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Positive active learning outcomes require student engagement with foundational, preparatory material prior to class. The current study involved thorough updating of cardiovascular physiology module format. It then examined preclinical medical student perceptions, and midterm exam performance, after using different preparatory module formats that were reviewed prior to participating in interactive classroom sessions. Modules that were initially created in Articulate Storyline were updated in Articulate Rise360 and introduced over a 3-year period. Both module styles contained the same cardiovascular physiology content, but updated Rise360 modules presented content in multiple formats to capture a variety of student learning preferences and divided each concept into several smaller topics to maintain student attention. Although midterm exam performance remained unchanged, student evaluations revealed that the updated Rise360 modules were more helpful with fewer technical issues indicating that students preferred the interactive online modules to prepare for collaborative classroom exercises. Students find updated preparatory modules to be more helpful and may therefore be more likely to engage with them before class and ultimately lead to a more productive interactive classroom learning experience.

Organismal invasions have repeatedly been cited as both a driving force behind global change and beneficiaries of that change. Although many drivers of these invasions have been well studied, few studies have addressed invasions through the perspective of native symbiont communities. In the Mountain Lake region of Virginia, crayfish host diverse assemblages of obligate cleaning symbionts known as branchiobdellida. This cleaning symbiosis has been found to result in significant fitness benefits for native crayfish. Historical survey work showed that invasive crayfish, known to be intolerant of symbionts, were introduced into the region by the 1960s. I carried out an extensive regional survey to determine how this invasion has progressed and what impacts it is having on the native crayfish-branchiobdellida symbiosis. Survey results show that invasive crayfish have successfully spread throughout the region, resulting in the displacement of native crayfish. Additionally, findings suggest that invasion results in significantly reductions in abundance and richness in native symbiont communities. To determine mechanisms contributing to observed impacts on native symbionts, I carried out a study that simulated displacement of native crayfish by invasive crayfish in a controlled setting. This study found that as native crayfish are increasingly displaced by invasive crayfish, both symbiont dispersal and survival are negatively affected. This potential loss of symbiosis caused by invasion may reduce symbionts on native crayfish below abundances necessary for fitness benefits, exacerbating the negative impacts of invasions and presenting a major conservation issue in invaded systems. M. S.

We have proposed that modified platelets could potentially be used to correct intrinsic platelet defects as well as for targeted delivery of therapeutic molecules to sights of vascular injury. Ectopic expression of proteins within α-granules prior to platelet activation has been achieved for several proteins, including urokinase, factor (F) VIII, and partially for FIX. Potential uses of platelet-directed therapeutics will be discussed, focusing on targeted delivery of urokinase as a thromboprophylactic agent and FVIII for the treatment of hemophilia A patients with intractable inhibitors. This presentation will discuss new strategies that may be useful in the care of patients with vascular injury as well as remaining challenges and limitations of these approaches.

