From the Cover: Conditional control of gene function by an invertible gene trap in zebrafish

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.High throughput functional genomic and informatic methods have been developed to interrogate the genome and extract functional predictions about many genes at a time. However, careful phenotypic analysis of genetic mutants remains the sine qua non of reductionist biological science. In most experimental organisms, random mutagenesis is the preferred or only mutagenic technique available. DNA alkylating agents, transposable elements, or retroviruses are traditionally used in these organisms. A major limitation of these traditional genetic methods is that they reveal only the earliest and/or most prominent function of a gene as later functions are masked by the earlier phenotype, which is often lethality. To assess later functions, for example in metabolism, aging, or behavior, conditional alleles are required.The development of conditional alleles has proven a boon to studying gene function in temporally or spatially restricted contexts. Traditional conditional alleles disrupt gene function by changing the environment, for example by increasing the temperature. Engineered conditional alleles disrupt gene function by activating a recombination-mediated molecular switch that ablates gene function in one state, but has no functional consequences in the other state (1, 2). In the mouse, engineered conditional alleles can be generated by homologous recombination to insert the molecular switch at defined loci or by retroviral-mediated random insertion of the molecular switch (3, 4). The second approach leverages the orientation-dependent gene disruption of a gene trap and the ability of Flp/Cre recombinases to stably invert the gene trap. By strategically arranging dimers of heterotypical flp- and cre-recombinase binding sites flanking the gene trap, stable inversion is achieved in cis by recombinase-mediated Flip and Excision (FlEx) (5). However, this conditional gene-trap mutagen has not been validated at the organismal level.A distinct advantage of FlEx-based conditional gene-trap mutations is the possibility of stage- and tissue-specific rescue or knockout of the mutated genes. In zebrafish, several gene-trap mutagenesis methods have been developed (6, 7), including the “gene-break” (6, 8) and “FlipTrap” (9) technologies. We set out to test whether the FlEx-based conditional gene-trap mutagenesis approach functions at the organismal level in zebrafish. We show here that a highly mutagenic transposable element can be used for conditional analysis of essential genes.     

Evaluation of Immunogenicity and Efficacy of Anthrax Vaccine Adsorbed for Postexposure Prophylaxis

Antimicrobials administered postexposure can reduce the incidence or progression of anthrax disease, but they do not protect against the disease resulting from the germination of spores that may remain in the body after cessation of the antimicrobial regimen. Such additional protection may be achieved by postexposure vaccination; however, no anthrax vaccine is licensed for postexposure prophylaxis (PEP). In a rabbit PEP study, animals were subjected to lethal challenge with aerosolized Bacillus anthracis spores and then were treated with levofloxacin with or without concomitant intramuscular (i.m.) vaccination with anthrax vaccine adsorbed (AVA) (BioThrax; Emergent BioDefense Operations Lansing LLC, Lansing, MI), administered twice, 1 week apart. A significant increase in survival rates was observed among vaccinated animals compared to those treated with antibiotic alone. In preexposure prophylaxis studies in rabbits and nonhuman primates (NHPs), animals received two i.m. vaccinations 1 month apart and were challenged with aerosolized anthrax spores at day 70. Prechallenge toxin-neutralizing antibody (TNA) titers correlated with animal survival postchallenge and provided the means for deriving an antibody titer associated with a specific probability of survival in animals. In a clinical immunogenicity study, 82% of the subjects met or exceeded the prechallenge TNA value that was associated with a 70% probability of survival in rabbits and 88% probability of survival in NHPs, which was estimated based on the results of animal preexposure prophylaxis studies. The animal data provide initial information on protective antibody levels for anthrax, as well as support previous findings regarding the ability of AVA to provide added protection to B. anthracis-infected animals compared to antimicrobial treatment alone.     

Developing and pretesting case studies in dental and dental hygiene education: using the diffusion of innovations model

Case-based learning offers exposure to clinical situations that health professions students may not encounter in their training. The purposes of this study were to apply the Diffusion of Innovations conceptual framework to 1) identify characteristics of case studies that would increase their adoption among dental and dental hygiene faculty members and 2) develop and pretest interactive web-based case studies on sensitive oral-systemic health issues. The formative study spanned two phases using mixed methods (Phase 1: eight focus groups and four interviews; Phase 2: ten interviews and satisfaction surveys). Triangulation of quantitative and qualitative data revealed the following positive attributes of the developed case studies: relative advantage of active learning and modeling; compatibility with a variety of courses; observability of case-related knowledge and skills; independent learning; and modifiability for use with other oral-systemic health issues. These positive attributes are expected to increase the likelihood that dental and dental hygiene faculty members will adopt the developed case study once it is available for use. The themes identified in this study could be applied to the development of future case studies and may provide broader insight that might prove useful for exploring differences in case study use across dental and dental hygiene curricula.     

