Faculty Bibliography

BACKGROUND/UNASSIGNED:The posterolateral approach to the ankle allows for reduction and fixation of the posterior and lateral malleoli through the same surgical incision. This can be accomplished via 1 or 2 surgical "windows." The purpose of this study is to compare outcomes including wound complications following direct fixation of unstable rotational ankle fracture through the posterolateral approach using either 1 or 2 surgical windows. METHODS/UNASSIGNED:One hundred sixty-four patients with bi- or trimalleolar ankle fractures treated using the single-window posterolateral approach (between the peroneal tendons and the flexor hallucis longus [FHL]) or the 2-window technique (between the peroneal tendons and the FHL for posterior malleolus fixation; lateral to the peroneal tendons for fibula fixation) were reviewed for demographics, radiographic details, and clinical outcomes. We were able to review these 164 at the 3-month follow-up and a subset of 104 at a minimum of 12-month follow-up. RESULTS/UNASSIGNED: = .021). We did not find a significant difference in nerve complications for these 2 cohorts. CONCLUSION/UNASSIGNED:In our study, we found the single-window posterolateral approach to be associated with fewer wound complications and better postoperative range of ankle motion when compared to the 2-window approach. LEVEL OF EVIDENCE/UNASSIGNED:Level III, retrospective cohort study.

For unknow reasons, the melanocyte stem cell (McSC) system fails earlier than other adult stem cell populations¹, which leads to hair greying in most humans and mice^2,3. Current dogma states that McSCs are reserved in an undifferentiated state in the hair follicle niche, physically segregated from differentiated progeny that migrate away following cues of regenerative stimuli^4-8. Here we show that most McSCs toggle between transit-amplifying and stem cell states for both self-renewal and generation of mature progeny, a mechanism fundamentally distinct from those of other self-renewing systems. Live imaging and single-cell RNA sequencing revealed that McSCs are mobile, translocating between hair follicle stem cell and transit-amplifying compartments where they reversibly enter distinct differentiation states governed by local microenvironmental cues (for example, WNT). Long-term lineage tracing demonstrated that the McSC system is maintained by reverted McSCs rather than by reserved stem cells inherently exempt from reversible changes. During ageing, there is accumulation of stranded McSCs that do not contribute to the regeneration of melanocyte progeny. These results identify a new model whereby dedifferentiation is integral to homeostatic stem cell maintenance and suggest that modulating McSC mobility may represent a new approach for the prevention of hair greying.

Glucocorticoid (GC) is one of the most prescribed medicines to treat various inflammatory and autoimmune diseases. However, high doses and long-term use of GCs lead to multiple adverse effects, particularly glucocorticoid-induced osteoporosis (GIO). Excessive GCs exert detrimental effects on bone cells, including osteoblasts, osteoclasts, and osteocytes, leading to impaired bone formation and resorption. The actions of exogenous GCs are considered to be strongly cell-type and dose dependent. GC excess inhibits the proliferation and differentiation of osteoblasts and enhances the apoptosis of osteoblasts and osteocytes, eventually contributing to reduced bone formation. Effects of GC excess on osteoclasts mainly include enhanced osteoclastogenesis, increased lifespan and number of mature osteoclasts, and diminished osteoclast apoptosis, which result in increased bone resorption. Furthermore, GCs have an impact on the secretion of bone cells, subsequently disturbing the process of osteoblastogenesis and osteoclastogenesis. This review provides timely update and summary of recent discoveries in the field of GIO, with a particular focus on the effects of exogenous GCs on bone cells and the crosstalk among them under GC excess.

General approaches for designing sequence-specific peptide-binding proteins would have wide utility in proteomics and synthetic biology. However, designing peptide-binding proteins is challenging, as most peptides do not have defined structures in isolation, and hydrogen bonds must be made to the buried polar groups in the peptide backbone^1-3. Here, inspired by natural and re-engineered protein-peptide systems^4-11, we set out to design proteins made out of repeating units that bind peptides with repeating sequences, with a one-to-one correspondence between the repeat units of the protein and those of the peptide. We use geometric hashing to identify protein backbones and peptide-docking arrangements that are compatible with bidentate hydrogen bonds between the side chains of the protein and the peptide backbone¹². The remainder of the protein sequence is then optimized for folding and peptide binding. We design repeat proteins to bind to six different tripeptide-repeat sequences in polyproline II conformations. The proteins are hyperstable and bind to four to six tandem repeats of their tripeptide targets with nanomolar to picomolar affinities in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including ladders of hydrogen bonds from protein side chains to peptide backbones. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating peptide sequences and for disordered regions of native proteins.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets ^1,2 . Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not transmit SARS-CoV-2 ³ . Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2) and omicron (B.1.1.529). We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing for the first time a role for an accessory protein this context.

Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Atherosclerosis evolves through dysregulated lipid metabolism interwoven with exaggerated inflammation. Previous work implicating the receptor for advanced glycation end products (RAGE) in atherosclerosis prompted us to explore if Diaphanous 1 (DIAPH1), which binds to the RAGE cytoplasmic domain and is important for RAGE signaling, contributes to these processes. We intercrossed atherosclerosis-prone Ldlr^-/- mice with mice devoid of Diaph1 and fed them Western diet for 16 weeks. Compared to male Ldlr^-/- mice, male Ldlr^-/- Diaph1^-/- mice displayed significantly less atherosclerosis, in parallel with lower plasma concentrations of cholesterol and triglycerides. Female Ldlr^-/- Diaph1^-/- mice displayed significantly less atherosclerosis compared to Ldlr^-/- mice and demonstrated lower plasma concentrations of cholesterol, but not plasma triglycerides. Deletion of Diaph1 attenuated expression of genes regulating hepatic lipid metabolism, Acaca, Acacb, Gpat2, Lpin1, Lpin2 and Fasn, without effect on mRNA expression of upstream transcription factors Srebf1, Srebf2 or Mxlipl in male mice. We traced DIAPH1-dependent mechanisms to nuclear translocation of SREBP1 in a manner independent of carbohydrate- or insulin-regulated cues but, at least in part, through the actin cytoskeleton. This work unveils new regulators of atherosclerosis and lipid metabolism through DIAPH1.

AIMS/OBJECTIVE:Acute myocardial infarction rapidly increases blood neutrophils (<2 hours). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 hours after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 minutes) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS/RESULTS:Here, we show that injury to the myocardium rapidly mobilises neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilised splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilisation of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS:Our findings show a novel EV-dependent mechanism for the rapid mobilisation of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells. TRANSLATIONAL PERSPECTIVE/UNASSIGNED:Peripheral blood neutrophils are rapidly elevated following acute myocardial infarction (AMI) and prior to alterations in systemic cytokines. Extracellular vesicles (EVs) are membrane enclosed particles that carry protein and miRNAs and are rapidly liberated from endothelial cells (EC). Here, we show that following AMI EC-derived-EVs (EC-EVs) mediate neutrophil mobilisation from the spleen via EC-EV-VCAM-1 and induce transcriptional activation of neutrophils in the blood to favour miRNA-126-mRNA targets; miRNA-126 antagomir treatment lowers infarct size. EC-EV-VCAM-1 and EC-EV-miRNA-126 are novel mechanisms that mobilise splenic reserve of neutrophils, a previously unidentified source of neutrophils in sterile ischaemic injury.

Connexins are crucial cardiac proteins that form hemichannels and gap junctions. Gap junctions are responsible for the propagation of electrical and chemical signals between myocardial cells and cells of the specialized conduction system in order to synchronize the cardiac cycle and steer cardiac pump function. Gap junctions are normally open, while hemichannels are closed, but pathological circumstances may close gap junctions and open hemichannels, thereby perturbing cardiac function and homeostasis. Current evidence demonstrates an emerging role of hemichannels in myocardial ischemia and arrhythmia, and tools are now available to selectively inhibit hemichannels without inhibiting gap junctions as well as to stimulate hemichannel incorporation into gap junctions. We review available experimental evidence for hemichannel contributions to cellular pro-arrhythmic events in ventricular and atrial cardiomyocytes, and link these to insights at the level of molecular control of connexin-43-based hemichannel opening. We conclude that a double-edged approach of both preventing hemichannel opening and preserving gap junctional function will be key for further research and development of new connexin-based experimental approaches for treating heart disease.