Seed bank dynamics govern persistence of Brassica hybrids in crop and natural habitats

Background and Aims Gene flow from crops to their wild relatives has the potential to alter population growth rates and demography of hybrid populations, especially when a new crop has been genetically modified (GM). This study introduces a comprehensive approach to assess this potential for altered population fitness, and uses a combination of demographic data in two habitat types and mathematical (matrix) models that include crop rotations and outcrossing between parental species.Methods Full life-cycle demographic rates, including seed bank survival, of non-GM Brassica rapa × B. napus F₁ hybrids and their parent species were estimated from experiments in both agricultural and semi-natural habitats. Altered fitness potential was modelled using periodic matrices including crop rotations and outcrossing between parent species.Key Results The demographic vital rates (i.e. for major stage transitions) of the hybrid population were intermediate between or lower than both parental species. The population growth rate (λ) of hybrids indicated decreases in both habitat types, and in a semi-natural habitat hybrids became extinct at two sites. Elasticity analyses indicated that seed bank survival was the greatest contributor to λ. In agricultural habitats, hybrid populations were projected to decline, but with persistence times up to 20 years. The seed bank survival rate was the main driver determining persistence. It was found that λ of the hybrids was largely determined by parental seed bank survival and subsequent replenishment of the hybrid population through outcrossing of B. rapa with B. napus.Conclusions Hybrid persistence was found to be highly dependent on the seed bank, suggesting that targeting hybrid seed survival could be an important management option in controlling hybrid persistence. For local risk mitigation, an increased focus on the wild parent is suggested. Management actions, such as control of B. rapa, could indirectly reduce hybrid populations by blocking hybrid replenishment.     

Product in indole detection by Ehrlich’s reagent

Ehrlich’s reagent (p-dimethylaminobenzaldehyde [DMAB, 1] in 95% EtOH with HCl as catalyst) was employed in spot tests of indoles, providing a diagnosis of, for example, liver diseases, hemolytic processes, occlusion of the common bile duct, and carcinoid syndrome. Although the reagent has been widely used for more than a century, it is not clear how many indole molecules react with a DMAB molecule and whether the reaction takes place at the α- or β-position of the indole molecule. Research here shows that the reaction of DMAB (1) with indole (2) in a 1:2 ratio gives β-bis(indolyl)methane (3). The reaction occurs at the β-position of indole under the conditions of the Ehrlich test, as confirmed by the crystal structure of 3.     

Role of the Cytosolic Loop C2 and the C Terminus of YidC in Ribosome Binding and Insertion Activity

Members of the YidC/Oxa1/Alb3 protein family mediate membrane protein insertion, and this process is initiated by the assembly of YidC·ribosome nascent chain complexes at the inner leaflet of the lipid bilayer. The positively charged C terminus of Escherichia coli YidC plays a significant role in ribosome binding but is not the sole determinant because deletion does not completely abrogate ribosome binding. The positively charged cytosolic loops C1 and C2 of YidC may provide additional docking sites. We performed systematic sequential deletions within these cytosolic domains and studied their effect on the YidC insertase activity and interaction with translation-stalled (programmed) ribosome. Deletions within loop C1 strongly affected the activity of YidC in vivo but did not influence ribosome binding or substrate insertion, whereas loop C2 appeared to be involved in ribosome binding. Combining the latter deletion with the removal of the C terminus of YidC abolished YidC-mediated insertion. We propose that these two regions play an crucial role in the formation and stabilization of an active YidC·ribosome nascent chain complex, allowing for co-translational membrane insertion, whereas loop C1 may be involved in the downstream chaperone activity of YidC or in other protein-protein interactions.     

On the Stability of Parainfluenza Virus 5 F Proteins

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.     

Discovery of a new class of glucosylceramide synthase inhibitors

A novel series of potent inhibitors of glucosylceramide synthase are described. The optimization of biochemical and cellular potency as well as ADME properties led to compound 23c. Broad tissue distribution was obtained following oral administration to mice. Thus 23c could be another useful tool compound for studying the effects of GCS inhibition in vitro and in vivo.     

