The ligands of the Bone Morphogenetic Protein (BMP) family of developmental signaling molecules are often under the control of complex cis-regulatory modules and play diverse roles in vertebrate development and evolution. Here, we investigated the cis-regulatory control of stickleback Bmp6. We identified a 190 bp enhancer ~2.5 kilobases 5’ of the Bmp6 gene that recapitulates expression in developing teeth and fins, with a core 72 bp sequence that is sufficient for both domains. By testing orthologous enhancers with varying degrees of sequence conservation from outgroup teleosts in transgenic reporter gene assays in sticklebacks and zebrafish, we found that the function of this regulatory element appears to have been conserved for over 250 million years of teleost evolution. We show that a predicted binding site for the TGFβ effector Smad3 in this enhancer is required for enhancer function and that pharmacological inhibition of TGFβ signaling abolishes enhancer activity and severely reduces endogenous Bmp6 expression. Finally, we used TALENs to disrupt the enhancer in vivo and find that Bmp6 expression is dramatically reduced in teeth and fins, suggesting this enhancer is necessary for expression of the Bmp6 locus. This work identifies a relatively short regulatory sequence that is required for expression in multiple tissues and, combined with previous work, suggests that shared regulatory networks control limb and tooth development.

Human disorders of the post-squalene cholesterol biosynthesis pathway frequently result in skeletal abnormalities, yet our understanding of the mechanisms involved is limited. In a forwardgenetic approach, we have found that a late-onset skeletal mutant, named koliber nu7 , is the result of a cis-acting regulatory mutation leading to loss of methylsterol monooxygenase 1 (msmo1) expression within pre-hypertrophic chondrocytes. Generated msmo1 nu81 knockdown mutation resulted in lethality at larval stage. We demonstrated that this is a result of both cholesterol deprivation and sterol intermediate accumulation by creating a mutation eliminating activity of Lanosterol synthase (Lss). Our results indicate that double lss nu60 ;msmo1 nu81 and single lss nu60 mutants survive significantly longer than msmo1 nu81 homozygotes. Liver-specific restoration of either Msmo1 or Lss in corresponding mutant backgrounds suppresses larval lethality. Rescued mutants develop dramatic skeletal abnormalities, with a loss of Msmo1 activity resulting in a more-severe patterning defect of a nearcomplete loss of hypertrophic chondrocytes marked by col10a1a expression. Our analysis suggests that hypertrophic chondrocytes depend on endogenous cholesterol synthesis, and blocking C4 demethylation exacerbates the cholesterol deficiency phenotype. Our findings offer new insight into the genetic control of bone development and provide new zebrafish models for human disorders of the cholesterol biosynthesis pathway.

Pulmonary fat embolism is a common phenomenon in cases of traumatic long bone fractures, with only a minority developing the more catastrophic Fat Embolism Syndrome (FES). Diagnosis is clinical and requires a high index of suspicion. Treatment remains under-investigated, with common interventions having low quality level-of-evidence and no mortality benefit. In severe cases, focus should be on supporting the failing right ventricle through use of inotropes, pulmonary vasodilators, and mechanical circulatory support. This requires a thorough understanding of the unique physiology through the pulmonary circulation.

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15 jection" used in the primary aim statement of our Introduction. By this 16 we mean generally "injection of medications" through either a 17 peripheral intravenous (PIV) or intraosseous (IO) device during car-18 diac arrest. We provide data only on epinephrine administration as 19 this is the most consistently used medication during cardiac arrest. 20 Additional medications were given to patients in this study with some 21 frequency. However, information regarding their relative use was 22 incomplete. Given this incomplete documentation, in combination 23 with their mostly undetermined or neutral effects on our study out-24 comes, we felt their comparative utilization would be difficult to inter-25 pret in the context of our study. 1 26 Second, we are asked to clarify the patient population in our 27 study. We use the term "acute care" to mean all those on the hos-28 pital floor, step-down unit (i.e. intermediate severity of illness 29 between ICU and floor), and observation unit (e.g. those patients 30 expected to stay inpatient for <72 hrs). As such, when included with 31 the intensive care and procedural patients, we feel our study popula-32 tion reasonably includes those thought of as being "in-hospital." 33 Third, we are asked to differentiate between those patients in the 34 "PIV group" who had pre-existing PIV access and those who had 35 PIV placed after IHCA. Patients with pre-existing PIV access could 36 have shorter times-to-epinephrine (TTE) and shorter times-to-ROSC 37 (TTR). Differentiating between these groups may also more accu-38 rately reflect the entirety of the in-hospital resuscitation process. 39 The authors additionally request information regarding time-to-40 access between PIV and IO. Unfortunately, this information was 41 not available with robust presence in our dataset. As a surrogate 42 for venous access times, we used TTE. This variable has been asso-43 ciated with differences in survival and neurologic outcomes. 2,3 As 44 discussed in our paper, the TTE was statistically longer in the IO 45 group compared to the PIV population and we adjusted for this vari-46 able during multivariate logistic regression.47 65 is a statistically significant difference in the number of witnessed car-66 diac arrest events between the PIV and IO group in favor of the PIV 67 group. In fact, several studies have shown an association between 68 witnessed status and increased odds of survival during IHCA. 5 Thus, 69 having a higher percentage of witnessed events in the PIV group 70 may skew towards more favourable outcomes. We have attempted 71 to correct for such a difference by adjusting for this variable using 72 multivariate logistic regression, rather than performing a subgroup 73 analysis as suggested. 6,7 74 Many of the limitations addressed here and previously should be 75 considered when interpreting the outcomes of our paper. Neverthe-76 less, we feel our analysis serves to add meaningful knowledge to 77 IHCA resuscitation literature, in addition to serving as a basis for fur-78 ther prospective ...

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