Monitoring settling of anaerobic digestates using low-field MRI profiling and NMR relaxometry measurements.

Dewatering of anaerobic digestate from red meat processing was assessed using low field MRI profiling and NMR relaxometry. Samples were flocculated using a cationic flocculant (EM640CT) at dosing range (0 to 1.6% v/v) and monitored during the initial 30 min of settling via MRI profiling to assess changes in water fraction, settling time and initial settling velocity. The profiles showed decreasing settling time and increasing initial settling velocity with increased dosing, while sample porosity was observed to increase up to the optimal dosing point (0.8% v/v). Significant increases in sample variability were observed past this point due to flocculant overdosing. The samples were then analysed in terms of turbidity and NMR relaxometry. Increasing flocculant concentration caused turbidity to decrease from 210 to 13 NTU. The relaxation rate of free water showed a strong positive correlation with turbidity. T2 peaks observed before overdosing could be assigned to different water structures (free, interstitial, vicinal and hydration). An additional T2 population emerged in the T2 distributions at the optimal dosing point. Multivariate exploratory data analysis (MEDA) showed that this T2 population from the solids layer was strongly correlated with the total solids layer height and turbidity of the watery layer. This T2 peak formation may therefore be used to study opaque flocculated solids to monitor for water structures associated with flocculant overdosing. Further studies using this technique will aim to assess the potential of low field T2 relaxometry monitoring inline before mechanical dewatering, to monitor optimal flocculant dosing during continuous operations on systems with high solids concentration.

Novel hyphenation of DGT in-situ passive sampling with YES assay to ascertain the potency of emerging endocrine disruptors in water systems in New Zealand.

This study represents the first attempt to investigate selected estrogenic compounds that include 17α-ethynylestradiol (EE2), 17ß-estradiol (E2) bisphenol A (BPA), and bisphenol AF (BPAF) along the drinkable water, from river-to-tap, and wastewater, from effluent-to-treated wastewater, treatment processes of the Hamilton City Council and the monitoring of the freshwater, from source-to-outfall, of the Waikato River in New Zealand. This was accomplished by the adoption of a novel combination of diffusive gradients in thin films (DGTs) in-situ passive sampling coupled with high-performance liquid chromatography/mass spectrometry analysis (HPLC/MS) and the Yeast Estrogen Screen (YES). Estradiol equivalency quantities, integrated in time, were evaluated theoretically (cEEQ) by DGT-HPLC/MS and experimentally (EEQ) by DGT-YES assay. cEEQ and EEQ highlighted that primary treatments are not suitable for estrogens and bisphenolic plastics removal both at drinkable and wastewater treatment plants in Hamilton where they worsen the water quality in terms of estrogenicity making these pollutants more available in the water phase. All downstream sites monitored along the Waikato River showed higher cEEQ and EEQ, moreover the Waikato River water quality showed a moderate worsening moving from Taupo (source) to Tuakau (outfall). The most polluted sites were downstream of Hamilton city and Huntly township wastewater treatment plants that serve the main conurbations in the area. cEEQ and EEQ generally showed good agreement at low concentrations but differed substantially at more polluted sites where cEEQ consistently underestimated estrogenic potency, possibly due to DGT accumulation of estrogenic compounds not quantified by HPLC/MS.

Low-field NMR relaxation-exchange measurements for the study of gas admission in microporous solids.

Understanding the uptake and storage of gases by microporous materials is important for our future energy security. As such, we demonstrate here the application of two-dimensional NMR relaxation experiments for probing the admission and corresponding exchange dynamics of methane within microporous zeolites. Specifically, we report low-field (12.7 MHz) 1H NMR relaxation-exchange correlation measurements of methane within commercial LTA zeolites (3A and 4A) at 25 and 35 bar and ambient temperature. Our results demonstrate the clear identification of bulk-pore and pore-pore exchange processes within zeolite 4A, facilitating the calculation and comparison of effective exchange rate dynamics across varying diffusion length scales and gas pressures. Additional data acquired for zeolite 3A reveals the sensitivity of NMR relaxation phenomena to size-exclusive gas admission phenomena, illustrating the potential of benchtop NMR protocols for material screening applications.

Three suturing techniques for closing fusiform excisions. A randomised controlled study.

