Extracellular pH regulation in microdomains of colonic crypts: effects of short-chain fatty acids.

It has been suggested that transepithelial gradients of short-chain fatty acids (SCFAs; the major anions in the colonic lumen) generate pH gradients across the colonic epithelium. Quantitative confocal microscopy was used to study extracellular pH in mouse distal colon with intact epithelial architecture, by superfusing tissue with carboxy SNARF-1 (a pH-sensitive fluorescent dye). Results demonstrate extracellular pH regulation in two separate microdomains surrounding colonic crypts: the crypt lumen and the subepithelial tissue adjacent to crypt colonocytes. Apical superfusion with (i) a poorly metabolized SCFA (isobutyrate), (ii) an avidly metabolized SCFA (n-butyrate), or (iii) a physiologic mixture of acetate/propionate/n-butyrate produced similar results: alkalinization of the crypt lumen and acidification of subepithelial tissue. Effects were (i) dependent on the presence and orientation of a transepithelial SCFA gradient, (ii) not observed with gluconate substitution, and (iii) required activation of sustained vectorial acid/base transport by SCFAs. Results suggest that the crypt lumen functions as a pH microdomain due to slow mixing with bulk superfusates and that crypts contribute significant buffering capacity to the lumen. In conclusion, physiologic SCFA gradients cause polarized extracellular pH regulation because epithelial architecture and vectorial transport synergize to establish regulated microenvironments.

Diffusion modeling of percutaneous absorption kinetics: 3. Variable diffusion and partition coefficients, consequences for stratum corneum depth profiles and desorption kinetics

Stratum corneum (SC) desorption experiments have yielded higher calculated steady-state fluxes than those obtained by epidermal penetration studies. A possible explanation of this result is a variable diffusion or partition coefficient across the SC. We therefore developed the diffusion model for percutaneous penetration and desorption to study the effects of either a variable diffusion coefficient or variable partition coefficient in the SC over the diffusion path length. Steady-state flux, lag time, and mean desorption time were obtained from Laplace domain solutions. Numerical inversion of the Laplace domain solutions was used for simulations of solute concentration-distance and amount penetrated (desorbed)-time profiles. Diffusion and partition coefficients heterogeneity were examined using six different models. The effect of heterogeneity on predicted flux from desorption studies was compared with that obtained in permeation studies. Partition coefficient heterogeneity had a more profound effect on predicted fluxes than diffusion coefficient heterogeneity. Concentration-distance profiles show even larger dependence on heterogeneity, which is consistent with experimental tape-stripping data reported for clobetasol propionate and other solutes. The clobetasol propionate tape-stripping data were most consistent with the partition coefficient decreasing exponentially for half the SC and then becoming a constant for the remaining SC. (C) 2004 Wiley-Liss, Inc.

Changes in body water distribution during treatment with inhaled steroid in pre-school children

Primary objective: The study aimed to examine the changes in water distribution in the soft tissue during systemic steroid activity. Research design: A three-way cross-over, randomized, placebo-controlled, double-blind trial was used, including 4 weeks of fluticasone propionate pMDI 200 mug b.i.d. delivered via Babyhaler(R), budesonide pressurized metered dose inhaler (pMDI) 200 mug b.i.d. delivered via Nebuchamber(R) and placebo. Spacers were primed before use. In total, 40 children aged 1-3 years, with mild intermittent asthma were included. Twenty-five of the children completed all three treatments. At the end of each treatment period body impedance and skin ultrasonography were measured. Methods and procedures: We measured changes in water content of the soft tissues by two methods. Skin ultrasonography was used to detect small changes in dermal water content, and bioelectrical impedance was used to assess body water content and distribution. Main outcomes and results: We found an increase in skin density of the shin from fluticasone as measured by ultrasonography (p = 0.01). There was a tendency for a consistent elevation of impedance parameters from active treatments compared to placebo although overall this effect was not statistically significant (0.1< p <0.2). However, sub-analyses indicated a significant effect on whole-body and leg impedance from budesonide treatment (p <0.05). Conclusion: Decreased growth during inhaled steroid treatment seems to partly reflect generalized changes in body water.

