Specific and sensitive quantitation of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in water, plasma and blood: application to toxicokinetic study in the rat after intravenous intoxication

A sensitive and specific capillary gas chromatographic method has been developed to measure trace amounts of 2,2′-dichlorodiethyl sulphide (sulphur mustard) in environmental or biological samples. Sulphur mustard was isolated from water or plasma by a solid-phase extraction procedure and from blood by liquid—liquid extraction. The accuracy and precision of the methods were demonstrated using replicate analyses of spiked water, plasma or blood: within-run and between-run variabilities were less than 20%. These analytical methods were used to evaluate the rate of sulphur mustard degradation in water or plasma. Good linear calibration curves, with a detection limit of 45 ng/ml, were obtained for quantitation and determination of sulphur mustard in blood following its intravenous administration to rats. Initial toxicokinetic data were obtained.     

All-or-none control of the bursting properties of the pacemaker neurons of the labster pyloric pattern generator

In Crustacea the central pattern generator for the pyloric motor rhythm (filtration to the midgut) is known to be located within the stomatogastric ganglion (STG); its cycling activity is known to be organized by three endogenous burster neurons acting as pacemakers and driving 11 follower neurons. In Homarus, recordings from the isolated stomatogastric nervous system (Fig. 1) indicate that (1) the pyloric output can be generated only when the STG is afferented (i.e., connected to the more rostral oesophageal and commissural ganglia) (Fig. 2) and (2) the deafferntation of the STG results in a complete loss of the bursting properties of the pacemaker neurons (Fig. 4). Manipulation of the STG inputs responsible for unmasking the properties of the pacemakers strongly suggests that (1) they are not phasic inputs (Fig. 5) and (2) they are long-term acting inputs (Fig. 6). These results provide evidence for a neural all-or-none control of the bursting properties of the pacemaker neurons of a motor pattern generator.     

In vivo activation of blood and arterial prophospholipase by an activator of blood platelet origin]

When partially purified platelet-rat lysate is injected in the rat aorta, transformation of prophospholipase into phospholipase is observed. Blood prophospholipases are activated almost entirely during about 15 minutes; aortic phospholipase are entirely activated for a longer time.     

Purification of rat adipose tissue lipoprotein lipase by affinity chromatography.

Lipoprotein lipase (EC 3.1.1.3) from rat adipose tissue was purified by affinity chromatography with heparin-Sepharose. Elution was carried out with buffered solutions of increasing NaCl molarity. Proteins without affinity for heparin were eluted with 0.5 M NaCl, while lipoprotein lipase activity was eluted as two peaks with 1.16 M NaCl (In earlier work on human adipose tissue (Etienne et al. (1974) C.R. Acad. Sc. Paris 279, 1487-1490) two fractions with lipoprotein lipase activity were also obtained). Phospholipase activity was detected in the fraction eluted with buffered 0.5 M NaCl and containing proteins without affinity for heparin. On feeding the fasting rats with fresh cream or glucose two peaks were also obtained, but the first peak had clearly increased while the second one had remained virtually unchanged.     

Pyronaridine–artesunate real-world safety,tolerability, and effectiveness in malaria patients in 5 African countries: A single-arm,open-label,cohort event monitoring study