BACKGROUND/OBJECTIVES: Australian doctors perform over one million skin excisions yearly. There are few randomised trials studying wound repair. The objective was to compare two suture techniques with controls for simple elliptical excisions in a prospective, randomised, single-blinded study. MATERIALS: One half of each wound was randomised to either one-layer closure with percutaneous nylon or modified two-layer closure where superficial closure was effected with adhesive tape. The control was the standard two-layer closure. Primary outcome measure was wound width at 6 months, with cosmesis as the secondary outcome. RESULTS: A total of 161 participants with 214 excisions were recruited from general practice. There was no significant difference in the primary outcome of wound width at 6 months between the groups. One-layer closure showed slight inferiority for cosmesis at 6 months versus the control. There was significant superiority for the modified two-layer closure versus the control for cosmesis at 6 months, and early wound erythema. Multivariate regression models adjusted for suture technique showed that younger age, wound infection and truncal location were predictors of increased scar width. Older age, wound erythema, and male gender were predictors of poorer patient-rated outcome. CONCLUSION: One-layer closure is an acceptable choice for elliptical excision wound closure. Modified two-layer closure may be preferable to standard two-layer closure for elliptical wounds.

Shear-induced emulsion droplet diffusion studies using NMR.

HYPOTHESIS: Shear-induced droplet diffusion of flowing hard spheres is relatively well understood and has been extensively studied both experimentally and via simulations. The same however is not true of soft spheres, specifically emulsions, despite their broad and extensive industrial relevance. Here we seek to demonstrate that appropriate NMR techniques can be used to quantitatively measure shear-induced droplet diffusion. Limited literature indicates that dilute dispersions of soft spheres experience significantly larger shear-induced droplet diffusion relative to otherwise equivalent hard sphere suspensions. Here we explore whether this effect persists to high concentrations. EXPERIMENTS: Nuclear Magnetic Resonance (NMR) pulsed field gradient (PFG) techniques were used to measure shear-induced droplet diffusion for capillary flow of various water-in-oil (w/o) emulsions in a direction transverse to flow. Two adaptations were necessary - the acquired signal was analyzed so as to quantitatively distinguish restricted molecular diffusion within the emulsion droplets from shear-induced diffusion of the droplets, whilst flow-compensated PFG pulse sequences were shown to be necessary to account for any erroneous effects due to flow. A range of w/o emulsions were considered to enable measurement of shear-induced droplet diffusion as a function of both water content and mean shear rate. The surfactant content of these emulsions was adjusted such that they presented similar (stationary) emulsion droplet size distributions (DSD) which were also measured using NMR PFG techniques. FINDINGS: The droplet shear-induced diffusion data for the emulsion systems were compared against relevant results from the literature. Consistent with predictions for dilute systems, significantly greater droplet diffusion was measured relative to hard sphere suspensions at all concentrations, and a quadratic dependence was found between droplet diffusion and mean droplet size. For more concentrated emulsions, a peak in the droplet diffusion-concentration relationship was observed for the first time in emulsions, prior to the onset of emulsion inversion.

A non-peptide oxytocin receptor agonist, WAY-267,464, alleviates novelty-induced hypophagia in mice: insights into changes in c-Fos immunoreactivity.

Anxiety caused by the novelty of food or of the environment where the food is presented leads to suppression of consumption (hyponeophagia) reflected by an increased latency to begin feeding and decreased food intake. Studies suggest that some anxiolytics, mainly benzodiazepines and SSRIs, resolve hyponeophagia. Though the neurohormone oxytocin (OT) affects both anxiety responsiveness and feeding-related homeostasis, the link between OT and hyponeophagia has not been established. The current experiments examined the effect of OT receptor stimulation on hyponeophagia in mice and associated changes in brain activity. We found that the OT receptor agonist, WAY-267,464, at 10 and 30 mg/kg b. wt. IP, reduced the latency to approach food and increased the amount of food eaten in hyponeophagia tests differing in animals' motivation to eat (hunger, reward) and the anxiogenic context of environmental novelty (illumination and type of the cage). This effect was abolished by the pretreatment with the OT receptor antagonist, L-368,899, at 10mg/kg b. wt. The antagonist also suppressed social transmission of preference for novel food. Mice subjected to novelty conditions causing hypophagia showed significant changes in c-Fos immunoreactivity in the hippocampus, lateral septum, cingulate and piriform cortex and in the bed nucleus of the stria terminalis, lateral division, posterolateral part (STLP). The pretreatment with WAY-267,464 restored c-Fos levels in the STLP to values detected in control animals subjected to non-anxiogenic conditions. We conclude that OT plays a role in shaping the magnitude of the novelty stress-provoked hypophagia and the activity of the relevant neural networks.