Short-term effects of carbon source on the competition of polyphosphate accumulating organisms and glycogen accumulating organisms

The effectiveness of enhanced biological phosphorus removal (ESPR) systems is directly affected by the competition of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs). This study investigated the short-term effects of carbon source on PAO and GAO performance. The tests were designed to clearly determine the impact of volatile fatty acid (VFA) composition on the performance of two types of biomass, one enriched for PAOs and the other for GAOs. The two populations were enriched in separate reactors using identical operating conditions and very similar influent compositions with acetate as the sole carbon source. The only difference was that a very tow level of phosphorus was present in the influent to the GAO reactor. The abundance of PAOs and GAOs was quantified using fluorescence in-situ hybridisation. The results clearly show that there are some very distinctive differences between PAOs and GAOs in their ability to utilise different carbon substrates. While both are able to take up acetate rapidly and completely, the GAOs are far slower at consuming propionate than the PAOs during short-term substrate changes. This provides a potentially highly valuable avenue to influence the competition between PAOs and GAOs. Other VFAs studied seem to be less usable in the short term by both PAOs and GAOs; as indicated by their much lower uptake rates.

Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs

There is an urgent need to treat restenosis, a major complication of the treatment of arteries blocked by atherosclerotic plaque, using local delivery techniques. We observed that cross-linked fibrin (XLF) is deposited at the site of surgical injury of arteries. An antibody to XLF, conjugated to anti-restenotic agents, should deliver the drugs directly and only to the site of injury. An anti-XLF antibody (H93.7C.1D2/48; 1D2) was conjugated to heparin (using N-succinimidyl 3-(2-pyridyldithio)-propionate), low molecular weight heparin (LMWH) (adipic acid dihydrazide) and rapamycin (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and the conjugates purified and tested for activity before use in vivo. Rabbits had their right carotid arteries de-endothelialised and then given a bolus of 1D2-heparin, 1D2-LMWH or 1D2-rapamycin conjugate or controls of saline, heparin, LMWH, rapamycin or 1D2 (+/-heparin bolus) and sacrificed after 2 or 4 weeks (12 groups, n=6/group). Rabbits given any of the conjugates had minimal neointimal development in injured arteries, with up to 59% fewer neointimal cells than those given control drugs. Rabbits given 1D2-heparin or 1D2-LMWH had an increased or insignificant reduction in luminal area, with positive remodelling, while the medial and total arterial areas of rabbits given 1D2-rapamycin were not affected by injury. Arteries exposed to 1D2-heparin or 1D2-rapamycin had more endothelial cells than rabbits given control drugs. Thus, XLF-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall, where the conjugates can influence remodelling, re-endothelialisation and neointimal cell density, with reduced neointimal formation. (C) 2004 Elsevier B.V. All rights reserved.

Monensin supplementation of lactating cows fed tropical grasses and cane molasses or grain

