A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible

Assessing pet owner and veterinarian perceptions of need for veterinary compounding services in a community pharmacy setting

Background: Pets, pet owners (referred to as clients in veterinary medicine and throughout this article), veterinarians, and community pharmacies may all benefit from veterinary compounding services provided in community pharmacies, but the benefits of this service are not well-documented in the literature. Objectives: This study identified perceived benefits and barriers and evaluated the need for veterinary compounding services in community pharmacies; it also evaluated current business practices related to veterinary compounding services. Methods: A cross-sectional survey was administered to three groups: 1) clients who filled a pet prescription at a study pharmacy, 2) clients who had not filled pet prescriptions, and 3) local veterinarians. Eligible participants were 18 or older; clients must have owned a pet in the past five years. The surveys collected demographic information and assessed benefits, barriers, need, and business practices regarding veterinary compounding services. Demographics were evaluated through descriptive statistics. Responses to Likert-scale items were compared between groups using the Mann-Whitney U test. Qualitative responses were assessed for emerging themes. Results: One hundred eighteen clients and 15 veterinarians participated in the study. Seventy-two of 116 clients (62%) and eight of 10 veterinarians (80%) agreed that clients would benefit from veterinary compounds provided in community pharmacies. Only 40% of veterinarians agreed that community pharmacists have the knowledge to compound pet medications, compared to 67% of clients (P=0.010). Similarly, 47% of veterinarians agreed that community pharmacists have the skills to compound pet medications, compared to 72% of clients (P=0.016). Forty-eight of 118 clients (41%) would travel 10 miles or more out of their way for veterinary compounding services at community pharmacies. Conclusions: This study assessed client and veterinarian perceptions of veterinary compounding service benefits, barriers, and need in community pharmacies. Clients identified more opportunities for veterinary compounding services in community pharmacies when compared to veterinarians. Both groups identified a need for veterinary compounding services and agreed community pharmacies providing these services would benefit pets and clients

Pharmacodynamics of ATI-2307 in a rabbit model of cryptococcal meningoencephalitis.

Cryptococcal meningoencephalitis (CM) is a devastating fungal disease with high morbidity and mortality. The current regimen that is standard-of-care involves a combination of three different drugs administered for up to one year. There is a critical need for new therapies due to both toxicity and inadequate fungicidal activity of the currently available antifungal drugs. ATI-2307 is a novel aryl amidine that disrupts the mitochondrial membrane potential and inhibits the respiratory chain complexes of fungi-it thus represents a new mechanism for direct antifungal action. Furthermore, ATI-2307 selectively targets fungal mitochondria via a fungal-specific transporter that is not present in mammalian cells. It has very potent in vitro anticryptococcal activity. In this study, the efficacy of ATI-2307 was tested in a rabbit model of CM. ATI-2307 demonstrated significant fungicidal activity at dosages between 1 and 2 mg/kg/d, and these results were superior to fluconazole and similar to amphotericin B treatment. When ATI-2307 was combined with fluconazole, the antifungal effect was greater than either therapy alone. While ATI-2307 has potent anticryptococcal activity in the subarachnoid space, its ability to reduce yeasts in the brain parenchyma was relatively less over the same study period. This new drug, with its unique mechanism of fungicidal action and ability to positively interact with an azole, has demonstrated sufficient anticryptococcal potential in this experimental setting to be further evaluated in clinical studies

Glycine and Folate Ameliorate Models of Congenital Sideroblastic Anemia

AbstractSideroblastic anemias are acquired or inherited anemias that result in a decreased ability tosynthesize hemoglobin in red blood cells and result in the presence of iron deposits in themitochondria of red blood cell precursors. A common subtype of congenital sideroblasticanemia is due to autosomal recessive mutations in the SLC25A38 gene. The current treatmentfor SLC25A38 congenital sideroblastic anemia is chronic blood transfusion coupledwith iron chelation. The function of SLC25A38 is not known. Here we report that theSLC25A38 protein, and its yeast homolog Hem25, are mitochondrial glycine transportersrequired for the initiation of heme synthesis. To do so, we took advantage of the fact thatmitochondrial glycine has several roles beyond the synthesis of heme, including the synthesisof folate derivatives through the glycine cleavage system. The data were consistent withHem25 not being the sole mitochondrial glycine importer, and we identify a second SLC25family member Ymc1, as a potential secondary mitochondrial glycine importer. Based onthese findings, we observed that high levels of exogenous glycine, or 5-aminolevulinic acid(5-Ala) a metabolite downstream of Hem25 in heme biosynthetic pathway, were able torestore heme levels to normal in yeast cells lacking Hem25 function. While neither glycinenor 5-Ala could ameliorate SLC25A38 congenital sideroblastic anemia in a zebrafishmodel, we determined that the addition of folate with glycine was able to restore hemoglobinlevels. This difference is likely due to the fact that yeast can synthesize folate, whereas inzebrafish folate is an essential vitamin that must be obtained exogenously. Given the tolerabilityof glycine and folate in humans, this study points to a potential novel treatment forSLC25A38 congenital sideroblastic anemia