Characterization of erythroferrone oligomerization and its impact on BMP antagonism.

Hepcidin, a peptide hormone that negatively regulates iron metabolism, is expressed by bone morphogenetic protein (BMP) signaling. Erythroferrone (ERFE) is an extracellular protein that binds and inhibits BMP ligands, thus positively regulating iron import by indirectly suppressing hepcidin. This allows for rapid erythrocyte regeneration after blood loss. ERFE belongs to the C1Q/TNF-related protein family and is suggested to adopt multiple oligomeric forms: a trimer, a hexamer, and a high molecular weight species. The molecular basis for how ERFE binds BMP ligands and how the different oligomeric states impact BMP inhibition are poorly understood. In this study, we demonstrated that ERFE activity is dependent on the presence of stable dimeric or trimeric ERFE and that larger species are dispensable for BMP inhibition. Additionally, we used an in silico approach to identify a helix, termed the ligand-binding domain, that was predicted to bind BMPs and occlude the type I receptor pocket. We provide evidence that the ligand-binding domain is crucial for activity through luciferase assays and surface plasmon resonance analysis. Our findings provide new insight into how ERFE oligomerization impacts BMP inhibition, while identifying critical molecular features of ERFE essential for binding BMP ligands.

Characterization of the molecular mechanisms that govern anti-Müllerian hormone synthesis and activity.

The roles of anti-Müllerian hormone (AMH) continue to expand, from its discovery as a critical factor in sex determination, through its identification as a regulator of ovarian folliculogenesis, its use in fertility clinics as a measure of ovarian reserve, and its emerging role in hypothalamic-pituitary function. In light of these actions, AMH is considered an attractive therapeutic target to address diverse reproductive needs, including fertility preservation. Here, we set out to characterize the molecular mechanisms that govern AMH synthesis and activity. First, we enhanced the processing of the AMH precursor to >90% by introducing more efficient proprotein convertase cleavage sites (RKKR or ISSRKKRSVSS [SCUT]). Importantly, enhanced processing corresponded with a dramatic increase in secreted AMH activity. Next, based on species differences across the AMH type II receptor-binding interface, we generated a series of human AMH variants and assessed bioactivity. AMHSCUT potency (EC50 4 ng/mL) was increased 5- or 10-fold by incorporating Gln484 Met/Leu535 Thr (EC50 0.8 ng/mL) or Gln484 Met/Gly533 Ser (EC50 0.4 ng/mL) mutations, respectively. Furthermore, the Gln484 Met/Leu535 Thr double mutant displayed enhanced efficacy, relative to AMHSCUT . Finally, we identified residues within the wrist pre-helix of AMH (Trp494 , Gln496 , Ser497 , and Asp498 ) that likely mediate type I receptor binding. Mutagenesis of these residues generated gain- (Trp494 Phe or Gln496 Leu) or loss- (Ser497 Ala) of function AMH variants. Surprisingly, combining activating type I and type II receptor mutations only led to modest additive increases in AMH potency/efficacy. Our study is the first to characterize AMH residues involved in type I receptor binding and suggests a step-wise receptor-complex assembly mechanism, in which enhancement in the affinity of the ligand for either receptor can increase AMH activity beyond the natural level.

Activin E is a transforming growth factor ß ligand that signals specifically through activin receptor-like kinase 7.

Activins are one of the three distinct subclasses within the greater Transforming growth factor ß (TGFß) superfamily. First discovered for their critical roles in reproductive biology, activins have since been shown to alter cellular differentiation and proliferation. At present, members of the activin subclass include activin A (ActA), ActB, ActC, ActE, and the more distant members myostatin and GDF11. While the biological roles and signaling mechanisms of most activins class members have been well-studied, the signaling potential of ActE has remained largely unknown. Here, we characterized the signaling capacity of homodimeric ActE. Molecular modeling of the ligand:receptor complexes showed that ActC and ActE shared high similarity in both the type I and type II receptor binding epitopes. ActE signaled specifically through ALK7, utilized the canonical activin type II receptors, ActRIIA and ActRIIB, and was resistant to the extracellular antagonists follistatin and WFIKKN. In mature murine adipocytes, ActE invoked a SMAD2/3 response via ALK7, like ActC. Collectively, our results establish ActE as a specific signaling ligand which activates the type I receptor, ALK7.

