Identifying links between athletic identity and risk factors related to youth sport participation.

Introduction: The development of identity formation occurs during adolescence through experiences, ideals and principle. With greater accessibility to sports, recent trends have shown increased rates of sports specialization over the past decade in youth athletes. Athletic identity measures the strength an individual is tied to the athlete role and can be formed in conjunction to adolescent identity formation. More specialized youth athletes may have stronger ties to their athletic identity during their adolescent identity formation period. Methods: Youth basketball athletes were surveyed on specialization levels and athletic identity via the Athletic Identity Measurement Scale (AIMS), including three submeasures: social identity, exclusivity, and negative affectivity. Results: Participants showed stronger identification to social identity items and the weakest identification with exclusivity items. Athletes reporting more time spent playing their primary sport presented higher scores across all measures of athletic identity, and total athletic identity was stronger in athletes reporting specialization at an earlier age. Exclusivity and negative affectivity tended to increase with specialization level which may primarily be driven by specialized athletes choosing to quit non-primary sports. Discussion: Athletic identity may be worth noting as a psychological indicator of potential risk of injury. The long-term goal of this work is to provide the research and clinical community a greater understanding of a potential psychosocial risk factor as youth athletes continue specializing and spending more time training in a singular sport.

Nanoparticle-Conjugated Toll-Like Receptor 9 Agonists Improve the Potency, Durability, and Breadth of COVID-19 Vaccines.

Development of effective vaccines for infectious diseases has been one of the most successful global health interventions in history. Though, while ideal subunit vaccines strongly rely on antigen and adjuvant(s) selection, the mode and time scale of exposure to the immune system has often been overlooked. Unfortunately, poor control over the delivery of many adjuvants, which play a key role in enhancing the quality and potency of immune responses, can limit their efficacy and cause off-target toxicities. There is a critical need for improved adjuvant delivery technologies to enhance their efficacy and boost vaccine performance. Nanoparticles have been shown to be ideal carriers for improving antigen delivery due to their shape and size, which mimic viral structures but have been generally less explored for adjuvant delivery. Here, we describe the design of self-assembled poly(ethylene glycol)-b-poly(lactic acid) nanoparticles decorated with CpG, a potent TLR9 agonist, to increase adjuvanticity in COVID-19 vaccines. By controlling the surface density of CpG, we show that intermediate valency is a key factor for TLR9 activation of immune cells. When delivered with the SARS-CoV-2 spike protein, CpG nanoparticle (CpG-NP) adjuvant greatly improves the magnitude and duration of antibody responses when compared to soluble CpG, and results in overall greater breadth of immunity against variants of concern. Moreover, encapsulation of CpG-NP into injectable polymeric-nanoparticle (PNP) hydrogels enhances the spatiotemporal control over codelivery of CpG-NP adjuvant and spike protein antigen such that a single immunization of hydrogel-based vaccines generates humoral responses comparable to those of a typical prime-boost regimen of soluble vaccines. These delivery technologies can potentially reduce the costs and burden of clinical vaccination, both of which are key elements in fighting a pandemic.

Differences in athletic identity, sport participation, and psychosocial factors following anterior cruciate ligament rehabilitation in youth athletes.

Introduction: While youth sports benefits the developing athlete, athletes may also be subject to injury and subsequent return-to-sport protocols. The current return-to-sport criteria emphasize physical measures; however, psychological measures may also be valuable to inform providers of an athlete's readiness. One such measure is athletic identity defined as the degree to which an individual identifies with the athlete role. To better understand athletic identity in return-to-sport, this study aimed to identify relationships and trends between the Athletic Identity Measurement Scale (AIMS), demographic variables, sport participation measures, and the Athletic Coping Skills Inventory-28 (ACSI-28) in youth athletes during rehabilitation following anterior cruciate ligament reconstruction (ACLR). Methods: A retrospective review was completed of patients who underwent ACLR at a sports medicine clinic between October 2019 and May 2021. Patients responded to a series of patient reported outcomes (PROs) regarding physical and psychological function at a pre-surgical baseline and after 1 year of rehabilitation. Patients were then divided into groups of high/low AIMS and an increased/decreased AIMS between 1 year and baseline for comparison. Independent samples t-tests and ANOVAs were performed as appropriate with a 95% confidence interval. Results: In the final sample, 87 patients (15.3 ± 1.8 years) were included, with 51.7% being females. Total AIMS scores decreased from 50.3 to 47.5 over rehabilitation (p = 0.019). Furthermore, results indicated that nearly all AIMS scores decreased during rehabilitation, with none showing an increase; however, not all domains were significant. Conversely, all sport participation and coping ability PROs increased over time points except for ACSI-Confidence and Achievement Motivation. Generally, those in the groups with high AIMS and an increase in AIMS also had higher scores in physical function and coping ability PROs, with the groups separated by high/low AIMS exhibiting more frequent statistical significance. Discussion: Given these results, it appears that athletes may lose identification with the athlete role after ACLR and struggle even 1 year for rehabilitation, but those who recover athletic identity the best may also be those able to cope most effectively with the stressors induced by injury.

