AtPAP2 is a tail-anchored protein in the outer membrane of chloroplasts and mitochondria

To date, Arabidopsis purple acid phosphatase 2 (AtPAP2) is the only known plant protein that is dual-targeted to chloroplasts and mitochondria by a C-terminal targeting signal. Using in vitro organelle import and green fluorescence protein (GFP) localization assays, we showed that AtPAP2 is located on, but not imported across the outer membrane (OM) of chloroplasts and mitochondria and exposed its N-terminal enzymatic domain to the cytosol. It was also found that a short stretch of 30 amino acids (a.a.) at the C-terminal region (a.a. 615-644) that contains a stretch of 18 hydrophobic residues, a WYAK motif and 8 hydrophilic residues is sufficient for dual-targeting. Mutation of WYAK to WYAE had no effect on dual-targeting ability suggesting that the charge within this flanking region alone is not an important determinant for dual-targeting.        

Simultaneous determination of key bioactive components in Hedyotis diffusa by capillary electrophoresis

A capillary zone electrophoresis (CZE) method based on systematic one-variable-at-a time approach was developed for the analysis of four important bioactive components (geniposidic acid, ursolic acid, quercetin and p-coumaric acid) in the extract of Hedyotis diffusa (HD). Separations were carried out in a fused-silica capillary tube with peak detection at 214 nm. Good separation was achieved using a 20 mM borate buffer containing 5% acetonitrile as organic modifier and pH adjusted to 10.0. Operating voltage was 15 kV and temperature was maintained at 25 degrees C while hydrodynamic injection was 5s. A good linearity, with correlation coefficients in the ranges of 0.997-0.999 was obtained in the calibration curves of each standard. Relative standard deviation (R.S.D.) of migration time was between 0.32 and 0.70% and deviation of corrected peak area was between 8.84 and 11.99%. These results indicate that this method could be used for rapid and simultaneous analysis of the bioactive components in HD and other herbal products.     

Contribution of conserved lysine residues in the alpha2-antiplasmin C terminus to plasmin binding and inhibition

α(2)-Antiplasmin is the physiological inhibitor of plasmin and is unique in the serpin family due to N- and C-terminal extensions beyond its core domain. The C-terminal extension comprises 55 amino acids from Asn-410 to Lys-464, and the lysine residues (Lys-418, Lys-427, Lys-434, Lys-441, Lys-448, and Lys-464) within this region are important in mediating the initial interaction with kringle domains of plasmin. To understand the role of lysine residues within the C terminus of α(2)-antiplasmin, we systematically and sequentially mutated the C-terminal lysines, studied the effects on the rate of plasmin inhibition, and measured the binding affinity for plasmin via surface plasmon resonance. We determined that the C-terminal lysine (Lys-464) is individually most important in initiating binding to plasmin. Using two independent methods, we also showed that the conserved internal lysine residues play a major role mediating binding of the C terminus of α(2)-antiplasmin to kringle domains of plasmin and in accelerating the rate of interaction between α(2)-antiplasmin and plasmin. When the C terminus of α(2)-antiplasmin was removed, the binding affinity for active site-blocked plasmin remained high, suggesting additional exosite interactions between the serpin core and plasmin.     

The impact of removing financial incentives and/or audit and feedback on chlamydia testing in general practice: A cluster randomised controlled trial (ACCEPt-able)

BackgroundFinancial incentives and audit/feedback are widely used in primary care to influence clinician behaviour and increase quality of care. While observational data suggest a decline in quality when these interventions are stopped, their removal has not been evaluated in a randomised controlled trial (RCT), to our knowledge. This trial aimed to determine whether chlamydia testing in general practice is sustained when financial incentives and/or audit/feedback are removed.Methods and findingsWe undertook a 2 × 2 factorial cluster RCT in 60 general practices in 4 Australian states targeting 49,525 patients aged 16–29 years for annual chlamydia testing. Clinics were recruited between July 2014 and September 2015 and were followed for up to 2 years or until 31 December 2016. Clinics were eligible if they were in the intervention group of a previous cluster RCT where general practitioners (GPs) received financial incentives (AU$5–AU$8) for each chlamydia test and quarterly audit/feedback reports of their chlamydia testing rates. Clinics were randomised into 1 of 4 groups: incentives removed but audit/feedback retained (group A), audit/feedback removed but incentives retained (group B), both removed (group C), or both retained (group D). The primary outcome was the annual chlamydia testing rate among 16- to 29-year-old patients, where the numerator was the number who had at least 1 chlamydia test within 12 months and the denominator was the number who had at least 1 consultation during the same 12 months. We undertook a factorial analysis in which we investigated the effects of removal versus retention of incentives (groups A + C versus groups B + D) and the effects of removal versus retention of audit/feedback (group B + C versus groups A + D) separately. Of 60 clinics, 59 were randomised and 55 (91.7%) provided data (group A: 15 clinics, 11,196 patients; group B: 14, 11,944; group C: 13, 11,566; group D: 13, 14,819). Annual testing decreased from 20.2% to 11.7% (difference −8.8%; 95% CI −10.5% to −7.0%) in clinics with incentives removed and decreased from 20.6% to 14.3% (difference −7.1%; 95% CI −9.6% to −4.7%) where incentives were retained. The adjusted absolute difference in treatment effect was −0.9% (95% CI −3.5% to 1.7%; p = 0.2267). Annual testing decreased from 21.0% to 11.6% (difference −9.5%; 95% CI −11.7% to −7.4%) in clinics where audit/feedback was removed and decreased from 19.9% to 14.5% (difference −6.4%; 95% CI −8.6% to −4.2%) where audit/feedback was retained. The adjusted absolute difference in treatment effect was −2.6% (95% CI −5.4% to −0.1%; p = 0.0336). Study limitations included an unexpected reduction in testing across all groups impacting statistical power, loss of 4 clinics after randomisation, and inclusion of rural clinics only.ConclusionsAudit/feedback is more effective than financial incentives of AU$5–AU$8 per chlamydia test at sustaining GP chlamydia testing practices over time in Australian general practice.Trial registrationAustralian New Zealand Clinical Trials Registry ACTRN12614000595617