Rosin as a Natural Alternative for the effective disinfection of ESKAPE Pathogens and Clostridioides difficile spores.

AIM: Hospital-acquired infections (HAIs) caused by antimicrobial-resistant ESKAPE pathogens are a significant concern for the healthcare industry, with an estimated cost of up to ${\$}$45 billion per year in the US alone. Clostridioides difficile is an additional opportunistic pathogen that also poses a serious threat to immunocompromised patients in hospitals. Infections caused by these pathogens lead to increased hospital stays and repeated readmission, resulting in a significant economic burden. Disinfectants and sporicidals are essential to reduce the risk of these pathogens in hospitals, but commercially available products can have a number of disadvantages including inefficacy, long contact times, short shelf lives, and operator health hazards. In this study we evaluated the effectiveness of Rosin (a natural substance secreted by coniferous trees as a defence mechanism against wounds in tree bark) and its commercial derivative Rosetax-21 as disinfectants and sporicidal against the six ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) and spore preparations from Clostridioides difficile. METHODS AND RESULTS: Both Rosin and Rosetax-21 were tested under simulated clean and dirty conditions (with BSA) against the ESKAPE pathogens, and C. difficile spore preparations. In clean conditions, Rosin (5% weight/volume: w/v) demonstrated significant efficacy against five of the ESKAPE pathogens, with A. baumannii and E. faecium being the most susceptible, and K. pneumoniae the most resistant, showing only a one-log reduction after a 5 min treatment. However, in dirty conditions, all pathogens including K. pneumoniae exhibited at least a 3-log reduction to Rosin within 5 min. Rosetax-21 (5% w/v) was found to be less effective than Rosin in clean conditions, a trend that was exacerbated in the presence of BSA. Additionally, both Rosin and Rosetax-21 at 2.5% (w/v) achieved complete eradication of C. difficile spores when combined with 0.5% glutaraldehyde, though their standalone sporicidal activity was limited. CONCLUSIONS: The findings from this study highlight the potential of Rosin and Rosetax-21 as both bactericidal and sporicidal disinfectants, with their efficacy varying based on the conditions and the pathogens tested. This presents an avenue for the development of novel healthcare disinfection strategies, especially against HAIs caused by antimicrobial-resistant ESKAPE pathogens and C. difficile.

Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-ß family.

Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-ß) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-ß family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.

Formation and characterization of BMP2/GDF5 and BMP4/GDF5 heterodimers.

BACKGROUND: Proteins of the TGFß family, which are largely studied as homodimers, are also known to form heterodimers with biological activity distinct from their component homodimers. For instance, heterodimers of bone morphogenetic proteins, including BMP2/BMP7, BMP2/BMP6, and BMP9/BMP10, among others, have illustrated the importance of these heterodimeric proteins within the context of TGFß signaling. RESULTS: In this study, we have determined that mature GDF5 can be combined with mature BMP2 or BMP4 to form BMP2/GDF5 and BMP4/GDF5 heterodimer. Intriguingly, this combination of a BMP2 or BMP4 monomer, which exhibit high affinity to heparan sulfate characteristic to the BMP class, with a GDF5 monomer with low heparan sulfate affinity produces a heterodimer with an intermediate affinity. Using heparin affinity chromatography to purify the heterodimeric proteins, we then determined that both the BMP2/GDF5 and BMP4/GDF5 heterodimers consistently signaled potently across an array of cellular and in vivo systems, while the activities of their homodimeric counterparts were more context dependent. These differences were likely driven by an increase in the combined affinities for the type 1 receptors, Alk3 and Alk6. Furthermore, the X-ray crystal structure of BMP2/GDF5 heterodimer was determined, highlighting the formation of two asymmetric type 1 receptor binding sites that are both unique relative to the homodimers. CONCLUSIONS: Ultimately, this method of heterodimer production yielded a signaling molecule with unique properties relative to the homodimeric ligands, including high affinity to multiple type 1 and moderate heparan binding affinity.

Exploring halophilic environments as a source of new antibiotics.