Effects of monensin (Mon) on performance of Holstein-Friesian cows fed tropical grasses and cane molasses (M) or cereal grain were examined in three experiments. In experiment I (incomplete 4 x 4 Latin square), three rumen-fistulated cows [188 I I days in milk (DIM)] were fed mixed diets based on rhodes grass (Chloris gayana cv. Callide) bay where M was substituted for wheat grain (W) at rates of 0 (MO), 125 (M 125) or 250 (M250) g/kg dry matter (DM). A fourth diet contained M250 plus 0.02 g Mon/kg DM (M250 + Mon). Substituting M for W tended (P < 0.10) to decrease the ratio of rumen molar proportions of acetate+butyrate (Bu):propionate (Pr) (4.3 versus 3.8 and 4.0 for M0, M125 and M250, respectively). There were no treatment effects (P> 0.10) on intake, organic matter digestibility, milk production or liveweight (LW) change. In experiment 2, 48 cows (173 &PLUSMN; 28.3 DIM) grazing kikuyu (Pennisetum clandestinum cv. common) pastures and supplemented with maize silage and a grain-based concentrate were offered either M (2.6 kg DM/(cow day)) or barley grain (B) (2.7 kg DM/(cow day)). Within each supplement type, half were fed 0 or 320 mg of Mon/(cow day). There were Mon x supplement interactions (Mon x S; P < 0.05) on the rumen molar proportion of Pr and Bu at 15:00 h, with B + Mon having the highest value for Pr (0.259 mmol/mmol) and lowest value for Bu (0.121 mmol/mmol). A Mon x S effect (P < 0.05) on milk fat content was noted with Mon causing a lower value regardless of energy source (31 and 36 g/l versus 40 and 38 g/l for B + Mon, M + Mon, B - Mon and M - Mon, respectively). As a main effect, M as opposed to B, reduced yields of milk (P < 0.05; 16.21/(cow day) versus 18.01/(cow day)) and protein (P < 0.05; 479 g/(cow day) versus 538 g/(cow day)). Monensin reduced milk fat yield (P < 0.05; 669 g/(cow day) versus 562 g/(cow day)), raised milk protein concentration (P < 0.05; 31 g/l versus 29 g/l) and caused LW gain rather than loss (P < 0.05; +0.06 kg/(cow day) versus -0.30 kg/(cow day)). No treatment effects on pasture intake were noted. In experiment 3, 48 cows (91 &PLUSMN; 16.1 DIM) grazing kikuyu pasture and supplemented with grain-based concentrate, sugar cane silage and 2.7 kg DM(cow day) of M were supplemented with either 0 or 320 mg Mon/(cow day). Monensin reduced (P < 0.05) milk fat content (33 g/l versus 30 g/l) and tended (P < 0.10) to reduce milk protein content (29 g/l versus 28 g/l). No effects of Mon on other milk production parameters, LW change or pasture intake were noted. Feeding monensin to mid-lactation Holstein-Friesian cows offered diets based on tropical grasses, and cane molasses or grain, improves rumen fermentation efficiency, thereby improving energy efficiency resulting in higher LW gain. Monensin had no effect on milk yield, but reduced milk fat concentration.

Doubling daily inhaled corticosteroid dose is ineffective in mild to moderately severe attacks of asthma in adults

Background: Asthma guidelines recommend increasing or doubling inhaled corticosteroid (ICS) dose to treat mild and moderate exacerbations of asthma in adults. Aim: To: (i) compare the effectiveness of doubling existing daily ICS dose (fluticasone) with maintaining usual ICS dose and usual daily ICS dose accompanied by oral steroids (OS) (dexamethasone) during mild and moderately severe exacerbations of asthma in adults; (ii) examine determinants of success and failure; and (iii) compare side-effect profiles. Methods: A randomized, double-blind, placebo-controlled (double-dummy), triple crossover trial. Participants acted as their own control. Outcome measures included treatment success/failure, peak expiratory flow (PEF) after 7 days therapy or at treatment failure, and side-effects. Results: From 22 participants (nine males and 13 females), 18 pairs of data were available for maintaining usual ICS versus doubling ICS and doubling ICS versus OS, and 19 for maintaining usual ICS versus OS. Median (fifth-95th percentile) age was 46.5 (32-64) years and forced expiratory volume in one second (FEV1) 73% (29-97%) predicted. The outcome after doubling ICS was not superior to maintaining usual ICS, with 11 (61%) failures in both arms (P = 0.66). OS, with only 5 (26%) failures, was superior to maintaining usual ICS with 12 (63%) failures (P = 0.04), and to doubling ICS with 5 (28%) versus 11 (61%) failures (P = 0.07). Median PEF (as percentage of run-in best) at end-points were 90.5% (57.1-177.1) for OS, 78.3% (39.5-103.1) for maintaining usual ICS and 77.9 (27.7-110.3) for doubling ICS. Neither gender nor PEF at exacerbation were predictive of failure. Although doubling ICS was not an effective therapy overall, ICS dose at exacerbation were predictive of success in the doubling ICS arm (P = 0.04). Treatment failures when doubling daily ICS dose were more common if achieved fluticasone dose was less than 2000 mu g (three of 11, 73%) compared to 2000 mu g or greater (eight of eight, 37.5%). Increasing age and the presence of an upper respiratory tract infection (URTI) were predictive of failure with OS. Side-effects were more commonly reported with OS (52.6%) than doubling ICS (42.1%) or maintaining usual ICS (19.1%) with the most common being mood changes (36.8%), sleep disturbance (31.6%) and changes in appetite (26.3%). Conclusions: Doubling daily ICS dose per se is not effective for the treatment of mild to moderately severe exacerbations of asthma in adults. Success may depend on achieved ICS dose. Oral steroids are effective, but side-effects are common. A review of current guidelines may be warranted.