Injectable Nanoparticle-Based Hydrogels Enable the Safe and Effective Deployment of Immunostimulatory CD40 Agonist Antibodies.

When properly deployed, the immune system can eliminate deadly pathogens, eradicate metastatic cancers, and provide long-lasting protection from diverse diseases. Unfortunately, realizing these remarkable capabilities is inherently risky as disruption to immune homeostasis can elicit dangerous complications or autoimmune disorders. While current research is continuously expanding the arsenal of potent immunotherapeutics, there is a technological gap when it comes to controlling when, where, and how long these drugs act on the body. Here, this study explored the ability of a slow-releasing injectable hydrogel depot to reduce dose-limiting toxicities of immunostimulatory CD40 agonist (CD40a) while maintaining its potent anticancer efficacy. A previously described polymer-nanoparticle (PNP) hydrogel system is leveraged that exhibits shear-thinning and yield-stress properties that are hypothesized to improve locoregional delivery of CD40a immunotherapy. Using positron emission tomography, it is demonstrated that prolonged hydrogel-based delivery redistributes CD40a exposure to the tumor and the tumor draining lymph node (TdLN), thereby reducing weight loss, hepatotoxicity, and cytokine storm associated with standard treatment. Moreover, CD40a-loaded hydrogels mediate improved local cytokine induction in the TdLN and improve treatment efficacy in the B16F10 melanoma model. PNP hydrogels, therefore, represent a facile, drug-agnostic method to ameliorate immune-related adverse effects and explore locoregional delivery of immunostimulatory drugs.

Delivery of CAR-T cells in a transient injectable stimulatory hydrogel niche improves treatment of solid tumors.

Adoptive cell therapy (ACT) has proven to be highly effective in treating blood cancers, but traditional approaches to ACT are poorly effective in treating solid tumors observed clinically. Novel delivery methods for therapeutic cells have shown promise for treatment of solid tumors when compared with standard intravenous administration methods, but the few reported approaches leverage biomaterials that are complex to manufacture and have primarily demonstrated applicability following tumor resection or in immune-privileged tissues. Here, we engineer simple-to-implement injectable hydrogels for the controlled co-delivery of CAR-T cells and stimulatory cytokines that improve treatment of solid tumors. The unique architecture of this material simultaneously inhibits passive diffusion of entrapped cytokines and permits active motility of entrapped cells to enable long-term retention, viability, and activation of CAR-T cells. The generation of a transient inflammatory niche following administration affords sustained exposure of CAR-T cells, induces a tumor-reactive CAR-T phenotype, and improves efficacy of treatment.

Youth athletes sleep more, practice less, and may lose interest in playing sports due to social distancing mandates.

In-person sport participation was suspended across the United States in the spring of 2020 to slow the spread of the novel coronavirus (COVID-19). The purpose of this study was to survey the impact of COVID-19 on young athletes during a period of social and organized sports restrictions. An anonymous cross-sectional survey study was conducted of youth athletes in the midst of social distancing mandates and consisted of six components: demographics, sport participation, changes in sport-related goals/aspirations, sleep habits, and measures of anxiety and depression. 711 individuals who accessed the survey link yielded 575 (81%) participants with responses available for analysis. All respondents (aged 13.0 years) played organized sports, 62% were single-sport athletes, and 74% considered high-level. Participants were training ∼3.3 h less per week, spending more time outside, and 86% of participants continued to train while social distancing. Sleep duration increased (∼1.2 h/night) and sleep quality improved in 29% of young athletes. Additionally, 22% and 28% reported PROMIS® anxiety and depression scores characterized as 'mild', 'moderate', or 'severe'. Older single-sport participants reported higher depression scores, while higher anxiety scores were seen in female participants with fewer years played. 10% of young athletes and 20% of teenagers changed their sports-related goals. Training style modifications, decreased training, and increased sleep quantity and quality were positive effects of COVID-19 restrictions, while athletic aspirational changes were undesirable effects. Single-sport athletes may be at greater risk for psychological symptoms when their routine is altered.