Development and validation of a homogeneous mobility shift assay for the measurement of infliximab and antibodies-to-infliximab levels in patient serum.

Antibody-based drugs such as infliximab (IFX) are effective for the treatment of inflammatory bowel disease (IBD) and other immune-mediated disorders. The development of antibodies against these drugs may result in unfavorable consequences, including the loss of drug efficacy, hypersensitivity reactions, and other adverse events. Therefore, accurate monitoring of serum drug and anti-drug antibody levels should be an important part of therapy for patients being treated with an antibody-based drug. Current methods for the assessment of anti-drug antibodies and drug levels, involving various bridging ELISA and radioimmunoassay techniques, are limited by their sensitivity, interference, and/or complexity. To overcome these limitations, we have developed a non-radiolabeled homogeneous mobility shift assay (HMSA) to measure the antibodies-to-infliximab (ATI) and IFX levels in serum samples. Full method validation was performed on both the ATI- and IFX-HMSA, and the clinical sample test results were also compared with those obtained from a bridging ELISA method to evaluate the difference in performance between the two assays. Validation of the ATI-HMSA revealed a lower limit of quantitation of 0.012 µg/mL in serum. The linear range of quantitation was 0.029-0.54 µg/mL. The intra- and inter-assay precision was less than 20% of coefficient of variation (CV), and the accuracy (% error) of the assay was less than 20%. In serum samples, ATI as low as 0.036 µg/mL can be measured, even in the presence of 60 µg/mL of IFX in the serum. Sera from 100 healthy subjects were tested to determine the cut point of the assay. ATI-positive samples that had been previously analyzed by using a bridging ELISA from 100 patients were also measured by the new method. There was a high correlation between the two methods for ATI levels (p<0.001). Significantly, the new method identified five false-positive samples from the bridging ELISA method. Validation of the mobility shift IFX assay also showed high assay sensitivity, precision and accuracy. The HMSA method may also be applied to other protein-based drugs to accurately detect serum drug and anti-drug antibody levels.

Flow cytometric characterization of freshwater crayfish hemocytes for the examination of physiological status in wild and captive animals.

Enumeration of invertebrate hemocytes is a potentially powerful tool for the determination of physiological effects of extrinsic stressors, such as hypoxia, disease, and toxicant exposure. A detailed flow cytometric method of broad application was developed for the objective characterization and enumeration of the hemocytes of New Zealand freshwater crayfish Paranephrops planifrons for the purpose of physiological health assessment. Hemocyte populations were isolated by flow cytometric sorting based on differential light scatter properties followed by morphological characterization via light microscopy and software image analysis. Cells were identified as hyaline, semigranular, and granular hemocytes based on established invertebrate hemocyte classification. A characteristic decrease in nuclear size, an increase in granularity between the hyaline and granular cells, and the eccentric location of nuclei in granular cells were also observed. The granulocyte subpopulations were observed to possess varying degrees of granularity. The developed methodology was used to perform total and differential hemocyte counts from three lake populations and between wild and captive crayfish specimens. Differences in total and differential hemocyte counts were not observed among the wild populations. However, specimens held in captivity for 14 d exhibited a significant 63% reduction in total hemocyte count, whereas the relative hemocyte proportions remained the same. These results demonstrate the utility of this method for the investigation of subacute stressor effects in selected decapod crustaceans.

Myostatin signals through Pax7 to regulate satellite cell self-renewal.

Myostatin, a Transforming Growth Factor-beta (TGF-beta) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells.

Substituted NDP-MSH peptides paired with mutant melanocortin-4 receptors demonstrate the role of transmembrane 6 in receptor activation.