The effect of pH on the competition between polyphosphate-accumulating organisms and glycogen-accumulating organisms

In enhanced biological phosphorus removal (EBPR) processes, glycogen-accumulating organisms (GAOs) may compete with polyphosphate-accumulating organisms (PAOs) for the often-limited carbon substrates, potentially resulting in disturbances to phosphorus removal. A detailed investigation of the effect of pH on the competition between PAOs and GAOs is reported in this study. The results show that a high external pH (similar to 8) provided PAOs with an advantage over GAOs in EBPR systems. The phosphorus removal performance improved due to a population shift favouring PAOs over GAOs, which was shown through both chemical and microbiological methods. Two lab-scale reactors fed with propionate as the carbon source were subjected to an increase in pH from 7 to 8. The phosphorus removal and PAO population (as measured by quantitative fluorescence in situ hybridisation analysis of Candidatus Accumulibacter phosphatis) increased in each system, where the PAOs appeared to out-compete a group of Alphaproteobacteria GAOs. A considerable improvement in the P removal was also observed in an acetate fed reactor, where the GAO population (primarily Candidatus Competibacter phosphatis) decreased substantially after a similar increase in the pH. The results from this study suggest that pH could be used as a control parameter to reduce the undesirable proliferation of GAOs and improve phosphorus removal in EBPR systems. (c) 2005 Elsevier Ltd. All rights reserved.

Optimisation of poly-b-hydroxyalkanoate analysis using gas chromatography for enhanced biological phosphorus removal systems

Poly-beta-hydroxyalkanoate (PHA) is a polymer commonly used in carbon and energy storage for many different bacterial cells. Polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs), store PHA anaerobically through metabolism of carbon substrates such as acetate and propionate. Although poly-beta-hydroxybutyrate (PHB)and poly-beta-hydroxyvalerate (PHV) are commonly quantified using a previously developed gas chromatography (GC) method, poly-beta-hydroxy-2-methyl valerate (PH2MV) is seldom quantified despite the fact that it has been shown to be a key PHA fraction produced when PAOs or GAOs metabolise propionate. This paper presents two GC-based methods modified for extraction and quantification of PHB, PHV and PH2MV from enhanced biological phosphorus removal (EBPR) systems. For the extraction Of PHB and PHV from acetate fed PAO and GAO cultures, a 3% sulfuric acid concentration and a 2-20 h digestion time is recommended, while a 10% sulfuric acid solution digested for 20 h is recommended for PHV and PH2MV analysis from propionate fed EBPR systems. (c) 2005 Elsevier B.V. All rights reserved.

Applicability of experience from laboratory reactors with biological phosphorus removal in full-scale plants

Enhanced biological phosphorus removal (EBPR) has been used at many wastewater treatment plants all over the world for many years. In this study a full-scale sludge with good EBPR was tested with P-release batch tests and combined FISH/MAR (fluorescence in situ hybridisation and microautoradiography). Proposed models of PAOs and GAOs (polyphosphate- and glycogen-accumulating organisms) and microbial methods suggested from studies of laboratory reactors were found to be applicable also on sludge from full-scale plants. Dependency of pH and the uptake of both acetate and propionate were studied and used for calculations for verifying the models and results from microbial methods. All rates found from the batch tests with acetate were higher than in the batch tests with propionate, which was explained by the finding that only those parts of the bacterial community that were able to take up acetate anaerobically were able to take up propionate anaerobically.