Hydrogel-Based Slow Release of a Receptor-Binding Domain Subunit Vaccine Elicits Neutralizing Antibody Responses Against SARS-CoV-2.

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum are found unable to elicit neutralizing responses following a prime-boost immunization. Here, it has been shown that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

Consistent tumorigenesis with self-assembled hydrogels enables high-powered murine cancer studies.

Preclinical cancer research is heavily dependent on allograft and xenograft models, but current approaches to tumor inoculation yield inconsistent tumor formation and growth, ultimately wasting valuable resources (e.g., animals, time, and money) and limiting experimental progress. Here we demonstrate a method for tumor inoculation using self-assembled hydrogels to reliably generate tumors with low variance in growth. The observed reduction in model variance enables smaller animal cohorts, improved effect observation and higher powered studies.

Modulation of injectable hydrogel properties for slow co-delivery of influenza subunit vaccine components enhance the potency of humoral immunity.

Vaccines are critical for combating infectious diseases across the globe. Influenza, for example, kills roughly 500,000 people annually worldwide, despite annual vaccination campaigns. Efficacious vaccines must elicit a robust and durable antibody response, and poor efficacy often arises from inappropriate temporal control over antigen and adjuvant presentation to the immune system. In this work, we sought to exploit the immune system's natural response to extended pathogen exposure during infection by designing an easily administered slow-delivery influenza vaccine platform. We utilized an injectable and self-healing polymer-nanoparticle (PNP) hydrogel platform to prolong the co-delivery of vaccine components to the immune system. We demonstrated that these hydrogels exhibit unique dynamic physical characteristics whereby physicochemically distinct influenza hemagglutinin antigen and a toll-like receptor 7/8 agonist adjuvant could be co-delivered over prolonged timeframes that were tunable through simple alteration of the gel formulation. We show a relationship between hydrogel physical properties and the resulting immune response to immunization. When administered in mice, hydrogel-based vaccines demonstrated enhancements in the magnitude and duration of humoral immune responses compared to alum, a widely used clinical adjuvant system. We found stiffer hydrogel formulations exhibited slower release and resulted in the greatest improvements to the antibody response while also enabling significant adjuvant dose sparing. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.

Full closed loop open-source algorithm performance comparison in pigs with diabetes.

Understanding how automated insulin delivery (AID) algorithm features impact glucose control under full closed loop delivery represents a critical step toward reducing patient burden by eliminating the need for carbohydrate entries at mealtimes. Here, we use a pig model of diabetes to compare AndroidAPS and Loop open-source AID systems without meal announcements. Overall time-in-range (70-180 mg/dl) for AndroidAPS was 58% ± 5%, while time-in-range for Loop was 35% ± 5%. The effect of the algorithms on time-in-range differed between meals and overnight. During the overnight monitoring period, pigs had an average time-in-range of 90% ± 7% when on AndroidAPS compared to 22% ± 8% on Loop. Time-in-hypoglycemia also differed significantly during the lunch meal, whereby pigs running AndroidAPS spent an average of 1.4% (+0.4/-0.8)% in hypoglycemia compared to 10% (+3/-6)% for those using Loop. As algorithm design for closed loop systems continues to develop, the strategies employed in the OpenAPS algorithm (known as oref1) as implemented in AndroidAPS for unannounced meals may result in a better overall control for full closed loop systems.

Hydrogel-based slow release of a receptor-binding domain subunit vaccine elicits neutralizing antibody responses against SARS-CoV-2.