The melanocortin-4 receptor (MC4R) is involved in regulating energy homeostasis and is a potential therapeutic target for obesity and cachexia. Molecular interactions between peptide ligands and MC4R have been studied in detail. Less is known regarding the role of these interactions in the mechanism of MC4R activation. The aim of this study was to investigate the molecular mechanism of human MC4R activation by [Nle4, d-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH), by first defining the role of the His6-d-Phe7-Arg8-Trp9 residues in receptor activation (Emax for stimulation of cAMP accumulation) using modified peptides, then understanding how their interaction with the receptor modulates activation using site-directed mutagenesis and a molecular model of NDP-MSH bound to the active state of the receptor. Alanine substitution indicated that the d-Phe7, Arg8, and Trp9 side chains contribute binding energy but are not essential for the receptor activation event. Conversely, His6 to Ala6 substitution reduced receptor activation but did not affect affinity. Chlorine substitutions on the d-Phe7 side chain also inhibited receptor activation. F261(6.51)A and F284(7.35)A receptor mutations acted as gain-of-function mutations, restoring efficacy to the His6 and d-Phe7 substituted peptides that had lost efficacy at the wild-type receptor. Based on a model of NDP-MSH and MC4R interaction, the antagonist behavior of these peptides is consistent with the prevention of transmembrane 6 (TM6) rotation. This data supports the hypothesis that increasing the size of d-Phe7 directly interferes with TM6 rotation, preventing receptor activation. We further propose that removing the interaction with the His6 side chain reorients the peptide within the binding pocket, indirectly impeding TM6 rotation by strengthening peptide interaction with F261(6.51) and F284(7.35). These findings refine the molecular basis for the mechanism of ligand-stimulated hMC4R activation and will be useful for the development of hMC4R agonists and antagonists.

Pharmacological characterization of a novel nonpeptide antagonist of the human gonadotropin-releasing hormone receptor, NBI-42902.

Suppression of the hypothalamic-pituitary-gonadal axis by peptides that act at the GnRH receptor has found widespread use in clinical practice for the management of sex-steroid-dependent diseases (such as prostate cancer and endometriosis) and reproductive disorders. Efforts to develop orally available GnRH receptor antagonists have led to the discovery of a novel, potent nonpeptide antagonist, NBI-42902, that suppresses serum LH concentrations in postmenopausal women after oral administration. Here we report the in vitro and in vivo pharmacological characterization of this compound. NBI-42902 is a potent inhibitor of peptide radioligand binding to the human GnRH receptor (K(i) = 0.56 nm). Tritiated NBI-42902 binds with high affinity (K(d) = 0.19 nm) to a single class of binding sites and can be displaced by a range of peptide and nonpeptide GnRH receptor ligands. In vitro experiments demonstrate that NBI-42902 is a potent functional, competitive antagonist of GnRH stimulated IP accumulation, Ca(2+) flux, and ERK1/2 activation. It did not stimulate histamine release from rat peritoneal mast cells. Finally, it is effective in lowering serum LH in castrated male macaques after oral administration. Overall, these data provide a benchmark of pharmacological characteristics required for a nonpeptide GnRH antagonist to effectively suppress gonadotropins in humans and suggest that NBI-42902 may have clinical utility as an oral agent for suppression of the hypothalamic-pituitary-gonadal axis.

NMR structural characterization of a minimal peptide antagonist bound to the extracellular domain of the corticotropin-releasing factor1 receptor.

Natural peptide agonists of corticotrophin-releasing factor (CRF) receptors bind to the receptor by a two-site mechanism as follows: the carboxyl end of the ligand binds the N-terminal extracellular domain (ECD) of the receptor and the amino portion of the ligand binds the extracellular face of the seven transmembrane region. Recently, peptide antagonists homologous to the 12 C-terminal residues of CRF have been derived, which bind the CRF(1) receptor through an interaction with the ECD. Here we characterized the binding of a minimal 12-residue peptide antagonist while bound to the isolated ECD of the CRF(1) receptor. We have expressed and purified soluble and properly folded ECD independent from the seven-transmembrane region as a thioredoxin fusion protein in Escherichia coli. A model of the peptide antagonist, cyclic corticotrophin-releasing factor residues 30-41 (cCRF(30-41)), was calculated while bound to the recombinant ECD using transferred nuclear Overhauser effect spectroscopy. Although the peptide is unstructured in solution, it adopts an alpha-helical conformation when bound to the ECD. Residues of cCRF(30-41) comprising the binding interface with the ECD were mapped using saturation transfer difference NMR. Two hydrophobic residues (Met(38) and Ile(41)) as well as two amide groups (Asn(34) and the C-terminal amide) on one face of the helix defined the binding epitope of the antagonist. This epitope may be used as a starting point for development of non-peptide antagonists targeting the ECD of this receptor.

Prolonged absence of myostatin reduces sarcopenia.

