] However, the structure of the NPS from B. japonicum proved to be quite different from that of its EPS. Methylation analysis of this NPS showed that it consists of 3-linked Gal, 3-linked Rha, 2,4-linked Rha, 4-linked Rha, and terminal 2-O-methyl GlcA in a 1:1:1:1:1 ratio. Stereochemical configurations of the glycosyl residues were determined by the preparation and analysis of trimethylsilyl (Me3Si) (-)-2-butyl glycosides. NMR spectroscopy (both 1H and 13C) showed that the Gal residue is alpha-linked, while all the other glycosyl residues are beta-linked. Oligosaccharides produced by periodate oxidation-Smith degradation were purified, as were oligosaccharides produced by partial acid hydrolysis. Characterization of the Smith degradation products by methylation analysis. NMR spectroscopy, electrospray-mass spectrometry, and characterization of the partial acid hydrolysate oligosaccharides showed that the repeating oligosaccharide unit of the NPS has the structure, [formula: see text]

The adrenal gland plays a fundamental role in the response to a variety of stress situations. After a stress condition, adrenal medullary chromaffin cells release, by exocytosis, high quantities of catecholamine (epinephrine, EP; norepinephrine, NE), especially EP. Once in the blood stream, catecholamines reach different target organs, and induce their biological actions through the activation of different adrenoceptors. Adrenal gland cells may also be activated by catecholamines, through hormonal, paracrine and/or autocrine system. The presence of functional adrenoceptors on human adrenal medulla and their involvement on catecholamines secretion was not previously evaluated. In the present study we investigated the role of β(1)-, β(2)- and β(3)-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture. We observed that the β-adrenoceptor agonist (isoproterenol) and β(2)-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β(2)-adrenoceptor antagonist (ICI 118,551; 100 nM) and β(3)-adrenoceptor antagonist (SR 59230A; 100 nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β(1)-adrenoceptor antagonist (atenolol; 100 nM) did not change the isoproterenol- neither the nicotine-evoked catecholamine release from human adrenal chromaffin cells. Moreover, our results show that the protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) are intracellular mechanisms involved in the catecholamine release evoked by salbutamol. In conclusion, our data suggest that the activation of β(2)- and β(3)-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.

Early-life exposure to microbial agents may play a protective role in asthma and allergies development. Geographical differences in the prevalence of these diseases exist, but the differences in early-life indoor microbial agent levels and their determinants have been hardly studied. We aimed to describe the early-life levels of endotoxin, extracellular polysaccharides (EPS), and β(1-3)-glucans in living room dust of four geographically spread European birth cohorts (LISA in Germany, PIAMA in the Netherlands, INMA in Spain, and LUKAS2 in Finland) and to assess their determinants. A total of 1572 dust samples from living rooms of participants were analyzed for endotoxin, Penicillium/Aspergillus EPS, and β(1-3)-glucans. Information on potential determinants was obtained through questionnaires. Concentrations of endotoxin, EPS, and β(1-3)-glucans were different across cohorts. Concentrations of endotoxin and EPS were respectively lower and higher in INMA than in other cohorts, while glucans were higher in LUKAS2. Season of sampling, dog ownership, dampness, and the number of people living at home were significantly associated with concentrations of at least one microbial agent, with heterogeneity of effect estimates of the determinants across cohorts. In conclusion, both early-life microbial exposure levels and exposure determinants differ across cohorts derived from diverse European countries.

CONCLUSIONS

This study adds evidence of variability in the levels of indoor endotoxin, extracellular polysaccharide, and β(1-3)-glucans across four geographically spread European regions. Furthermore, we observed heterogeneity across regions in the effect of exposure determinants. We hypothesize that the variations observed in our study may play a role in the differences in asthma and allergies prevalences across countries.

Aspergillus fumigatus biofilms represent a problematic clinical entity, especially because of their recalcitrance to antifungal drugs, which poses a number of therapeutic implications for invasive aspergillosis, the most difficult-to-treat Aspergillus-related disease. While the antibiofilm activities of amphotericin B (AMB) deoxycholate and its lipid formulations (e.g., liposomal AMB [LAMB]) are well documented, the effectiveness of these drugs in combination with nonantifungal agents is poorly understood. In the present study, in vitro interactions between polyene antifungals (AMB and LAMB) and alginate lyase (AlgL), an enzyme degrading the polysaccharides produced as extracellular polymeric substances (EPSs) within the biofilm matrix, against A. fumigatus biofilms were evaluated by using the checkerboard microdilution and the time-kill assays. Furthermore, atomic force microscopy (AFM) was used to image and quantify the effects of AlgL-antifungal combinations on biofilm-growing hyphal cells. On the basis of fractional inhibitory concentration index values, synergy was found between both AMB formulations and AlgL, and this finding was also confirmed by the time-kill test. Finally, AFM analysis showed that when A. fumigatus biofilms were treated with AlgL or polyene alone, as well as with their combination, both a reduction of hyphal thicknesses and an increase of adhesive forces were observed compared to the findings for untreated controls, probably owing to the different action by the enzyme or the antifungal compounds. Interestingly, marked physical changes were noticed in A. fumigatus biofilms exposed to the AlgL-antifungal combinations compared with the physical characteristics detected after exposure to the antifungals alone, indicating that AlgL may enhance the antibiofilm activity of both AMB and LAMB, perhaps by disrupting the hypha-embedding EPSs and thus facilitating the drugs to reach biofilm cells. Taken together, our results suggest that a combination of AlgL and a polyene antifungal may prove to be a new therapeutic strategy for invasive aspergillosis, while reinforcing the EPS as a valuable antibiofilm drug target.