The development of effective vaccines that can be rapidly manufactured and distributed worldwide is necessary to mitigate the devastating health and economic impacts of pandemics like COVID-19. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein, which mediates host cell entry of the virus, is an appealing antigen for subunit vaccines because it is efficient to manufacture, highly stable, and a target for neutralizing antibodies. Unfortunately, RBD is poorly immunogenic. While most subunit vaccines are commonly formulated with adjuvants to enhance their immunogenicity, we found that clinically-relevant adjuvants Alum, AddaVax, and CpG/Alum were unable to elicit neutralizing responses following a prime-boost immunization. Here we show that sustained delivery of an RBD subunit vaccine comprising CpG/Alum adjuvant in an injectable polymer-nanoparticle (PNP) hydrogel elicited potent anti-RBD and anti-spike antibody titers, providing broader protection against SARS-CoV-2 variants of concern compared to bolus administration of the same vaccine and vaccines comprising other clinically-relevant adjuvant systems. Notably, a SARS-CoV-2 spike-pseudotyped lentivirus neutralization assay revealed that hydrogel-based vaccines elicited potent neutralizing responses when bolus vaccines did not. Together, these results suggest that slow delivery of RBD subunit vaccines with PNP hydrogels can significantly enhance the immunogenicity of RBD and induce neutralizing humoral immunity.

Prolonged Codelivery of Hemagglutinin and a TLR7/8 Agonist in a Supramolecular Polymer-Nanoparticle Hydrogel Enhances Potency and Breadth of Influenza Vaccination.

The sustained release of vaccine cargo has been shown to improve humoral immune responses to challenging pathogens such as influenza. Extended codelivery of antigen and adjuvant prolongs germinal center reactions, thus improving antibody affinity maturation and the ability to neutralize the target pathogen. Here, we develop an injectable, physically cross-linked polymer-nanoparticle (PNP) hydrogel system to prolong the local codelivery of hemagglutinin and a toll-like receptor 7/8 agonist (TLR7/8a) adjuvant. By tethering the TLR7/8a to a NP motif within the hydrogels (TLR7/8a-NP), the dynamic mesh of the PNP hydrogels enables codiffusion of the adjuvant and protein antigen (hemagglutinin), therefore enabling sustained codelivery of these two physicochemically distinct molecules. We show that subcutaneous delivery of PNP hydrogels carrying hemagglutinin and TLR7/8a-NP in mice improves the magnitude and duration of antibody titers in response to a single injection vaccination compared to clinically used adjuvants. Furthermore, the PNP gel-based slow delivery of influenza vaccines led to increased breadth of antibody responses against future influenza variants, including a future pandemic variant, compared to clinical adjuvants. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of influenza subunit vaccines.

Injectable Hydrogels for Sustained Codelivery of Subunit Vaccines Enhance Humoral Immunity.

Vaccines aim to elicit a robust, yet targeted, immune response. Failure of a vaccine to elicit such a response arises in part from inappropriate temporal control over antigen and adjuvant presentation to the immune system. In this work, we sought to exploit the immune system's natural response to extended pathogen exposure during infection by designing an easily administered slow-delivery vaccine platform. We utilized an injectable and self-healing polymer-nanoparticle (PNP) hydrogel platform to prolong the codelivery of vaccine components to the immune system. We demonstrated that these hydrogels exhibit unique delivery characteristics, whereby physicochemically distinct compounds (such as antigen and adjuvant) could be codelivered over the course of weeks. When administered in mice, hydrogel-based sustained vaccine exposure enhanced the magnitude, duration, and quality of the humoral immune response compared to standard PBS bolus administration of the same model vaccine. We report that the creation of a local inflammatory niche within the hydrogel, coupled with sustained exposure of vaccine cargo, enhanced the magnitude and duration of germinal center responses in the lymph nodes. This strengthened germinal center response promoted greater antibody affinity maturation, resulting in a more than 1000-fold increase in antigen-specific antibody affinity in comparison to bolus immunization. In summary, this work introduces a simple and effective vaccine delivery platform that increases the potency and durability of subunit vaccines.

Nanoparticles Presenting Potent TLR7/8 Agonists Enhance Anti-PD-L1 Immunotherapy in Cancer Treatment.

