Comparative Genomics of Hypoxia and Barotrauma Tolerance in Diving Mammals

Hypoxia and barotrauma (pressure) tolerance are two main challenges for marine mammal evolution. Marine mammals have evolved many changes in their anatomy and physiology to combat hypoxia and barotrauma risks they are exposed to with their diving lifestyle. Changes in structures across vascular, pulmonary, musculoskeletal, and several other systems work together to prevent and delay injuries that are typically fatal to land mammals. Genomics studies on hypoxia tolerance in marine mammals have focused so far on a limited number of species or consider a limited number of genes. At the same time, genomic studies of barotrauma tolerance are almost absent due to limited physiological understanding of the relevant marine mammal phenotypes. This comparative genomic analysis explored the possibility of convergent evolution of members of pinnipeds, cetaceans and sirenians based on their diving patterns. Shared genes were extracted from complete genomes of 25 marine mammal species and used to infer a phylogenetic tree. I then assessed the evidence for positive selection between the deeper-diving members and their shallow-diving counterparts. Evidence of selection was detected in 315 genes in deeper-diving species, and these genes were significantly enriched for functions relating to hematopoietic (blood cell production) and immunological pathways. This result agrees with existing physiological studies of hypoxia and barotrauma tolerance. It supports existing working theories, such as preventing embolism caused by free-floating fibrin fragments in blood vessels and the intense, prolonged onset of the inflammatory response during dives and surfacing

Structure of Gremlin-1 and analysis of its interaction with BMP-2.

Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β (TGF-β) signalling family and has a very broad biological role in development. Its signalling is regulated by many effectors: transmembrane proteins, membrane-attached proteins and soluble secreted antagonists such as Gremlin-1. Very little is known about the molecular mechanism by which Gremlin-1 and other DAN (differential screening-selected gene aberrative in neuroblastoma) family proteins inhibit BMP signalling. We analysed the interaction of Gremlin-1 with BMP-2 using a range of biophysical techniques, and used mutagenesis to map the binding site on BMP-2. We have also determined the crystal structure of Gremlin-1, revealing a similar conserved dimeric structure to that seen in other DAN family inhibitors. Measurements using biolayer interferometry (BLI) indicate that Gremlin-1 and BMP-2 can form larger complexes, beyond the expected 1:1 stoichiometry of dimers, forming oligomers that assemble in alternating fashion. These results suggest that inhibition of BMP-2 by Gremlin-1 occurs by a mechanism that is distinct from other known inhibitors such as Noggin and Chordin and we propose a novel model of BMP-2-Gremlin-1 interaction yet not seen among any BMP antagonists, and cannot rule out that several different oligomeric states could be found, depending on the concentration of the two proteins.We would like to thank for members of the Hyvönen lab for the help and advice, in particular Ms Katharina Ravn for the original wild-type BMP-2 preparation and Dr Gerhard Fischer for his help with crystallography and SAXS data processing. We are grateful to Dr Katri Koli for providing us with the cDNA clone of Gremlin-1. We also acknowledge Dr. Grahame McKenzie, MRC Cancer Unit, University of Cambridge, who provided the C2C12 mouse myoblast cells. We thank the Diamond Light Source and the beamline staff for access to beamline I04 (proposal mx9537) and beamline I22 for SAXS measurements. This work was supported by Cambridge European Trust through a postgraduate scholarship to MK and by China Scholarship Council scholarship to XW.This is the author accepted manuscript. The final version is available from Portland Press via http://dx.doi.org/10.1042/BCJ2016025

Structure and activation of pro-activin A.

Activins are growth factors with multiple roles in the development and homeostasis. Like all TGF-β family of growth factors, activins are synthesized as large precursors from which mature dimeric growth factors are released proteolytically. Here we have studied the activation of activin A and determined crystal structures of the unprocessed precursor and of the cleaved pro-mature complex. Replacing the natural furin cleavage site with a HRV 3C protease site, we show how the protein gains its bioactivity after proteolysis and is as active as the isolated mature domain. The complex remains associated in conditions used for biochemical analysis with a dissociation constant of 5 nM, but the pro-domain can be actively displaced from the complex by follistatin. Our high-resolution structures of pro-activin A share features seen in the pro-TGF-β1 and pro-BMP-9 structures, but reveal a new oligomeric arrangement, with a domain-swapped, cross-armed conformation for the protomers in the dimeric protein