Microbial natural products from microbes in extreme environments, including haloarchaea, and halophilic bacteria, possess a huge capacity to produce novel antibiotics. Additionally, enhanced isolation techniques and improved tools for genomic mining have expanded the efficiencies in the antibiotic discovery process. This review article provides a detailed overview of known antimicrobial compounds produced by halophiles from all three domains of life. We summarize that while halophilic bacteria, in particular actinomycetes, contribute the vast majority of these compounds the importance of understudied halophiles from other domains of life requires additional consideration. Finally, we conclude by discussing upcoming technologies- enhanced isolation and metagenomic screening, as tools that will be required to overcome the barriers to antimicrobial drug discovery. This review highlights the potential of these microbes from extreme environments, and their importance to the wider scientific community, with the hope of provoking discussion and collaborations within halophile biodiscovery. Importantly, we emphasize the importance of bioprospecting from communities of lesser-studied halophilic and halotolerant microorganisms as sources of novel therapeutically relevant chemical diversity to combat the high rediscovery rates. The complexity of halophiles will necessitate a multitude of scientific disciplines to unravel their potential and therefore this review reflects these research communities.

Collection and processing of hematopoietic progenitor cell products at risk of presenting with cold agglutination.

BACKGROUND AIMS: Cold agglutinins are commonly identified in transfusion laboratories and are defined by their ability to agglutinate erythrocytes at 3-4°C, with most demonstrating a titer >64. Similarly, cryoglobulins can precipitate from plasma when temperatures drop below central body temperature, resulting in erythrocyte agglutination. Thankfully, disease associated from these autoantibodies is rare, but unfortunately, such temperature ranges are routinely encountered outside of the body's circulation, as in an extracorporeal circuit during hematopoietic progenitor cell (HPC) collection or human cell therapy laboratory processing. When agglutination occurs ex vivo, complications with the collection and product may be encountered, resulting in adverse events or product loss. Here, we endeavor to share our experience in preventing and responding to known cases at risk of or spontaneous HPC agglutination in our human cell therapy laboratory. CASE REPORTS: Four cases of HPC products at risk for, or spontaneously, agglutinating were seen at our institution from 2018 to 2020. Planned modifications occurred, including ambient room temperature increases, tandem draw and return blood warmers, warm product transport and extended post-thaw warming occurred. In addition, unplanned modifications were undertaken, including warm HPC product processing and plasma replacement of the product when spontaneous agglutination of the product was identified. All recipients successfully engrafted after infusion. CONCLUSIONS: While uncommon, cold agglutination of HPC products can disrupt standard processes of collection and processing. Protocol modifications can circumvent adverse events for the donor and minimize product loss. Such process modifications should be considered in individuals with known risks for agglutination going to HPC donation/collection.

In situ generation of cold atmospheric plasma-activated mist and its biocidal activity against surrogate viruses for COVID-19.

AIMS: To provide an alternative to ultra violet light and vapourized hydrogen peroxide to enhance decontamination of surfaces as part of the response to the COVID-19 pandemic. METHODS AND RESULTS: We developed an indirect method for in situ delivery of cold plasma and evaluated the anti-viral activity of plasma-activated mist (PAM) using bacteriophages phi6, MS2, and phiX174, surrogates for SARS-CoV-2. Exposure to ambient air atmospheric pressure derived PAM caused a 1.71 log10 PFU ml-1 reduction in phi6 titer within 5 min and a 7.4 log10 PFU ml-1 reduction after 10 min when the the PAM source was at 5 and 10 cm. With MS2 and phiX174, a 3.1 and 1.26 log10 PFU ml-1 reduction was achieved, respectively, after 30 min. The rate of killing was increased with longer exposure times but decreased when the PAM source was further away. Trace amounts of reactive species, hydrogen peroxide and nitrite were produced in the PAM, and the anti-viral activity was probably attributable to these and their secondary reactive species. CONCLUSIONS: PAM exhibits virucidal activity against surrogate viruses for COVID-19, which is time and distance from the plasma source dependent.

Therapeutic plasma exchange for steroid refractory idiopathic inflammatory myopathies with interstitial lung disease.