Sarcopenia is a progressive age-related loss of skeletal muscle mass and strength. Parabiotic experiments show that circulating factors positively influence the proliferation and regenerative capacity of satellite cells in aged mice. In addition, we believe that negative regulators of muscle mass also serve to balance the signals that influence satellite cell activation and regeneration capacity with ageing. Myostatin, a negative regulator of pre- and postnatal myogenesis, inhibits satellite cell activation and muscle regeneration postnatally. To investigate the role of myostatin during age-related sarcopenia, we examined muscle mass and regeneration in young and old myostatin-null mice. Young myostatin-null muscle fibers were characterized by massive hypertrophy and hyperplasia and an increase in type IIB fibers, resulting in a more glycolytic muscle. With ageing, wild-type muscle became increasingly oxidative and fiber atrophy was prominent. In contrast no fiber type switching was observed and atrophy was minimal in aged myostatin-null muscle. The effect of ageing on satellite cell numbers appeared minimal, however, satellite cell activation declined significantly in both wild-type and myostatin-null muscles. In young mice, lack of myostatin resulted in increased satellite cell number and activation compared to wild-type, suggesting a greater propensity to undergo myogenesis, a difference maintained in the aged mice. In addition, muscle regeneration of myostatin-null muscle following notexin injury was accelerated and fiber hypertrophy and type were recovered with regeneration, unlike in wild-type muscle. In conclusion, a lack of myostatin appears to reduce age-related sarcopenia and loss of muscle regenerative capacity.

The N-terminal domain of CCL21 reconstitutes high affinity binding, G protein activation, and chemotactic activity, to the C-terminal domain of CCL19.

CC chemokine receptor 7 (CCR7), which regulates the trafficking of leucocytes to the secondary lymphoid organs, has two endogenous chemokine ligands: CCL19 and CCL21. Although both ligands possess similar affinities for the receptor and similar abilities to promote G protein activation and chemotaxis, they share only 25% sequence identity. Here, we show that substituting N-terminal six amino acids of CCL21 (SDGGAQ) for the corresponding N-terminal domain of CCL19 (GTNDAE) results in a chimeric chemokine that exhibits high affinity binding and G protein activation of CCR7. These data demonstrate that despite dissimilar sequences, the amino terminal hexapeptide of these two chemokines is capable of performing similar roles resulting in receptor activation.

Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism.

Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.

Excising basal cell carcinoma in general practice.

BACKGROUND: Currently there is much interest in the general practice management of basal cell carcinoma (BCC). Published research regarding its surgical management mainly pertains to tumours located on the head and neck. However, in my general practice experience, the majority of excisions came from other sites. METHODS: A retrospective audit of histopathology reports from 91 excisions of BCCs. RESULTS: Thirty-nine percent of BCCs in this series were located on the head or neck, compared with 75-94% from other series; 50% of BCCs from the head/neck contained an aggressive histological subtype, compared with only 10% from other sites; four out of 139 papers found regarding surgical management of BCCs were from primary care practice. DISCUSSION: Significant differences exist between the location mix and histological types of BCCs treated in general practice with those reported in the research literature. Treatments advocated by this literature may not be readily transferable to the general practice setting.

Cumulative impacts assessment along a large river, using brown bullhead catfish (Ameiurus nebulosus) populations.

The effects of point-source and diffuse discharges on resident populations of brown bullhead catfish (Ameiurus nebulosus (LeSueur, 1819)) in the Waikato River (New Zealand) were assessed at sites both upstream and downstream of point-source discharges. At each site, the population parameters, relative abundance, age structure, and individual indices, such as condition factor, organ (gonad, liver, and spleen) to somatic weight ratios, and number and size of follicles per female, were assessed. Physiological (blood), biochemical (hepatic ethoxyresorufin-O-deethylase [EROD] and plasma steroids), and other indicators (bile chemistry and liver metals) of exposure or response also were measured. No impacts on brown bullhead health were obvious at individual geothermal, municipal sewage, or thermal discharge sites or cumulatively along the river. Brown bullhead from the bleached kraft mill effluent site showed elevated levels of EROD, decreased numbers of red blood cells, increased numbers of white blood cells, and depressed levels of sex steroids. However, growth rates, condition factor, age structure, and gonadosomatic index suggest that discharges with significant heat or nutrients benefit catfish despite physiological impairment at one site. Consideration of brown bullhead population-level responses to discharges in a monitoring framework revealed three different population-level response patterns resulting from the point-source discharges.

Combined effects of pulp and paper effluent, dehydroabietic acid, and hypoxia on swimming performance, metabolism, and hematology of rainbow trout.

