The androgen receptor (AR) has a critical role in promoting androgen-dependent and -independent apoptosis in testicular cells. However, the molecular mechanisms that underlie the ligand-independent apoptosis, including the activity of AR in testicular stem cells, are not completely understood. In the present study, we generated induced pluripotent stem cells (iPSCs) from bovine testicular cells by electroporation of octamer-binding transcription factor 4 (OCT4). The cells were supplemented with leukemia inhibitory factor and bone morphogenetic protein 4, which maintained and stabilized the expression of stemness genes and pluripotency. The iPSCs were used to assess the apoptosis activity following exposure to phthalate esters, including di (2-ethyhexyl) phthalates, di (n-butyl) phthalate, and butyl benzyl phthalate. Phthalate esters significantly reduced the expression of AR in iPSCs and induced a higher ratio of BAX/BCL-2, thereby favoring apoptosis. Phthalate esters also increased the expression of cyclin-dependent kinase inhibitor 1 (p21Cip1) in a p53-dependent manner and enhanced the transcriptional activity of p53. The forced expression of AR and knockdown of p21Cip1 led to the rescue of the phthalate-mediated apoptosis. Overall, this study suggests that testicular iPSCs are a useful system for screening the toxicity of environmental disruptors and examining their effect on the maintenance of stemness and pluripotency, as well as for identifying the iPSC signaling pathway(s) that are deregulated by these chemicals.

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This study assessed the effectiveness of education reforms on student-reported learning outcomes at the end of the 5-year medical school (M5) and 1-year internship (HO) in 2006, 2007 and 2008. A self-administered anonymous survey with 17 learning outcomes assessed, derived from Harden’s Three-Circle Outcomes Model for outcomes-based education, was administered to 683 students at the end of medical school (M5) and internship (HO) from 2006, 2007 and 2008. We identified learning outcomes which changed significantly for internship (Cohorts A, B and C) and medical school (Cohorts B, C and D) between cohorts from 2006 to 2008, and compared learning outcomes between medical school and internship within cohorts (i.e. Cohort B which was M5 in 2006 and HO in 2007; Cohort C which was M5 in 2007 and HO in 2008). The proportion of students who agreed that medical school helped them achieve learning outcomes increased significantly from 2006 to 2008 for 15 out of 17 learning outcomes assessed. The proportion of students who agreed that internship helped them achieve learning outcomes increased significantly from 2006 to 2008 for 6 learning outcomes assessed. For Cohorts B and C, internship was more effective than medical school in achieving 8 learning outcomes. Cohort C reported that internship was more effective than medical school in 3 additional learning outcomes than Cohort B: patient management, humility and dedication. We conclude that a successful journey of education reform is an ongoing process that needs to comprehensively address multifaceted components such as faculty, administration and curriculum. Key words: Harden’s Three-Circle Outcomes Model, Housemanship, Internship

Enteric microbial pathogens, including Escherichia coli, Shigella and Cryptosporidium species, take a particularly heavy toll in low-income countries and are highly associated with infant mortality. We describe here a means to display anti-infective agents on the surface of a probiotic bacterium. Because of their stability and versatility, VHHs, the variable domains of camelid heavy-chain-only antibodies, have potential as components of novel agents to treat or prevent enteric infectious disease. We isolated and characterized VHHs targeting several enteropathogenic Escherichia.coli (EPEC) virulence factors: flagellin (Fla), which is required for bacterial motility and promotes colonization; both intimin and the translocated intimin receptor (Tir), which together play key roles in attachment to enterocytes; and E. coli secreted protein A (EspA), an essential component of the type III secretion system (T3SS) that is required for virulence. Several VHHs that recognize Fla, intimin, or Tir blocked function in vitro. The probiotic strain E. coli Nissle 1917 (EcN) produces on the bacterial surface curli fibers, which are the major proteinaceous component of E. coli biofilms. A subset of Fla-, intimin-, or Tir-binding VHHs, as well as VHHs that recognize either a T3SS of another important bacterial pathogen (Shigella flexneri), a soluble bacterial toxin (Shiga toxin or Clostridioides difficile toxin TcdA), or a major surface antigen of an important eucaryotic pathogen (Cryptosporidium parvum) were fused to CsgA, the major curli fiber subunit. Scanning electron micrographs indicated CsgA-VHH fusions were assembled into curli fibers on the EcN surface, and Congo Red binding indicated that these recombinant curli fibers were produced at high levels. Ectopic production of these VHHs conferred on EcN the cognate binding activity and, in the case of anti-Shiga toxin, was neutralizing. Taken together, these results demonstrate the potential of the curli-based pathogen sequestration strategy described herein and contribute to the development of novel VHH-based gut therapeutics.