Determination of thiopurine S-methyltransferase activity in erythrocytes using 6-thioguanine as substrate and a non-extraction liquid chromatographic technique

A non-extraction high-performance liquid chromatographic (HPLC) method has been developed for the determination of 6-methylthioguanine (6-MTG), as part of the determination of thiopurine S-methyltransferase activity (TPMT) in erythrocytes. Erythrocyte lysate is added to a glass vial containing substrates and incubation buffer, which is then sealed for the rest of the analysis. Enzyme incubation, sample preparation, and analysis are then undertaken without further sample-handling steps. The need for a solvent extraction step has been overcome by heating the incubate to 85 degrees C to stop the enzyme reaction. The heat inactivation step precipitates protein which upon centrifugation forms a thin film in the bottom of the glass vial enabling the supernatant to be injected directly onto the HPLC system. The assay shows excellent precision and recovery with a within-batch imprecision giving a co-efficient of variation of 2.9% (mean=41.5 nmol 6-MTG/gHb/h, n=10) and 5.1% (mean=12.6 nmol 6-MTG/g Hb/h, n=10). The between-batch imprecision gives a co-efficient of variation of 8.2% (mean=11.1 nmol 6-MTG/gHb/h, n=11) and 7.3% (mean=41.0 nmol 6-MTG/gHb/h, n=16). Determination of the TPMT activity in 120 people shows a range of enzyme activity of 11.3-63.8 nmol 6-MTG/gHb/h with a mean and median activity of 34.8 and 34.2 nmol 6-MTG/gHb/h, respectively. TPMT is increasingly used in clinical practice to ensure optimisation of treatment with thioguanine drugs. This direct HPLC method minimises sample-handling, reduces inherent imprecision, the possibility of laboratory error and with the potential for further automation, makes it ideal for use in a regional referral laboratory.     

Methotrexate in rheumatoid arthritis is frequently effective, even if re-employed after a previous failure

Effectiveness of therapy with individual disease-modifying antirheumatic drugs (DMARDs) in rheumatoid arthritis (RA) is limited, and the number of available DMARDs is finite. Therefore, at some stage during the lengthy course of RA, institution of traditional DMARDs that have previously been applied may have to be reconsidered. In the present study we investigated the effectiveness of re-employed methotrexate in patients with a history of previous methotrexate failure (original course). A total of 1,490 RA patients (80% female, 59% rheumatoid factor positive) were followed from their first presentation, yielding a total of 6,470 patient-years of observation. We identified patients in whom methotrexate was re-employed after at least one intermittent course of a different DMARD. We compared reasons for discontinuation, improvement in acute phase reactants, and cumulative retention rates of methotrexate therapy between the original course of methotrexate and its re-employment. Similar analyses were peformed for other DMARDs. Methotrexate was re-employed in 86 patients. Compared with the original courses, re-employment was associated with a reduced risk for treatment termination because of ineffectiveness (P = 0.02, by McNemar test), especially if the maximum methotrexate dose of the original course had been low (<12.5 mg/week; P = 0.02, by logistic regression). In a Cox regression model, re-employed MTX was associated with a significantly reduced hazard of treatment termination compared with the original course of methotrexate, adjusting for dose and year of employment (hazard ratio 0.64, 95% confidence interval 0.42-0.97; P = 0.04). These findings were not recapitulated in analyses of re-employment of other DMARDs. Re-employment of MTX despite prior inefficacy, but not re-employment of other DMARDs, is an effective therapeutic option, especially in those patients in whom the methotrexate dose of the original course was low.     

Biosorption of Lead from Industrial Wastewater Using Chrysophyllum albidum Seed Shell

The shell of the seed of Chrysophyllum albidum carbon was used to adsorb lead (Pb) from aqueous solution, the sorption process with respect to its equilibria and kinetics as well as the effects of pH, contact time, adsorbent mass, adsorbate concentration, and particle size on adsorption were also studied. The most effective pH range was found to be between 4.5 and 5 for the sorption of the metal ion. The first-order rate equation by Lagergren was tested on the kinetic data and the adsorption process followed first-order rate kinetics. Isotherm data were analyzed for possible agreement with the Langmuir and Freundlich adsorption isotherms; the Freundlich and Langmuir models for dynamics of metal ion uptake proposed in this work fitted the experimental data reasonably well. However, equilibrium sorption data were better represented by Langmuir model than Freundlich. The adsorption capacity calculated from Langmuir isotherm was 72.1 mg Pb (II) g^{- 1} at initial pH of 5.0 at 30°C for the particle size of 1.00 to 1.25 mm with the use of 2.0 g/100 ml adsorbent mass. The structural features of the adsorbent were characterized by Fourier transform infrared (FTIR) spectrometry; the presence of hydroxyl, carbonyl, amide, and phosphate groups confirms the potential mechanism adsorption of the adsorbent. This readily available adsorbent is efficient in the uptake of Pb (II) ion in aqueous solution, thus, it could be an excellent alternative for the removal of heavy metals and organic matter from water and wastewater.     