Experiments were conducted to examine the effects of a thermomechanical (TMP)/bleached kraft pulp and paper mill effluent (BKME), dehydroabietic acid (DHAA), hypoxia, and combinations of hypoxia and effluent on juvenile rainbow trout. In the first two experiments, trout were exposed for 4 weeks to 0%, 10%, 30%, and 70% TMP/BKME or 0, 35, 110, and 250 microgL(-1) DHAA, respectively. Endpoints of those dose-response studies included critical swimming speed, oxygen consumption, and hematology. Reduced swimming performance was found for fish exposed to 70% TMP/BKME. Moderate increases in mean cell hemoglobin concentration at 70% TMP/BKME and blood glucose at 30% and 70% TMP/BKME were also seen. The opposite trend for glucose was found for DHAA-exposed fish, where a slight decrease in glucose was seen at 110 and 250 microgL(-1) DHAA. The third experiment examined the effects of 15% v/v TMP/BKME exposure at 2.5 and 5.0 mgL(-1) dissolved oxygen (DO) for 4 weeks. This experiment found no effect of low DO on swimming ability. An interactive effect between DO and effluent exposure was seen only on hematocrit, where effluent caused an increase in hematocrit at 5 mgL(-1) and a decrease at 2.5 mgL(-1) DO. Effluent exposure in this experiment resulted in a greater number of smaller red blood cells. The current study demonstrated physiological effects in rainbow trout exposed to varying concentrations (15-70% v/v) of a TMP/BKME and no substantial effects of DHAA exposure. With the exception of the reduced swimming performance in fish exposed to TMP/BKME, the observed effects are considered relatively small in magnitude but are occurring at concentrations of effluent that occur in the receiving environment.

Myostatin negatively regulates the expression of the steroid receptor co-factor ARA70.

Myostatin is a transforming growth factor-beta (TGF-beta) superfamily member and a key negative regulator of embryonic and postnatal muscle growth. In order to identify downstream target genes regulated by Myostatin, we performed suppressive subtraction hybridization (SSH) on cDNA generated from the biceps femoris muscle of wild-type and myostatin-null mice. Sequence analysis identified several known and unknown genes as Myostatin downstream target genes. Here, we have investigated the regulation of gene expression of an androgen receptor (AR) binding co-factor, androgen receptor associated protein-70 (ARA70), by Myostatin. We show that in mouse there are two isoforms of ARA70 with high homology (79%) to human ARA70; an alpha-isoform which is a canonical ARA70 and a beta-isoform which has a 9 consecutive amino acid deletion and 6 amino acid substitutions in the carboxyl-terminal portion. Reverse Northern analysis on the differentially expressed cDNA library indicated that there is increased expression of ARA70 in the muscles of myostatin-null mice. In addition, Northern blot, together with semi-quantitative PCR analysis, confirmed that there is increased expression of ARA70 in myostatin-null biceps femoris muscle when compared to wild-type muscle. In corroboration of these results, addition of exogenous Myostatin results in down-regulation of ARA70 expression confirming that Myostatin is a negative regulator of ARA70 gene expression. Expression analysis further confirmed that ARA70 is up-regulated during myogenesis and that peak expression of ARA70 is observed following the peak expression of MyoD in differentiating myoblasts. Given that lack of Myostatin and increased expression of AR leads to hypertrophy, we propose that absence of Myostatin, at least in part, induces the hypertrophy phenotype by increasing the activity of AR by up-regulating the expression of ARA70, a known stimulating co-factor of AR.

Atropisomeric property of 1-(2,6-difluorobenzyl)-3-[(2R)-amino-2-phenethyl]-5-(2-fluoro-3-methoxyphenyl)-6-methyluracil.

1-(2,6-Difluorobenzyl)-3-[(2R)-amino-2-phenethyl]-5-(2-fluoro-3-methoxyphenyl)-6-methyluracil (6), a potent and orally active antagonist of the human gonadotropin-releasing hormone receptor, exists as a pair of atropisomers in solution, which was detected by NMR spectroscopy, and separable by HPLC. In addition to a (R)-configured benzylamine, there is a second stereogenic element due to the presence of a chiral axis between the substituted 5-phenyl group and the uracil core. The rate constant of the interconversion (k = 5.07 x 10(-5) s(-1)) of these two atropisomers was determined by proton NMR analysis of a diastereoisomer-enriched sample in aqueous solution at 25 degrees C, and the corresponding Gibbs free energy DeltaG(#) of rotation barrier (97.4 kJ mol(-1)) was calculated using the Eyring equation. The diastereoisomer half-life at physiological temperature (37 degrees C) in aqueous media was estimated to be about 46 min.