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.

Comprehensive workflow evaluation to improve medication distribution to the emergency department in an academic medical center.

PURPOSE: The fast-paced environment of the emergency department (ED), with frequent admissions, discharges, and transfers, poses a challenge for pharmacy departments to effectively distribute and store medications. The purpose of this study is to propose a unique workflow of patient-specific medication delivery to the ED from a hospital pharmacy to reduce the number of missing medications resulting in medication messages and redispenses. METHODS: The medication delivery workflow proposed in this study consists of batching the preparation and distribution of patient-specific medications sent from the pharmacy to the ED in the 1 to 2 hours prior to their administration time. Chi-square analysis was completed to compare medication redispenses and "missing medication" messages before and after the intervention, with the significance level set at P < 0.05. RESULTS: The percentage of redispensed medications was effectively decreased following implementation of the workflow change from 21.6% to 9.2% (P < 0.001), with unit doses having the greatest reduction (25.8% vs 6.1%, P < 0.001). Benefits of this workflow change were also illustrated through a reduction in the percentage of missing-medication messages sent by nursing staff from 97.7% to 93.9% (P < 0.001). CONCLUSION: This study showed that implementation of standard, hourly batches of medications dispensed from the pharmacy to the ED resulted in a significant reduction in the total percentage of redispensed medications and missing-medication messages. The overall reduction in redispensed medications as a result of this innovative workflow change not only benefited nursing and pharmacy staff but can reduce medication waste and improve patient care through timely administration of medications.

Functional substitutions of amino acids that differ between GDF11 and GDF8 impact skeletal development and skeletal muscle.

Growth differentiation factor 11 (GDF11) and GDF8 (MSTN) are closely related TGF-ß family proteins that interact with nearly identical signaling receptors and antagonists. However, GDF11 appears to activate SMAD2/3 more potently than GDF8 in vitro and in vivo. The ligands possess divergent structural properties, whereby substituting unique GDF11 amino acids into GDF8 enhanced the activity of the resulting chimeric GDF8. We investigated potentially distinct endogenous activities of GDF11 and GDF8 in vivo by genetically modifying their mature signaling domains. Full recoding of GDF8 to that of GDF11 yielded mice lacking GDF8, with GDF11 levels ∼50-fold higher than normal, and exhibiting modestly decreased muscle mass, with no apparent negative impacts on health or survival. Substitution of two specific amino acids in the fingertip region of GDF11 with the corresponding GDF8 residues resulted in prenatal axial skeletal transformations, consistent with Gdf11-deficient mice, without apparent perturbation of skeletal or cardiac muscle development or homeostasis. These experiments uncover distinctive features between the GDF11 and GDF8 mature domains in vivo and identify a specific requirement for GDF11 in early-stage skeletal development.

Exogenous GDF11, but not GDF8, reduces body weight and improves glucose homeostasis in mice.

Insulin resistance is associated with aging in mice and humans. We have previously shown that administration of recombinant GDF11 (rGDF11) to aged mice alters aging phenotypes in the brain, skeletal muscle, and heart. While the closely related protein GDF8 has a role in metabolism, limited data are available on the potential metabolic effects of GDF11 or GDF8 in aging. To determine the metabolic effects of these two ligands, we administered rGDF11 or rGDF8 protein to young or aged mice fed a standard chow diet, short-term high-fat diet (HFD), or long-term HFD. Under nearly all of these diet conditions, administration of exogenous rGDF11 reduced body weight by 3-17% and significantly improved glucose tolerance in aged mice fed a chow (~30% vs. saline) or HF (~50% vs. saline) diet and young mice fed a HFD (~30%). On the other hand, exogenous rGDF8 showed signifcantly lesser effect or no effect at all on glucose tolerance compared to rGDF11, consistent with data demonstrating that GFD11 is a more potent signaling ligand than GDF8. Collectively, our results show that administration of exogenous rGDF11, but not rGDF8, can reduce diet-induced weight gain and improve metabolic homeostasis.

Variation in zygotic CRISPR/Cas9 gene editing outcomes generates novel reporter and deletion alleles at the Gdf11 locus.

Recent advances in CRISPR/Cas gene editing technology have significantly expanded the possibilities and accelerated the pace of creating genetically engineered animal models. However, CRISPR/Cas-based strategies designed to precisely edit the genome can often yield unintended outcomes. Here, we report the use of zygotic CRISPR/Cas9 injections to generate a knock-in GFP reporter mouse at the Gdf11 locus. Phenotypic and genomic characterization of founder animals from these injections revealed a subset that contained the correct targeting event and exhibited GFP expression that, within the hematopoietic system, was restricted predominantly to lymphoid cells. Yet, in another subset of founder mice, we detected aberrant integration events at the target site that dramatically and inaccurately shifted hematopoietic GFP expression from the lymphoid to the myeloid lineage. Additionally, we recovered multiple Gdf11 deletion alleles that modified the C-terminus of the GDF11 protein. When bred to homozygosity, most of these alleles recapitulated skeletal phenotypes reported previously for Gdf11 knockout mice, suggesting that these represent null alleles. However, we also recovered one Gdf11 deletion allele that encodes a novel GDF11 variant protein ("GDF11-WE") predicted to contain two additional amino acids (tryptophan (W) and glutamic acid (E)) at the C-terminus of the mature ligand. Unlike the other Gdf11 deletion alleles recovered in this study, homozygosity for the Gdf11WE allele did not phenocopy Gdf11 knockout skeletal phenotypes. Further investigation using in vivo and in vitro approaches demonstrated that GDF11-WE retains substantial physiological function, indicating that GDF11 can tolerate at least some modifications of its C-terminus and providing unexpected insights into its biochemical activities. Altogether, our study confirms that one-step zygotic injections of CRISPR/Cas gene editing complexes provide a quick and powerful tool to generate gene-modified mouse models. Moreover, our findings underscore the critical importance of thorough characterization and validation of any modified alleles generated by CRISPR, as unintended on-target effects that fail to be detected by simple PCR screening can produce substantially altered phenotypic readouts.