DNA-based population analysis was applied in combination with Raman spectrometry and Environmental Scanning Electron Microscopy for the characterisation of natural biofilms from sand and activated carbon filters operated for a long term at a municipal waterworks. Whereas the molecular biology polymerase chain reaction combined with denaturing gradient gel electrophoresis approach provides a deeper insight into the bacterial biofilm diversities, Raman spectrometry analyses the chemical composition of the extracellular polymer substances (EPS), microorganisms embedded in EPS as well as other substances inside biofilm (inorganic compounds and humic substances). Microscopy images the spatial distribution of biofilms on the two different filter materials. In addition, bacterial bulk water populations were compared with biofilm consortia using the molecular fingerprint technique mentioned. Population analysis demonstrated the presence of more diverse bacterial species embedded in a matrix of EPS (polysaccharides, peptides, and nucleic acids) on the sand filter materials. In contrast to this, activated carbon granules were colonised by reduced numbers of bacterial species in biofilms. Besides alpha-, beta-, and gamma-Proteobacteria, a noticeable specific colonisation with Actinobacteria was found on activated carbon particles. Here, the reduced biofilm formation came along with a decreased EPS synthesis. The taxonomy profiles of the different biofilms revealed up to 60% similarity on the same filter materials and 32% similarity of different materials. Similarity of adherent communities from filter materials and bulk water populations from the filter effluent varied between 36% and 58% in sand filters and 6-40% in granular activated carbon filters. The biofilm investigation protocols are most crucial to subsequent acquisition of knowledge on biofilm dynamics and bacterial contributions to transformation or adsorption processes in waterworks facilities.

A strain of Bacillus licheniformis displaying a ropy phenotype was isolated from a French ropy cider. The influence of culture conditions on the production of exopolysaccharide (EPS) was investigated. When B. licheniformis was grown in Man, Rogosa and Sharpe (MRS) medium, the highest amount of EPS was observed at mid exponential growth phase whatever the carbon source, glucose, fructose or sucrose. Interestingly at mid exponential growth phase, EPS amounts did not increase with increasing sugar concentrations. Incubation of B. licheniformis cells in media supplemented with ethanol (1-7%, v/v) revealed that EPS production was enhanced by the presence of ethanol, in exponential as well as in stationary phase. High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR) analysis of EPS composition indicated that it was a heteropolymer in which mannose was the predominant monosaccharide as it constituted more than 80% of total polysaccharide.

The effect of chronic ethanol exposure on proopiomelanocortin (POMC) mRNA accumulation, beta-endorphin (beta-EP) levels as well as incorporation of [3H]-phenylalanine into beta-EP-related peptides was investigated in the rat hypothalamus. Animals were treated with an ethanol-containing liquid diet for 15 days. Both sucrose pair-fed and ad libitum-fed control groups were included. The levels of immunoreactive beta-EP, as well as the relative proportions of nonacetyl and acetyl forms of beta-EP in the hypothalami of the ethanol-treated rats were not significantly different from those in the sucrose and lab chow fed control rats. Northern blot analysis of total hypothalamic RNA indicated that ethanol-treated rats had higher POMC mRNA levels than sucrose pair-fed and control rats. Quantitation of the in vitro incorporation of [3H]-phenylalanine into POMC, beta-lipotropin and beta-EP by the hypothalamus, using immunoprecipitation and polyacrylamide disc-gel electrophoresis with sodium dodecyl sulfate, revealed that the increased hypothalamic POMC mRNA content was associated with increased incorporation of [3H]-phenylalanine into POMC by the hypothalami of the ethanol-treated rats. It is concluded that chronic ethanol can alter the expression of the POMC gene in the hypothalamus.

Male Sprague Dawley rats were chronically pair-fed with liquid diets containing 6.5% (vol/vol) ethanol, or equicaloric sucrose. After 21 days the ethanol-containing diet was discontinued and both groups were fed the sucrose diet. Groups of animals were killed on day 22 (0 day of ethanol withdrawal) and 1, 3, 8, and 15 days after ethanol withdrawal and the neurointermediate lobes (NILs) were removed and incubated with [3H]phenylalanine for 3 h. Chronic ethanol treatment induced an increase in the biosynthesis and release of beta-endorphin-like peptides by the rat NIL. After ethanol withdrawal the beta-endorphin-like immunoreactivity content in the NIL and the in vitro release of immunoreactive beta-endorphin (beta EP) by the NIL were significantly lower than in the controls on the first day, whereas no significant difference was found on days 3, 8, and 15 after ethanol withdrawal. The in vitro incorporation of [3H]phenylalanine into POMC, beta-lipotropin and beta EP was found to be higher in the ethanol-treated animals than in the controls on days 0, 1, and 3 after ethanol withdrawal, with no significant difference on days 8 and 15 after ethanol withdrawal. Furthermore, in both the ethanol-treated animals and their pair-fed controls the rate of incorporation of [3H]phenylalanine into total proteins, POMC, beta-lipotropin, and beta EP was significantly higher on days 8 and 15 after ethanol withdrawal than on the day of ethanol withdrawal (day 0), suggesting the implication of a nutritional factor. HPLC analysis of the beta EP peptides indicated that the percentage of acetylated forms of beta EP was higher in the NIL of the alcohol-treated animals, especially on days 8 and 15 after ethanol withdrawal. This observation suggests that though the rates of biosynthesis and release of beta EP-related peptides have returned to normal at 15 days after ethanol treatment, the activity of the enzyme responsible for the acetylation of beta EP remained elevated.