BackgroundIn Phase II/III randomized controlled clinical trials for the treatment of acute uncomplicated malaria, pyronaridine–artesunate demonstrated high efficacy and a safety profile consistent with that of comparators, except that asymptomatic, mainly mild-to-moderate transient increases in liver aminotransferases were reported for some patients. Hepatic safety, tolerability, and effectiveness have not been previously assessed under real-world conditions in Africa.Methods and findingsThis single-arm, open-label, cohort event monitoring study was conducted at 6 health centers in Cameroon, Democratic Republic of Congo, Gabon, Ivory Coast, and Republic of Congo between June 2017 and April 2019. The trial protocol as closely as possible resembled real-world clinical practice for the treatment of malaria at the centers. Eligible patients were adults or children of either sex, weighing at least 5 kg, with acute uncomplicated malaria who did not have contraindications for pyronaridine–artesunate treatment as per the summary of product characteristics. Patients received fixed-dose pyronaridine–artesunate once daily for 3 days, dosed by body weight, without regard to food intake. A tablet formulation was used in adults and adolescents and a pediatric granule formulation in children and infants under 20 kg body weight. The primary outcome was the hepatic event incidence, defined as the appearance of the clinical signs and symptoms of hepatotoxicity confirmed by a >2× rise in alanine aminotransferase/aspartate aminotransferase (ALT/AST) versus baseline in patients with baseline ALT/AST >2× the upper limit of normal (ULN). As a secondary outcome, this was assessed in patients with ALT/AST >2× ULN prior to treatment versus a matched cohort of patients with normal baseline ALT/AST. The safety population comprised 7,154 patients, of mean age 13.9 years (standard deviation (SD) 14.6), around half of whom were male (3,569 [49.9%]). Patients experienced 8,560 malaria episodes; 158 occurred in patients with baseline ALT/AST elevations >2×ULN. No protocol-defined hepatic events occurred following pyronaridine–artesunate treatment of malaria patients with or without baseline hepatic dysfunction. Thus, no cohort comparison could be undertaken. Also, as postbaseline clinical chemistry was only performed where clinically indicated, postbaseline ALT/AST levels were not systematically assessed for all patients. Adverse events of any cause occurred in 20.8% (1,490/7,154) of patients, most frequently pyrexia (5.1% [366/7,154]) and vomiting (4.2% [303/7,154]). Adjusting for Plasmodium falciparum reinfection, clinical effectiveness at day 28 was 98.6% ([7,369/7,746] 95% confidence interval (CI) 98.3 to 98.9) in the per-protocol population. There was no indication that comorbidities or malnutrition adversely affected outcomes. The key study limitation was that postbaseline clinical biochemistry was only evaluated when clinically indicated.ConclusionsPyronaridine–artesunate had good tolerability and effectiveness in a representative African population under conditions similar to everyday clinical practice. These findings support pyronaridine–artesunate as an operationally useful addition to the management of acute uncomplicated malaria.Trial registrationClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT03201770","term_id":"NCT03201770"}}NCT03201770.

Gaston Tona Lutete and co-workers report on safety and effectiveness of the antimalarial drug pyronaridine-artesunate in African countries.     

Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the formation of a complex with ch-TOG-clathrin that crosslinks and stabilises kinetochore microtubules. Here we map the Aurora-A-binding interface in TACC3 and show that TACC3 potently activates Aurora-A through a domain centered on F525. Vertebrate cells carrying homozygous F525A mutation in the endogenous TACC3 loci exhibit defects in TACC3 function, namely perturbed localization, reduced phosphorylation and weakened interaction with clathrin. The most striking feature of the F525A cells however is a marked shortening of mitosis, at least in part due to rapid spindle assembly. F525A cells do not exhibit chromosome missegregation, indicating that they undergo fast yet apparently faithful mitosis. By contrast, mutating the phosphorylation site S558 to alanine in TACC3 causes aneuploidy without a significant change in mitotic duration. Our work has therefore defined a regulatory role for the Aurora-A-TACC3 interaction beyond the act of phosphorylation at S558. We propose that the regulatory relationship between Aurora-A and TACC3 enables the transition from the microtubule-polymerase activity of TACC3-ch-TOG to the microtubule-crosslinking activity of TACC3-ch-TOG-clathrin complexes as mitosis progresses. Aurora-A-dependent control of TACC3 could determine the balance between these activities, thereby influencing not only spindle length and stability but also the speed of spindle formation with vital consequences for chromosome alignment and segregation.     