Range-wide fragmentation in a threatened fish associated with post-European settlement modification in the Murray–Darling Basin,Australia

Distinguishing the relative influence of historic (i.e. natural) versus anthropogenic factors in metapopulation structure is an important but often overlooked step in management programs of threatened species. Biotas in freshwater wetlands and floodplains, such as those in the Murray–Darling Basin (MDB)—one of Australia’s most impacted ecosystems, are particularly susceptible to anthropogenic fragmentation. Here we present a comprehensive multilocus assessment of genetic variation in the threatened southern pygmy perch Nannoperca australis (578 individuals; 45 localities; microsatellite, allozyme and mitochondrial DNA datasets), an ecological specialist with low dispersal potential. We assess patterns of spatial structure and genetic diversity in populations spanning the highly fragmented MDB and test whether recent anthropogenic modification has disrupted range-wide connectivity. We detected strong and hierarchical population structure, very low genetic diversity and lack of contemporary gene flow across the MDB. In contrast, the apparent absence of pronounced or long-term phylogeographic structure suggests that observed population divergences generally do not reflect deeply historic natural fragmentation. Coalescent-based analyses supported this inference, revealing that divergence times between populations from the upper and lower MDB fall into the period of European settlement. It appears that the observed contemporary isolation of populations is partly explained by the severe modification of the MDB post-dating the onset of European settlement. Our integrated approach substantially improves the interpretation of how fragmentation impacts present-day biodiversity. It also provides novel contributions for risk-assessing management actions in the context of captive breeding and translocations of small freshwater fishes, a group of increasing global conservation concern.     

Reconciliation and third‐party affiliation in carrion crows

Conflicts are costly because they can damage social relationships. To buffer conflicts, various species use post‐conflict behaviour, such as reconciliation or third‐party affiliation. Both behaviours have predominantly been studied in non‐human primates. However, recently, studies revealed post‐conflict behaviour in other mammalian and some bird species (e.g., corvids). While third‐party affiliation has been reported in several corvid species, reconciliation has only rarely been observed. The social structure of the studied groups has been postulated as a reason for the absence of reconciliation. Here, we investigated whether post‐conflict behaviours in corvids indeed mirror the relationship structure. We studied the behaviour of a newly established group of juvenile carrion crows (Corvus corone corone), where pair bonds had not yet been established. We applied a combination of observations and food monopolisation experiments to quantify the use of post‐conflict behaviours. Provisioning food in one or two pieces induced different patterns of aggression during feeding and differently affected the affiliation patterns after feeding. Specifically, victims of severe aggression affiliated with third parties after conflicts in the two‐piece condition, while aggressors affiliated with victims of mild aggression in the one‐piece condition. We thus provide the first evidence that a corvid species, crows, flexibly engage in both third‐party affiliation and reconciliation.     

The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms

Receptors for interleukin-4 (IL-4) are expressed at low levels on a wide variety of primary cells and cultured cell lines. Fluorescence-activated sorting of CTLL-2 cells resulted in the isolation of a subclone, CTLL 19.4, which expressed 10(6) IL-4 receptors per cell. These cells were used for the purification of IL-4 receptor protein and to prepare a hybrid-subtracted cDNA probe for isolation of cDNA clones. Three classes of IL-4 receptor cDNA were identified. The first encoded a 140 kd membrane bound IL-4 receptor containing extracellular, transmembrane, and cytoplasmic domains. The second class lacked the cytoplasmic region, and the third encoded a secreted form of the receptor. All cDNA clones expressed in COS-7 cells had IL-4 binding properties comparable to the native IL-4 receptor. The soluble form of the IL-4 receptor blocked the ability of IL-4 to induce CTLL cell proliferation and may represent a regulatory molecule specific for IL-4-dependent immune responses.     

Sequentially Deposited versus Conventional Nonfullerene Organic Solar Cells: Interfacial Trap States,Vertical Stratification,and Exciton Dissociation

Bulk heterojunction (BHJ) nonfullerene organic solar cells prepared from sequentially deposited donor and acceptor layers (sq‐BHJ) have recently been shown to be highly efficient, environmentally friendly, and compatible with large area and roll‐to‐roll fabrication. However, the related photophysics at donor‐acceptor interface and the vertical heterogeneity of donor‐acceptor distribution, critical for exciton dissociation and device performance, have been largely unexplored. Herein, steady‐state and time‐resolved optical and electrical techniques are employed to characterize the interfacial trap states. Correlating with the luminescent efficiency of interfacial states and its nonradiative recombination, interfacial trap states are characterized to be about 40% more populated in the sq‐BHJ devices than the as‐cast BHJ (c‐BHJ), which probably limits the device voltage output. Cross‐sectional energy‐dispersive X‐ray spectroscopy and ultraviolet photoemission spectroscopy depth profiling directly visualize the donor–acceptor vertical stratification with a precision of 1–2 nm. From the proposed “needle” model, the high exciton dissociation efficiency is rationalized. This study highlights the promise of sequential deposition to fabricate efficient solar cells, and points toward improving the voltage output and overall device performance via eliminating interfacial trap states.     

Analysis of Clostridium botulinum serotype E strains by using multilocus sequence typing, amplified fragment length polymorphism, variable-number tandem-repeat analysis, and botulinum neurotoxin gene sequencing

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA, involved with bont/E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont/E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont/E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont/E result in toxin variants or subtypes within strains.