We and others have shown that FVIII expressed ectopically in platelets (pFVIII) is stored in α-granules, released at sites of vascular injury and restores hemostasis in FVIIInull mice, even in the presence of neutralizing antibodies to FVIII. These studies support the concept that unlike therapeutic interventions that correct plasma FVIII, pFVIII may be a useful therapy in hemophilia A with intractable inhibitors and significant bleeds. We have also demonstrated this approach has several limitations that may make pFVIII gene therapy bone marrow transplantation (BMT) strategies problematic: 1) pFVIII is not equivalent to plasma FVIII and its efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors, and 2) pFVIII expressed during megakaryopoiesis can cause injury to the Mks, potentially exacerbating post-BMT thrombocytopenia. We propose an alternative strategy: interval prophylactic infusions of FVIII-containing platelets generated from patient-specific iMks expressing either human B-domain-deleted (BDD) FVIII or variants of this FVIII that have greater stability and longer half-lives; making them especially efficacious as pFVIII as we previously demonstrated. iPSCs are a renewable source of cells that can be pre-screened prior to clinical usage for lines that express optimal levels of pFVIII and also release optimal numbers of platelets after differentiation into iMks. Such iPSCs were transfected with a self-inactivating lentivirus containing cDNA for one of three FVIII variants: wildtype BDD FVIII (WT FVIII), R1645H PACE/furin cleavage site FVIII (FVIIIR1645H), and amino acid 1645 to 1648 deletion FVIII (FVIIIΔ). FVIIIR1645H and FVIIIΔ show greater stability and consequently greater specific activity with no increase in injuring Mks. All FVIII variants were expressed using the MK-specific Cxcl4 promoter and were shown to be effective in several bleeding models in FVIIInull mice. Differentiated and transduced iMKs were analyzed for RNA and protein expression. All of the FVIII variant iMKs expressed at least forty-fold higher levels of mRNA compared to the non-transduced control (n=6) and protein was expressed at >550 pg/106 CD42b+ iMKs (n=6). Transduced MKs released FVIII into the supernatant when activated by thrombin showing the pFVIII was likely stored in α-granules. Annexin staining was the same between FVIII-expressing iMKs and control iMks suggesting that the level of pFVIII did not cause the iMks to become apoptotic. To test the ability of FVIII-expressing iMKs to correct the coagulopathy in hemophilia A, 5x105 iMKs were added to FVIIInull murine whole blood and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each FVIII variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (n=4). These data show that iMKs expressing FVIII variants can improve hemostasis in a whole blood clotting assay. Our next goal is to generate sufficient platelets from these iMKs to test for correction of the bleeding diathesis in immunodeficient FVIIInull mice and to determine their efficacy in improving hemostasis in a number of clinically relevant hemostatic models. Disclosures Arruda: Pfizer: Consultancy, Patents & Royalties, Research Funding; Spark Therapeutics: Patents & Royalties. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; NovoNordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Jacobsen syndrome is a rare, inherited hemizygous deletion of chromosome 11q that is often associated with a dysmegakaryopoiesis and macrothrombocytopenia termed Paris Trousseau syndrome (PTSx). Among the genes involved in the chromosomal deletion are FLI1 and ETS1, both of which belong to the ETS family of transcription factors and have been associated with megakaryopoiesis. One prior study using primary human hematopoietic stem cells suggested that the defect in PTSx was due to FLI1 allelic exclusion resulting in the generation of two distinct megakaryocyte (MK) populations, one immature and one mature. More consistent with the clinical course of this disorder, we hypothesize that PTSx is caused by FLI1 haploinsufficiency, where all MKs are affected and do not mature properly. The goal of our studies was to better understand MK development by investigating the role of FLI1 during megakaryopoiesis, including in PTSx. However, Fli1 deletions in mice did not replicate the defect observed in humans, so we used genome-engineered human induced pluripotent stem cell (iPSC) lines. We have established an iPSC line from a PTSx patient and derived from this a FLI1 overexpressing (OE) line in which FLI1 cDNA was cloned into the AAVS1 "safe harbor" locus with MK-specific expression driven by the GP1bα promoter. In parallel, we have a healthy control line, a control-derived FLI1 OE line, and a homozygous FLI1+/- line. In the control- and PTSx-FLI1 OE lines, FLI1 mRNA levels in MKs were 2X higher than control levels. The FLI1+/- line was generated using TALENs and expressed RNA at levels comparable to the PTSx line. To analyze MK progenitor potential, the iPSC lines were differentiated to hematopoietic progenitor cells (HPCs) and analyzed using Megacult colony assays. The PTSx line generated 4- to 6-fold less CFU-MK colonies per 1000 plated CD41+CD235+ cells compared to control (P=0.1) and PTSx-FLI1 OE (P=0.002). Likewise, the FLI1+/- line had less colonies compared to control (P=0.2) and control-FLI1 OE (P=0.005). The control-FLI1 OE line generated 70% more colonies (P=0.22) than the control line. To analyze MK generation, identical numbers of HPCs were expanded in liquid culture containing MK-specific cytokines and the numbers of CD41+CD42a+ cells were quantitated. The PTSx line had Disclosures No relevant conflicts of interest to declare.