Dispersal, edaphic fidelity and speciation in species-rich Western Australian shrublands: evaluating a neutral model of biodiversity

Over evolutionary time, the number of species in a community reflects the balance between the rate of speciation and the rate of extinction. Over shorter time‐scales local species richness is also affected by how often species move into and out of the local community. These processes are at the heart of Hubbell's ‘unified neutral theory of biodiversity’ ( Hubbell 2001 ). Hubbell's spatially implicit, dispersal‐limited neutral model is the most widely used of the many implementations of neutral theory and it provides an estimate of the rate of speciation in a metacommunity (if metacommunity size is known) and the rate at which species migrate into the local community from the wider metacommunity. Recently, this neutral model has been used to compare rates of speciation and migration in the species‐rich fynbos of South Africa and in neotropical forests. Here we use new analytical methods for estimating the neutral model's parameters to infer speciation and dispersal rates for three sites in species‐rich sclerophyll shrublands (equivalent to fynbos) in Western Australia (WA). Our estimates suggest that WA shrublands are intermediate between fynbos and tropical rainforest in terms of speciation and dispersal. Although a weak test, the model predicts species abundance distributions and species accumulation curves similar to those observed at the three sites. The neutral model's predictions also remain plausible when confronted with independent data describing: (1) known edaphic relationships between sites, (2) estimates of metacommunity species richness and (3) rates of speciation among resprouters and nonsprouters. Two of the site pairs, however, show species turnovers significantly different from those predicted by the spatially implicit form of the neutral model that we use. This suggests that non‐neutral processes, in this case probably edaphic specialisation, are important in the WA shrubland metacommunity. The neutral model predicts similar rates of speciation in resprouter and sprouter taxa, a finding supported by recent molecular phylogenies. Finally, when converted into temporally scaled speciation rates and species longevities, the estimates produced by the neutral model seem implausible. The apparent departure from neutrality in the turnover of species between some sites and the implausible temporal dynamics may be due to the particular model chosen and does not reduce the significance of our other results, which confirm that local dispersal limitation, coupled with broader scale edaphic fidelity, combine to structure this biodiverse metacommunity.     

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.     

Protein-Protein Interactions in the β-Oxidation Part of the Phenylacetate Utilization Pathway: CRYSTAL STRUCTURE OF THE PaaF-PaaG HYDRATASE-ISOMERASE COMPLEX*

Microbial anaerobic and so-called hybrid pathways for degradation of aromatic compounds contain β-oxidation-like steps. These reactions convert the product of the opening of the aromatic ring to common metabolites. The hybrid phenylacetate degradation pathway is encoded in Escherichia coli by the paa operon containing genes for 10 enzymes. Previously, we have analyzed protein-protein interactions among the enzymes catalyzing the initial oxidation steps in the paa pathway (Grishin, A. M., Ajamian, E., Tao, L., Zhang, L., Menard, R., and Cygler, M. (2011) J. Biol. Chem. 286, 10735–10743). Here we report characterization of interactions between the remaining enzymes of this pathway and show another stable complex, PaaFG, an enoyl-CoA hydratase and enoyl-Coa isomerase, both belonging to the crotonase superfamily. These steps are biochemically similar to the well studied fatty acid β-oxidation, which can be catalyzed by individual monofunctional enzymes, multifunctional enzymes comprising several domains, or enzymatic complexes such as the bacterial fatty acid β-oxidation complex. We have determined the structure of the PaaFG complex and determined that although individually PaaF and PaaG are similar to enzymes from the fatty acid β-oxidation pathway, the structure of the complex is dissimilar from bacterial fatty acid β-oxidation complexes. The PaaFG complex has a four-layered structure composed of homotrimeric discs of PaaF and PaaG. The active sites of PaaF and PaaG are adapted to accept the intermediary components of the Paa pathway, different from those of the fatty acid β-oxidation. The association of PaaF and PaaG into a stable complex might serve to speed up the steps of the pathway following the conversion of phenylacetyl-CoA to a toxic and unstable epoxide-CoA by PaaABCE monooxygenase.