BACKGROUND: Idiopathic inflammatory myopathies (IIMs) encompass many rheumatologic diseases characterized by inflammatory muscle disease, typically unified by proximal muscle weakness. A subset of patients with IIM present with interstitial lung disease (ILD) with identifiable antibodies such as in anti-synthetase syndrome (AS) with antibodies to aminoacyl-tRNA synthetases, and clinically amyopathic dermatomyositis (CADM) with anti-melanoma differentiation-associated protein 5 (MDA5). Recent case reports demonstrate response to therapeutic plasma exchange (TPE) or column filtration plasmapheresis in IIM with ILD resistant to medical management. We present our experience with eight patients with IIM with ILD undergoing TPE at a large US-based hospital system. PATIENT CHARACTERISTICS: Eight patients with IIM with ILD were treated with TPE over the last 10 years. The therapy consisted of 5-7 one plasma volume exchanges every other day to daily. Seven of eight patients had identifiable antibodies. RESULTS: Following completion of TPE, seven of eight demonstrated improvement in pulmonary function despite lack of improvement of pulmonary function with standard therapy. CONCLUSION: In antibody-mediated, treatment refractory IIM with ILD, TPE may be a viable intervention. This is a disease for which the role of apheresis is evolving. CLINICAL TRIAL REGISTRATION: Not application.

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-ß1. 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.

How do I warm HPC(A) products to maximize cell viability in the setting of cold agglutinin disease?

BACKGROUND: High titers of cold agglutinins jeopardize the quality of an apheresis product meant for autologous or allogeneic transplant. Management of transplant patients with cold agglutinin disease (CAD) is often experience-based and under reported, yet decisions must be made quickly to optimize product management and patient outcomes. There remains a lack of data quantifying cell recovery and viability when using various warming methodologies. STUDY DESIGN AND METHODS: To expand the published experimental data on this subject, our human cellular therapy lab compared cellular recoveries and viabilities after manipulation of cryopreserved apheresis products through various warming methodologies: (1) extended warming in a water bath, (2) warming via blood warmer and infusion pump, and (3) warming in a water bath followed by infusion pump as a control to assess potential shear stress effects. RESULTS: The presented studies demonstrate that all methods of product warming produce the same rates of recovery of total and viable cells across vital cell types prior to patient administration. Statistically, use of an extended water bath protocol provided a marginal benefit in recovery of total nucleated cells, though this effect is diminished when products are held for an extended period to simulate a delay in administration. DISCUSSION: These results can inform decisions to improve patient care and minimize product manipulation and loss. Centers are encouraged to use this information to guide proactive measures to establish a standard operating procedure to manage CAD cases.

Microbial communities of halite deposits and other hypersaline environments.

Large regions of Earth's surface are underlain by salt deposits that evaporated from ancient oceans and are populated by extreme halophilic microbes. While the microbiology of ancient evaporites has been well studied, the ecology of halite deposits and more recently formed NaCl "salticle" stalactite structures (speleothems) in a Triassic halite mine are less well characterized. The microbiome of Kilroot Salt Mine was profiled using conventional and enhanced culturing techniques. From this, 89 halophilic archaeal isolates from six known genera, and 55 halophilic or halotolerant bacterial isolates from 18 genera were obtained. Culture-independent metagenomic approaches also revealed that culturing techniques were inadvertently biased toward specific taxa, and the need for optimized isolation procedures are required to enhance cultivation diversity. Speleothems formed from saturated brines are unique structures that have the potential to entomb haloarchaea cells for thousands of years within fluid inclusions. The presence of such fluid inclusions, alongside the high abundance of genes related to glycerol metabolism, biofilm formation, and persister cell formation is highly suggestive of an environmental niche that could promote longevity and survivability. Finally, previous studies reporting the discovery of novel biocatalysts from the Kilroot mine microbiome, suggests that this environment may be an untapped source of chemical diversity with high biodiscovery potential.

Characterization of tolloid-mediated cleavage of the GDF8 procomplex.