Design and synthesis of long acting inhaled corticosteroids for the treatment of asthma

In this Letter we present data for a novel series of ICS for the treatment of asthma. 'Inhalation by design' principles have been applied to a series of highly potent steroidal GR agonists, with a focus on optimising the potential therapeutic index in human. Pharmacokinetic properties were tuned with high intrinsic clearance and low oral bioavailability in mind, to minimise systemic exposure and reduce systemically driven adverse events. High CYP mediated clearance as well as glucuronidation were targeted to achieve high intrinsic clearance coupled with multiple routes of clearance to minimise drug-drug interactions. Furthermore, pharmaceutical properties such as stability, crystallinity and solubility were considered to ensure compatibility with a dry powder inhaler. This work culminated in the identification of the clinical candidate 15, which demonstrates preclinically the desired efficacy and safety profiles confirming its potential as an inhaled agent for the treatment of asthma.     

In vitro and in vivo evaluation of 6-aminopyrazolyl-pyridine-3-carbonitriles as JAK2 kinase inhibitors

Synthesis and biological evaluation of a series of 6-aminopyrazolyl-pyridine-3-carbonitriles as JAK2 kinase inhibitors was reported. Biochemical screening, followed by profile optimization, resulted in JAK2 inhibitors exhibiting good kinase selectivity, pharmacokinetic properties, physical properties and pharmacodynamic effects.     

Activation of volume regulated chloride channels protects myocardium from ischemia/reperfusion damage in second-window ischemic preconditioning

Activation of volume regulated chloride channels (VRCCs) has been shown to be cardioprotective in ischemic preconditioning (IPC) of isolated hearts but the underlying molecular mechanisms remain unclear. Recent independent studies support that ClC-3, a ClC voltage-gated chloride channel, may function as a key component of the VRCCs. Thus, ClC-3 knockout (Clcn3(-/-)) mice and their age-matched heterozygous (Clcn3(+/-)) and wild-type (Clcn3(+/+)) littermates were used to test whether activation of VRCCs contributes to cardioprotection in early and/or second-window IPC. Targeted disruption of ClC-3 gene caused a decrease in the body weight but no changes in heart/body weight ratio. Telemetry ECG and echocardiography revealed no differences in ECG and cardiac function under resting conditions among all groups. Under treadmill stress (10 m/min for 10 min), the Clcn3(-/-) mice had significant slower heart rate (648±12 bpm) than Clcn3(+/+) littermates (737±19 bpm, n=6, P<0.05). Ex vivo IPC in the isolated working-heart preparations protected cardiac function during reperfusion and significantly decreased apoptosis and infarct size in all groups. In vivo early IPC significantly reduced infarct size in all groups including Clcn3(-/-) mice (22.7±3.7% vs control 40.1±4.3%, n=22, P=0.004). Second-window IPC significantly reduced apoptosis and infarction in Clcn3(+/+) (22.9±3.2% vs 45.7±5.4%, n=22, P<0.001) and Clcn3(+/-) mice (27.5±4.1% vs 42.2±5.7%, n=15, P<0.05) but not in Clcn3(-/-) littermates (39.8±4.9% vs 41.5±8.2%, n=13, P>0.05). Impaired cell volume regulation of the Clcn3(-/-) myocytes may contribute to the failure of cardioprotection by second-window IPC. These results strongly support that activation of VRCCs may play an important cardioprotective role in second-window IPC.     

Charge-surrounded pockets and electrostatic interactions with small ions modulate the activity of retroviral fusion proteins

Refolding of viral class-1 membrane fusion proteins from a native state to a trimer-of-hairpins structure promotes entry of viruses into cells. Here we present the structure of the bovine leukaemia virus transmembrane glycoprotein (TM) and identify a group of asparagine residues at the membrane-distal end of the trimer-of-hairpins that is strikingly conserved among divergent viruses. These asparagines are not essential for surface display of pre-fusogenic envelope. Instead, substitution of these residues dramatically disrupts membrane fusion. Our data indicate that, through electrostatic interactions with a chloride ion, the asparagine residues promote assembly and profoundly stabilize the fusion-active structures that are required for viral envelope-mediated membrane fusion. Moreover, the BLV TM structure also reveals a charge-surrounded hydrophobic pocket on the central coiled coil and interactions with basic residues that cluster around this pocket are critical to membrane fusion and form a target for peptide inhibitors of envelope function. Charge-surrounded pockets and electrostatic interactions with small ions are common among class-1 fusion proteins, suggesting that small molecules that specifically target such motifs should prevent assembly of the trimer-of-hairpins and be of value as therapeutic inhibitors of viral entry.