The relationship between early speech and later speech and language performance for children with cleft lip and palate

This study examined the relationship between speech measures at presurgery/9 months and postsurgery/13 months and speech and language performance at 21 months for children with cleft lip and palate and their noncleft peers. Comparisons were also made between the speech and lexical development of children with cleft lip and palate and noncleft children at 21 months of age. The participants included 30 children; 15 with cleft lip and palate and 15 noncleft children. Results revealed differences between the groups for several measures of speech and lexical development at 21 months. For the children with cleft palate, correlational analyses suggested that true stop production, both immediately before and after palatal surgery, was positively correlated with a majority of the speech production measures at 21 months. At postsurgery/13 months, true stop production was related to later vocabulary development, and size of true consonant inventory was related to all measure of speech production and one measure of lexical development at 21 months. For the noncleft group, true canonical babbling ratio at 13 months was the only measure that was significantly correlated with any of the speech and/or language measures at 21 months. The impact of clefting on prelinguistic and later speech and language skills is discussed.     

Caregivers' perceived comfort regarding oral care delivery in group homes: a pilot study

The purpose of this study was to investigate the perceived comfort, behaviors, and barriers reported by group home caregivers while providing oral health care to individuals with special healthcare needs (SHCN). A 23-item survey was sent to 428 caregivers in two group homes in Iowa. Bivariate and logistic regression models were used to analyze data (p ≤ 0.05). The overall response rate was 32%. An analysis of the bivariate and multivariate logistic regression indicated that caregivers who felt more comfortable providing care for individuals who verbally and physically resisted oral health care had worked more than 2 years at their current location (p = 0.0323), felt "neutral to very comfortable" brushing (p = 0.0020) and flossing (p < 0.0001) the teeth of individuals with SHCN, and reported "sometimes to always" experiencing these individuals not opening their mouths (p = 0.0127). Comfort in providing oral care to individuals with SHCN appears to be linked to experience and length of time working with this population.     

Phase 3 evaluation of HP802‐247 in the treatment of chronic venous leg ulcers

In 2012 we reported promising results from a phase 2 clinical trial of HP802‐247, a novel spray‐applied investigational treatment for chronic venous leg ulcers consisting of human, allogeneic fibroblasts and keratinocytes. We now describe phase 3 clinical testing of HP802‐247, its failure to detect efficacy, and subsequent investigation into the root causes of the failure. Two randomized, controlled trials enrolled a total of 673 adult outpatients at 96 centers in North America and Europe. The primary endpoint was the proportion of ulcers with confirmed closure at the end of 12 weeks of treatment. An investigation into the root cause for the failure of HP802‐247 to show efficacy in these two phase 3 trials was initiated immediately following the initial review of the North American trial results. Four hundred twenty‐one patients were enrolled in the North American (HP802‐247, 211; Vehicle 210) and 252 in the European (HP802‐247, 131; Vehicle 121) trials. No difference in proportion of closed ulcers at week 12 was observed between treatment groups for either the North American (HP802‐247, 61.1%; Vehicle 60.0%; p = 0.5896) or the European (HP802‐247, 47.0%; Vehicle 50.0%; p = 0.5348) trials. Thorough investigation found no likelihood that design or execution of the trials contributed to the failure. Variability over time during the trials in the clinical response implicated the quality of the cells comprising HP802‐247. Concordance between the two separate, randomized, controlled trials with distinct, nonoverlapping investigative sites and independent monitoring teams renders the possibility of a Type II error vanishingly small and provides strong credibility for the unexpected lack of efficacy observed. The most likely causative factors for the efficacy failure in phase 3 was phenotypic change in the cells (primarily keratinocytes) leading to batch to batch variability due to the age of the cell banks.     

Potential neuroprotective role of astroglial exosomes against smoking-induced oxidative stress and HIV-1 replication in the central nervous system

Introduction: HIV-1-infected smokers are at risk of oxidative damage to neuronal cells in the central nervous system by both HIV-1 and cigarette smoke. Since neurons have a weak antioxidant defense system, they mostly depend on glial cells, particularly astrocytes, for protection against oxidative damage and neurotoxicity. Astrocytes augment the neuronal antioxidant system by supplying cysteine-containing products for glutathione synthesis, antioxidant enzymes such as SOD and catalase, glucose for antioxidant regeneration via the pentose-phosphate pathway, and by recycling of ascorbic acid.

Areas covered: The transport of antioxidants and energy substrates from astrocytes to neurons could possibly occur via extracellular nanovesicles called exosomes. This review highlights the neuroprotective potential of exosomes derived from astrocytes against smoking-induced oxidative stress, HIV-1 replication, and subsequent neurotoxicity observed in HIV-1-positive smokers.

Expert opinion: During stress conditions, the antioxidants released from astrocytes either via extracellular fluid or exosomes to neurons may not be sufficient to provide neuroprotection. Therefore, we put forward a novel strategy to combat oxidative stress in the central nervous system, using synthetically developed exosomes loaded with antioxidants such as glutathione and the anti-aging protein Klotho.