Effects of solids concentration, pH and carbon addition on the production rate and composition of volatile fatty acids in prefermenters using primary sewage sludge

Increasing evidence is emerging that the performance of enhanced biological phosphorus removal (EBPR) systems relies on not only the total amount but also the composition of volatile fatty acids (VFAs). Domestic wastewater often contains limited amounts of VFAs with acetic acid typically being the dominating species. Consequently, prefermenters are often employed to generate additional VFAs to meet the demand for carbon by EBPR and/or denitrification processes. Limited knowledge is currently available on the effects of operational conditions on the production rate and composition of VFAs in prefermenters. In this study, a series of controlled batch experiments were conducted with sludge from a full-scale prefermenter to determine the impact of solids concentration, pH and addition of molasses on prefermentation processes. It was found that an increase in solids concentration enhanced total VFA production with an increased propionic acid fraction. The optimal pH for prefermentation was in the range of 6-7 with significant productivity loss when pH was below 5.5. Molasses addition significantly increased the production of VFAs particularly the propionic acid. However, the fermentation rate was likely limited by the biological activity of the sludge rather than by the amount of molasses added.

Solution properties of star and linear poly(N-isopropylacrylamide)

The LCST transitions of novel N-isopropylacrylamide ( NIPAM) star polymers, prepared using the four-armed RAFT agent pentaerythritoltetrakis(3-(S-benzyltrithiocarbonyl) propionate) (PTBTP) and their hydrolyzed linear arms were studied using H-1 NMR, PFG-NMR, and DLS. The aim was to determine the effect of polymer architecture and the presence of end groups derived from RAFT agents on the LCST. The LCST transitions of star PNIPAM were significantly depressed by the presence of the hydrophobic star core and possibly the benzyl end groups. The effect was molecular weight dependent and diminished once the number of repeating units per arm >= 70. The linear PNIPAM exhibited an LCST of 35 degrees C, regardless of molecular weight; the presence of both hydrophilic and hydrophobic end groups after hydrolysis from the star core was suggested to cancel effects on the LCST. A significant decrease in R-H was observed below the LCST for star and linear PNIPAM and was attributed to the formation of n-clusters. Application of a scaling law to the linear PNIPAM data indicated the cluster size n = 6. Tethering to the hydrophobic star core appeared to inhibit n-cluster formation in the lowest molecular weight stars; this may be due to enhanced stretching of the polymer chains, or the presence of larger numbers of n-clusters at temperatures below those measured.

Obtaining highly enriched cultures of candidatus accumulibacter phosphates through alternating carbon sources

Candidatus Accumulibacter Phosphatis is widely considered to be a polyphosphate accumulating organism (PAO) of prime importance in enhanced biological phosphorus removal (EBPR) systems. This organism has yet to be isolated, despite many attempts. Previous studies on the biochemical and physiological aspects of this organism, as well as its response to different EBPR operational conditions, have generally relied on the use of mixed culture enrichments. One frequent problem in obtaining highly enriched cultures of this organism is the proliferation of glycogen accumulating organisms (GAO) that can compete with PAOs for limited carbon sources under similar operational conditions. In this study, Candidatus Accumulibacter Phosphatis has been enriched in a lab-scale bioreactor to a level greater than 90% as quantified by fluorescence in situ hyrbridisation (FISH). This is the highest enrichment of this organism that has been reported thus far, and was obtained by alternating the sole carbon source in the feed between acetate and propionate every one to two sludge ages, and operating the bioreactor within a pH range of 7.0-8.0. Simultaneously, the presence of two known groups of GAOs was eliminated under these operational conditions. Excellent phosphorus removal performance and stability were maintained in this system, where the phosphorous concentration in the effluent was below 0.2 mg/L for more than 7 months. When a disturbance was introduced to this system by adding sludge from an enriched GAO culture, Candidatus Accumulibacter Phosphatis once again became highly enriched, while the GAOs were out-competed. This feeding strategy is recommended for future studies focused on describing the physiology and biochemistry of Accumulibacter, where a highly-enriched culture of this organism is of high importance. (c) 2006 Elsevier Ltd. All rights reserved.