Growth differentiation factor 8 (GDF8), a.k.a. myostatin, is a member of the larger TGFß superfamily of signaling ligands. GDF8 has been well characterized as a negative regulator of muscle mass. After synthesis, GDF8 is held latent by a noncovalent complex between the N-terminal prodomain and the signaling ligand. Activation of latent GDF8 requires proteolytic cleavage of the prodomain at residue D99 by a member of the tolloid family of metalloproteases. While tolloid proteases cleave multiple substrates, they lack a conserved consensus sequence. Here, we investigate the tolloid cleavage site of the GDF8 prodomain to determine what residues contribute to tolloid recognition and subsequent proteolysis. Using sequential alanine mutations, we identified several residues adjacent to the scissile bond, including Y94, that when mutated, abolish tolloid-mediated activation of latent GDF8. Using the astacin domain of Tll1 (Tolloid Like 1) we determined that prodomain mutants were more resistant to proteolysis. Purified latent complexes harboring the prodomain mutations, D92A and Y94A, impeded activation by tolloid but could be fully activated under acidic conditions. Finally, we show that co-expression of GDF8 WT with prodomain mutants that were tolloid resistant, suppressed GDF8 activity. Taken together our data demonstrate that residues towards the N-terminus of the scissile bond are important for tolloid-mediated activation of GDF8 and that the tolloid-resistant version of the GDF8 prodomain can function dominant negative to WT GDF8.

Structural characterization of an activin class ternary receptor complex reveals a third paradigm for receptor specificity.

TGFß family ligands, which include the TGFßs, BMPs, and activins, signal by forming a ternary complex with type I and type II receptors. For TGFßs and BMPs, structures of ternary complexes have revealed differences in receptor assembly. However, structural information for how activins assemble a ternary receptor complex is lacking. We report the structure of an activin class member, GDF11, in complex with the type II receptor ActRIIB and the type I receptor Alk5. The structure reveals that receptor positioning is similar to the BMP class, with no interreceptor contacts; however, the type I receptor interactions are shifted toward the ligand fingertips and away from the dimer interface. Mutational analysis shows that ligand type I specificity is derived from differences in the fingertips of the ligands that interact with an extended loop specific to Alk4 and Alk5. The study also reveals differences for how TGFß and GDF11 bind to the same type I receptor, Alk5. For GDF11, additional contacts at the fingertip region substitute for the interreceptor interactions that are seen for TGFß, indicating that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe84), when mutated, abolishes GDF11 signaling, but has little impact on TGFß signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFß family, different mechanisms regulate type I receptor binding and specificity, providing a molecular explanation for how the activin class accommodates low-affinity type I interactions without the requirement of cooperative receptor interactions.

Microbiology of a NaCl stalactite 'salticle' in Triassic halite.

Large regions of Earth's surface are underlain by salt deposits that evaporated from ancient oceans and are populated by extreme halophilic microbes. Some of these halophiles may have been preserved over geological timescales within hypersaline fluid inclusions, but ingresses of water and/or anthropogenic activities can lead to the formation of alternative habitats, including NaCl stalactites or other speleothems. While the microbiology of ancient evaporites has been well studied, the ecology of these recently formed structures is less-well understood. Here, the microbiology of a NaCl stalactite ('salticle') in a Triassic halite mine is characterized. The specific aims were to determine the presence of fluid inclusions, determine the microbial structure of the salticle compared with a nearby brine-pool and surficial soil, and characterize the ecophysiological capabilities of this unique ecosystem. The salticle contained fluid inclusions, and their microbiome was composed of Euryarchaetota, Proteobacteria, and Actinobacteria, with Haloarchaea in greater abundance than brine-pool or soil microbiomes. The salticle metagenome exhibited a greater abundance of genes involved in osmoregulation, anaerobic respiration, UV resistance, oxidative stress, and stress-protein synthesis relative to the soil microbiome. We discuss the potential astrobiological implications of salticles as enclosed salt-saturated habitats that are protected from ionizing radiation and have a stable water activity.

Deletion of Gremlin-2 alters estrous cyclicity and disrupts female fertility in mice†.

