Mutations in the Parainfluenza Virus 5 Fusion Protein Reveal Domains Important for Fusion Triggering and Metastability

Paramyxovirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxovirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in parainfluenza virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger.     

A novel ATM‐dependent checkpoint defect distinct from loss of function mutation promotes genomic instability in melanoma

Melanomas have high levels of genomic instability that can contribute to poor disease prognosis. Here, we report a novel defect of the ATM‐dependent cell cycle checkpoint in melanoma cell lines that promotes genomic instability. In defective cells, ATM signalling to CHK2 is intact, but the cells are unable to maintain the cell cycle arrest due to elevated PLK1 driving recovery from the arrest. Reducing PLK1 activity recovered the ATM‐dependent checkpoint arrest, and over‐expressing PLK1 was sufficient to overcome the checkpoint arrest and increase genomic instability. Loss of the ATM‐dependent checkpoint did not affect sensitivity to ionizing radiation demonstrating that this defect is distinct from ATM loss of function mutations. The checkpoint defective melanoma cell lines over‐express PLK1, and a significant proportion of melanomas have high levels of PLK1 over‐expression suggesting this defect is a common feature of melanomas. The inability of ATM to impose a cell cycle arrest in response to DNA damage increases genomic instability. This work also suggests that the ATM‐dependent checkpoint arrest is likely to be defective in a higher proportion of cancers than previously expected.     

Decoupled genomic elements and the evolution of partner quality in nitrogen‐fixing rhizobia

Understanding how mutualisms evolve in response to a changing environment will be critical for predicting the long‐term impacts of global changes, such as increased N (nitrogen) deposition. Bacterial mutualists in particular might evolve quickly, thanks to short generation times and the potential for independent evolution of plasmids through recombination and/or HGT (horizontal gene transfer). In a previous work using the legume/rhizobia mutualism, we demonstrated that long‐term nitrogen fertilization caused the evolution of less‐mutualistic rhizobia. Here, we use our 63 previously isolated rhizobium strains in comparative phylogenetic and quantitative genetic analyses to determine the degree to which variation in partner quality is attributable to phylogenetic relationships among strains versus recent genetic changes in response to N fertilization. We find evidence of distinct evolutionary relationships between chromosomal and pSym genes, and broad similarity between pSym genes. We also find that nifD has a unique evolutionary history that explains much of the variation in partner quality, and suggest MoFe subunit interaction sites in the evolution of less‐mutualistic rhizobia. These results provide insight into the mechanisms behind the evolutionary response of rhizobia to long‐term N fertilization, and we discuss the implications of our results for the evolution of the mutualism.     

Signaling Dynamics Control Cell Fate in the Early Drosophila Embryo

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Energetic implications of floodplain wetland restoration strategies for waterfowl

Modifications of the Illinois River and associated tributaries have resulted in altered hydrologic cycles and persistent river‐floodplain connections during the growing season that frequently impede the establishment of hydrophytic vegetation and have reduced value for migratory waterfowl and other waterbirds. To help guide floodplain restoration, we compared energetic carrying capacity for waterfowl in two wetland complexes along the Illinois River under different management regimes during 2012–2015. The south pool of Chautauqua National Wildlife Refuge (CNWR) was seasonally flooded due to a partial river connection and managed for moist‐soil vegetation. Emiquon Preserve was hydrologically isolated from the Illinois River by a high‐elevation levee and managed as a semipermanently flooded emergent marsh. Semipermanent emergent marsh management at Emiquon Preserve produced 5,495 energetic use‐days (EUD)/ha for waterfowl and other waterbirds across wetland cover types and years, and seasonal moist‐soil management at CNWR produced 6,199 EUD/ha in one of 4 years. At Emiquon Preserve, the aquatic bed cover type produced 9,660 EUD/ha, followed by 5,261 EUD/ha in moist‐soil, 1,398 EUD/ha in persistent emergent, 1,185 EUD/ha in hemi‐marsh, and 12 EUD/ha in open water cover types. At CNWR, the annual grass and sedge cover type produced 7,031 EUD/ha, followed by 5,618 EUD/ha in annual broadleaf and 1,305 EUD/ha in perennial grass cover types. Restoration of floodplain wetlands in isolation from frequent flood pulses during the growing season can produce hemi‐marsh and aquatic bed vegetation communities that provide high‐quality habitat for waterfowl and which have been mostly eliminated from large river systems in the Midwest, U.S.A.     

The 2-oxoacid dehydrogenase multi-enzyme complex of the archaeon Thermoplasma acidophilum - recombinant expression, assembly and characterization

The aerobic archaea possess four closely spaced, adjacent genes that encode proteins showing significant sequence identities with the bacterial and eukaryal components comprising the 2-oxoacid dehydrogenase multi-enzyme complexes. However, catalytic activities of such complexes have never been detected in the archaea, although 2-oxoacid ferredoxin oxidoreductases that catalyze the equivalent metabolic reactions are present. In the current paper, we clone and express the four genes from the thermophilic archaeon, Thermoplasma acidophilum, and demonstrate that the recombinant enzymes are active and assemble into a large (M(r) = 5 x 10(6)) multi-enzyme complex. The post-translational incorporation of lipoic acid into the transacylase component of the complex is demonstrated, as is the assembly of this enzyme into a 24-mer core to which the other components bind to give the functional multi-enzyme system. This assembled complex is shown to catalyze the oxidative decarboxylation of branched-chain 2-oxoacids and pyruvate to their corresponding acyl-CoA derivatives. Our data constitute the first proof that the archaea possess a functional 2-oxoacid dehydrogenase complex.