Cancer immunotherapy can be augmented with toll-like receptor agonist (TLRa) adjuvants, which interact with immune cells to elicit potent immune activation. Despite their potential, use of many TLRa compounds has been limited clinically due to their extreme potency and lack of pharmacokinetic control, causing systemic toxicity from unregulated systemic cytokine release. Herein, we overcome these shortcomings by generating poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) nanoparticles (NPs) presenting potent TLR7/8a moieties on their surface. The NP platform allows precise control of TLR7/8a valency and resulting surface presentation through self-assembly using nanoprecipitation. We hypothesize that the pharmacokinetic profile of the NPs minimizes systemic toxicity, localizing TLR7/8a presentation to the tumor bed and tumor-draining lymph nodes. In conjunction with antiprogrammed death-ligand 1 (anti-PD-L1) checkpoint blockade, peritumoral injection of TLR7/8a NPs slows tumor growth, extends survival, and decreases systemic toxicity in comparison to the free TLR7/8a in a murine colon adenocarcinoma model. These NPs constitute a modular platform for controlling pharmacokinetics of immunostimulatory molecules, resulting in increased potency and decreased toxicity.

An ultrafast insulin formulation enabled by high-throughput screening of engineered polymeric excipients.

Insulin has been used to treat diabetes for almost 100 years; yet, current rapid-acting insulin formulations do not have sufficiently fast pharmacokinetics to maintain tight glycemic control at mealtimes. Dissociation of the insulin hexamer, the primary association state of insulin in rapid-acting formulations, is the rate-limiting step that leads to delayed onset and extended duration of action. A formulation of insulin monomers would more closely mimic endogenous postprandial insulin secretion, but monomeric insulin is unstable in solution using present formulation strategies and rapidly aggregates into amyloid fibrils. Here, we implement high-throughput-controlled radical polymerization techniques to generate a large library of acrylamide carrier/dopant copolymer (AC/DC) excipients designed to reduce insulin aggregation. Our top-performing AC/DC excipient candidate enabled the development of an ultrafast-absorbing insulin lispro (UFAL) formulation, which remains stable under stressed aging conditions for 25 ± 1 hours compared to 5 ± 2 hours for commercial fast-acting insulin lispro formulations (Humalog). In a porcine model of insulin-deficient diabetes, UFAL exhibited peak action at 9 ± 4 min, whereas commercial Humalog exhibited peak action at 25 ± 10 min. These ultrafast kinetics make UFAL a promising candidate for improving glucose control and reducing burden for patients with diabetes.

A co-formulation of supramolecularly stabilized insulin and pramlintide enhances mealtime glucagon suppression in diabetic pigs.

Treatment of patients with diabetes with insulin and pramlintide (an amylin analogue) is more effective than treatment with insulin only. However, because mixtures of insulin and pramlintide are unstable and have to be injected separately, amylin analogues are only used by 1.5% of people with diabetes needing rapid-acting insulin. Here, we show that the supramolecular modification of insulin and pramlintide with cucurbit[7]uril-conjugated polyethylene glycol improves the pharmacokinetics of the dual-hormone therapy and enhances postprandial glucagon suppression in diabetic pigs. The co-formulation is stable for over 100 h at 37 °C under continuous agitation, whereas commercial formulations of insulin analogues aggregate after 10 h under similar conditions. In diabetic rats, the administration of the stabilized co-formulation increased the area-of-overlap ratio of the pharmacokinetic curves of pramlintide and insulin from 0.4 ± 0.2 to 0.7 ± 0.1 (mean ± s.d.) for the separate administration of the hormones. The co-administration of supramolecularly stabilized insulin and pramlintide better mimics the endogenous kinetics of co-secreted insulin and amylin, and holds promise as a dual-hormone replacement therapy.

Injectable supramolecular polymer-nanoparticle hydrogels enhance human mesenchymal stem cell delivery.

Stem cell therapies have emerged as promising treatments for injuries and diseases in regenerative medicine. Yet, delivering stem cells therapeutically can be complicated by invasive administration techniques, heterogeneity in the injection media, and/or poor cell retention at the injection site. Despite these issues, traditional administration protocols using bolus injections in a saline solution or surgical implants of cell-laden hydrogels have highlighted the promise of cell administration as a treatment strategy. To address these limitations, we have designed an injectable polymer-nanoparticle (PNP) hydrogel platform exploiting multivalent, noncovalent interactions between modified biopolymers and biodegradable nanoparticles for encapsulation and delivery of human mesenchymal stem cells (hMSCs). hMSC-based therapies have shown promise due to their broad differentiation capacities and production of therapeutic paracrine signaling molecules. In this work, the fundamental hydrogel mechanical properties that enhance hMSC delivery processes are elucidated using basic in vitro models. Further, in vivo studies in immunocompetent mice reveal that PNP hydrogels enhance hMSC retention at the injection site and retain administered hMSCs locally for upwards of 2 weeks. Through both in vitro and in vivo experiments, we demonstrate a novel scalable, synthetic, and biodegradable hydrogel system that overcomes current limitations and enables effective cell delivery.