In a cluster randomized trial, Jane S Hocking and colleagues investigate the impact of removing financial incentives and/or audit and feedback on chlamydia testing in general practice in Australia.     

Diversification of Lupine Bradyrhizobium Strains: Evidence from Nodulation Gene Trees

Bradyrhizobium strains isolated in Europe from Genisteae and serradella legumes form a distinct lineage, designated clade II, on nodulation gene trees. Clade II bradyrhizobia appear to prevail also in the soils of Western Australia and South Africa following probably accidental introduction with seeds of their lupine and serradella hosts. Given this potential for dispersal, we investigated Bradyrhizobium isolates originating from a range of native New World lupines, based on phylogenetic analyses of nodulation (nodA, nodZ, noeI) and housekeeping (atpD, dnaK, glnII, recA) genes. The housekeeping gene trees revealed considerable diversity among lupine bradyrhizobia, with most isolates placed in the Bradyrhizobium japonicum lineage, while some European strains were closely related to Bradyrhizobium canariense. The nodA gene tree resolved seven strongly supported groups (clades I to VII) that correlated with strain geographical origins and to some extent with major Lupinus clades. All European strains were placed in clade II, whereas only a minority of New World strains was placed in this clade. This work, as well as our previous studies, suggests that clade II diversified predominately in the Old World, possibly in the Mediterranean. Most New World isolates formed subclade III.2, nested in a large “pantropical” clade III, which appears to be New World in origin, although it also includes strains originating from nonlupine legumes. Trees generated using nodZ and noeI gene sequences accorded well with the nodA tree, but evidence is presented that the noeI gene may not be required for nodulation of lupine and that loss of this gene is occurring.     

Fertile forests produce biomass more efficiently

Trees with sufficient nutrition are known to allocate carbon preferentially to aboveground plant parts. Our global study of 49 forests revealed an even more fundamental carbon allocation response to nutrient availability: forests with high-nutrient availability use 58?±?3% (mean?±?SE; 17 forests) of their photosynthates for plant biomass production (BP), while forests with low-nutrient availability only convert 42?±?2% (mean?±?SE; 19 forests) of annual photosynthates to biomass. This nutrient effect largely overshadows previously observed differences in carbon allocation patterns among climate zones, forest types and age classes. If forests with low-nutrient availability use 16?±?4% less of their photosynthates for plant growth, what are these used for? Current knowledge suggests that lower BP per unit photosynthesis in forests with low- versus forests with high-nutrient availability reflects not merely an increase in plant respiration, but likely results from reduced carbon allocation to unaccounted components of net primary production, particularly root symbionts.     

Meandering worms: mechanics of undulatory burrowing in muds

Recent work has shown that muddy sediments are elastic solids through which animals extend burrows by fracture, whereas non-cohesive granular sands fluidize around some burrowers. These different mechanical responses are reflected in the morphologies and behaviours of their respective inhabitants. However, Armandia brevis, a mud-burrowing opheliid polychaete, lacks an expansible anterior consistent with fracturing mud, and instead uses undulatory movements similar to those of sandfish lizards that fluidize desert sands. Here, we show that A. brevis neither fractures nor fluidizes sediments, but instead uses a third mechanism, plastically rearranging sediment grains to create a burrow. The curvature of the undulating body fits meander geometry used to describe rivers, and changes in curvature driven by muscle contraction are similar for swimming and burrowing worms, indicating that the same gait is used in both sediments and water. Large calculated friction forces for undulatory burrowers suggest that sediment mechanics affect undulatory and peristaltic burrowers differently; undulatory burrowing may be more effective for small worms that live in sediments not compacted or cohesive enough to extend burrows by fracture.     

Aortic endothelial cells damaged by a nitric oxide donor and protected by flavonoids.

Cultured porcine aortic endothelial cells (PAEC) were exposed to four concentrations (0.00 mM - 5.00 mM) of 3-Morpholino-sydnonimine-hydrochloride (SIN-1, a nitric oxide donor). SIN-1 demonstrated a dose dependent cytotoxicity against PAEC as indicated by the thiobarbituric acid (TBA) assay. Morphologically and biochemically, the presence of selected flavonoids (morin, quercetin, or catechin) was shown to protect the PAEC from SIN-1 toxicity. Protection levels determined from the TBA assay were significant (p<0.05) for all flavonoids, with morin at 72+/-8%. Quercetin and catechin had comparable protective activities of 54+/-6% and 43+/-3%, respectively. This study supports the contention that SIN-1 is cytotoxic to PAEC and that antioxidants such as flavonoids may attenuate such toxicity.