Members of the differential screening-selected gene aberrative in neuroblastoma (DAN) protein family are developmentally conserved extracellular binding proteins that antagonize bone morphogenetic protein (BMP) signaling. This protein family includes the Gremlin proteins, GREM1 and GREM2, which have key functions during embryogenesis and adult physiology. While BMPs play essential roles in ovarian follicle development, the role of the DAN family in female reproductive physiology is less understood. We generated mice null for Grem2 to determine its role in female reproduction in addition to screening patients with primary ovarian insufficiency (POI) for variants in GREM2. Grem2-/- mice are viable, but female Grem2-/- mice have diminished fecundity and irregular estrous cycles. This is accompanied by significantly reduced production of ovarian anti-Müllerian hormone (AMH) from small growing follicles, leading to a significant decrease in serum AMH. Surprisingly, as AMH is a well-established marker of the ovarian reserve, morphometric analysis of ovarian follicles showed maintenance of primordial follicles in Grem2-/- mice like wild-type (WT) littermates. While Grem2 mRNA transcripts were not detected in the pituitary, Grem2 is expressed in hypothalami of WT female mice, suggesting the potential for dysfunction in multiple tissues composing the hypothalamic-pituitary-ovarian axis that contribute to the subfertility phenotype. Additionally, screening 106 women with POI identified one individual with a heterozygous variant in GREM2 that lies within the predicted BMP-GREM2 interface. In total, these data suggest that Grem2 is necessary for female fecundity by playing a novel role in regulating the HPO axis and contributing to female reproductive disease.

Resource selection and movement by northern bobwhite broods varies with age and explains survival.

Resource selection is a dynamic process driven by habitat valuation and risk avoidance in heterogeneous landscapes. Resource selection and movement decisions of individuals may be sensitive to intrinsic factors, such as body condition, and variation in these choices may have consequences on subsequent survival. We evaluated northern bobwhite (Colinus virginianus) brood resource selection patterns to quantify utility of different cover types during the development period using integrated step-selection analysis in a Bayesian hierarchical modeling framework with three brood stages: flightless broods ≤ 14 days old, dependent broods 15-35 days old, and independent broods over 35 days old. Broods showed strongest selection for native grasslands that were burned and grazed at least once in the previous two years, and agricultural fields. Brood mobility improved with age; broods > 35 days old travelled farther on average and took daily steps > 200 m more frequently than younger broods. Young broods ≤ 14 days old did not select for idle native grasslands, while broods > 35 days old did select for that cover type. Young broods also selected areas farther from trees compared to older broods. We evaluated the survival consequences of resource selection by comparing patterns in choices of broods that succeeded to choices of broods that failed to survive to 35 days. Successful broods chose habitats with more shrub cover and areas farther from trees compared to failed broods. Our results suggest that conservation planning should consider age-specific patterns in habitat use and demographic consequences of habitat choice for greatest effectiveness.

Characterization of the different oligomeric states of the DAN family antagonists SOSTDC1 and SOST.

The DAN (differential screening-selected gene aberrative in neuroblastoma) family are a group of secreted extracellular proteins which typically bind to and antagonize BMP (bone morphogenetic protein) ligands. Previous studies have revealed discrepancies between the oligomerization state of certain DAN family members, with SOST (a poor antagonist of BMP signaling) forming a monomer while Grem1, Grem2, and NBL1 (more potent BMP antagonists) form non-disulfide linked dimers. The protein SOSTDC1 (Sclerostin domain containing protein 1) is sequentially similar to SOST, but has been shown to be a better BMP inhibitor. In order to determine the oligomerization state of SOSTDC1 and determine what effect dimerization might have on the mechanism of DAN family antagonism of BMP signaling, we isolated the SOSTDC1 protein and, using a battery of biophysical, biochemical, and structural techniques, showed that SOSTDC1 forms a highly stable non-covalent dimer. Additionally, this SOSTDC1 dimer was shown, using an in vitro cell based assay system, to be an inhibitor of multiple BMP signaling growth factors, including GDF5, while monomeric SOST was a very poor antagonist. These results demonstrate that SOSTDC1 is distinct from paralogue SOST in terms of both oligomerization and strength of BMP inhibition.

Molecular characterization of latent GDF8 reveals mechanisms of activation.

Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-ß. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or "triggered" state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is "spring-loaded." Structural analysis shows that the prodomain:GDF8 complex adopts an "open" configuration, distinct from the latency state of TGF-ß and more similar to the open state of Activin A and BMP9 (nonlatent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared with WT GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.