Doublesex and mab-3-related transcription factor 5 promotes midbrain dopaminergic identity in pluripotent stem cells by enforcing a ventral-medial progenitor fate.

Understanding the control of cell-fate choices during embryonic stem cell (ESC) differentiation is crucial for harnessing strategies for efficient production of desired cell types for pharmaceutical drug screening and cell transplantation. Here we report the identification of the zinc finger-like doublesex and mab-3-related transcription factor 5 (Dmrt5) as a marker for mammalian ventral-medial mesencephalic neuroepithelium that give rise to dopamine neurons. Gain- and loss-of-function studies in ESC demonstrate that Dmrt5 is critically involved in the specification of ventral-medial neural progenitor cell fate and the subsequent generation of dopamine neurons expressing essential midbrain characteristics. Genome-wide analysis of Dmrt5-mediated transcriptome changes and expression profiling of ventral-medial and ventral-lateral mesencephalic neuroepithelium revealed suppressive and inductive regulatory roles for Dmrt5 in the transcription program associated with the ventral-medial neural progenitor fates. Together, these data identify Dmrt5 as an important player in ventral mesencephalic neural fate specification.

Effects of in ovo electroporation on endogenous gene expression: genome-wide analysis.

BACKGROUND: In ovo electroporation is a widely used technique to study gene function in developmental biology. Despite the widespread acceptance of this technique, no genome-wide analysis of the effects of in ovo electroporation, principally the current applied across the tissue and exogenous vector DNA introduced, on endogenous gene expression has been undertaken. Here, the effects of electric current and expression of a GFP-containing construct, via electroporation into the midbrain of Hamburger-Hamilton stage 10 chicken embryos, are analysed by microarray. RESULTS: Both current alone and in combination with exogenous DNA expression have a small but reproducible effect on endogenous gene expression, changing the expression of the genes represented on the array by less than 0.1% (current) and less than 0.5% (current + DNA), respectively. The subset of genes regulated by electric current and exogenous DNA span a disparate set of cellular functions. However, no genes involved in the regional identity were affected. In sharp contrast to this, electroporation of a known transcription factor, Dmrt5, caused a much greater change in gene expression. CONCLUSIONS: These findings represent the first systematic genome-wide analysis of the effects of in ovo electroporation on gene expression during embryonic development. The analysis reveals that this process has minimal impact on the genetic basis of cell fate specification. Thus, the study demonstrates the validity of the in ovo electroporation technique to study gene function and expression during development. Furthermore, the data presented here can be used as a resource to refine the set of transcriptional responders in future in ovo electroporation studies of specific gene function.

FGF signaling is required for myofibroblast differentiation during alveolar regeneration.

Normal alveolarization has been studied in rodents using detailed morphometric techniques and loss of function approaches for growth factors and their receptors. However, it remains unclear how these growth factors direct the formation of secondary septae. We have previously developed a transgenic mouse model in which expression of a soluble dominant-negative FGF receptor (dnFGFR) in the prenatal period results in reduced alveolar septae formation and subsequent alveolar simplification. Retinoic acid (RA), a biologically active derivative of vitamin A, can induce regeneration of alveoli in adult rodents. In this study, we demonstrate that RA induces alveolar reseptation in this transgenic mouse model and that realveolarization in adult mice is FGF dependent. Proliferation in the lung parenchyma, an essential prerequisite for lung regrowth was enhanced after 14 days of RA treatment and was not influenced by dnFGFR expression. During normal lung development, formation of secondary septae is associated with the transient presence of alpha-smooth muscle actin (alphaSMA)-positive interstitial myofibroblasts. One week after completion of RA treatment, alphaSMA expression was detected in interstitial fibroblasts, supporting the concept that RA-initiated realveolarization recapitulates aspects of septation that occur during normal lung development. Expression of dnFGFR blocked realveolarization with increased PDGF receptor-alpha (PDGFRalpha)-positive cells and decreased alphaSMA-positive cells. Taken together, our data demonstrate that FGF signaling is required for the induction of alphaSMA in the PDGFRalpha-positive myofibroblast progenitor and the progression of alveolar regeneration.