Selective Autophagy of BES1 Mediated by DSK2 Balances Plant Growth and Survival

Plants encounter a variety of stresses and must fine-tune their growth and stress-response programs to best suit their environment. BES1 functions as a master regulator in the brassinosteroid (BR) pathway that promotes plant growth. Here, we show that BES1 interacts with the ubiquitin receptor protein DSK2 and is targeted to the autophagy pathway during stress via the interaction of DSK2 with ATG8, a ubiquitin-like protein directing autophagosome formation and cargo recruitment. Additionally, DSK2 is phosphorylated by the GSK3-like kinase BIN2, a negative regulator in the BR pathway. BIN2 phosphorylation of DSK2 flanking its ATG8 interacting motifs (AIMs) promotes DSK2-ATG8 interaction, thereby targeting BES1 for degradation. Accordingly, loss-of-function dsk2 mutants accumulate BES1, have altered global gene expression profiles, and have compromised stress responses. Our results thus reveal that plants coordinate growth and stress responses by integrating BR and autophagy pathways and identify the molecular basis of this crosstalk

Editorial : An update on brassinosteroids : homeostasis, crosstalk, and adaptation to environmental stress

Over the last three decades, there have been significant advances in the understanding of brassinosteroid (BR) biosynthesis and signaling, particularly in the model plant species Arabidopsis thaliana. BRs regulate a variety of morphogenetic and physiological processes throughout plant life. Notably, BR biosynthesis and signaling are interconnected with the signaling pathways of other phytohormones and environmental stresses. Gathering knowledge about these aspects in monocot and dicot crops is of particular importance as it may allow modulation of these processes and enable the development cultivars better adapted to ongoing climate change. This Research Topic, providing An Update on Brassinosteroids: Homeostasis, Crosstalk, and Adaptation to Environmental Stress is aimed at introducing the latest findings in the regulation of BR metabolism, the interconnection of the BR signalosome with phytohormonal and stress signaling pathways, and the BR-mediated adaptation of plants to environmental conditions. The Research Topic includes five reviews and one original research article. [fragm. tekstu

PCR Techniques and Their Clinical Applications

Kary B. Mullis developed a revolutionary method name polymerase chain reaction (PCR) in 1983, which can synthesize new strand of DNA complementary to the template strand of DNA and produce billions of copies of a DNA fragment only in few hours. Denaturation, annealing, and extension are the three primary steps involved in the PCR process, which generally requires thermocyclers, DNA template, a pair of primers, Taq polymerase, nucleotides, buffers, etc. With the development of PCR, from traditional PCR, quantitative PCR, to next digital PCR, PCR has become a powerful tool in life sciences and medicine. Applications of PCR techniques for infectious diseases include specific or broad-spectrum pathogen detection, assessment and surveillance of emerging infections, early detection of biological threat agents, and antimicrobial resistance analysis. Applications of PCR techniques for genetic diseases include prenatal diagnosis and screening of neonatal genetic diseases. Applications of PCR techniques for cancer research include tumor-related gene detection. This chapter aimed to discuss about the different types of PCR techniques, including traditional PCR, quantitative PCR, digital PCR, etc., and their applications for rapid detection, mutation screen or diagnosis in infectious diseases, inherited diseases, cancer, and other diseases

Water-soluble, stable and azide-reactive strained dialkynes for biocompatible double strain-promoted click chemistry.

The Sondheimer dialkyne is extensively used in double strain-promoted azide-alkyne cycloadditions. This reagent suffers with poor water-solubility and rapidly decomposes in aqueous solutions. This intrinsically limits its application in biological systems, and no effective solutions are currently available. Herein, we report the development of novel highly water-soluble, stable, and azide-reactive strained dialkyne reagents. To demonstrate their extensive utility, we applied our novel dialkynes to a double strain-promoted macrocyclisation strategy to generate functionalised p53-based stapled peptides for inhibiting the oncogenic p53-MDM2 interaction. These functionalised stapled peptides bind MDM2 with low nanomolar affinity and show p53 activation in a cellular environment. Overall, our highly soluble, stable and azide-reactive dialkynes offer significant advantages over the currently used Sondheimer dialkyne, and could be utilised for numerous biological applications

Structure of the human myostatin precursor and determinants of growth factor latency.

Myostatin, a key regulator of muscle mass in vertebrates, is biosynthesised as a latent precursor in muscle and is activated by sequential proteolysis of the pro-domain. To investigate the molecular mechanism by which pro-myostatin remains latent, we have determined the structure of unprocessed pro-myostatin and analysed the properties of the protein in its different forms. Crystal structures and SAXS analyses show that pro-myostatin adopts an open, V-shaped structure with a domain-swapped arrangement. The pro-mature complex, after cleavage of the furin site, has significantly reduced activity compared with the mature growth factor and persists as a stable complex that is resistant to the natural antagonist follistatin. The latency appears to be conferred by a number of distinct features that collectively stabilise the interaction of the pro-domains with the mature growth factor, enabling a regulated stepwise activation process, distinct from the prototypical pro-TGF-β1. These results provide a basis for understanding the effect of missense mutations in pro-myostatin and pave the way for the design of novel myostatin inhibitors