Expression of three P4-phospholipid flippases-atp11a, atp11b, and atp11c in zebrafish (Danio rerio).

Cellular membrane asymmetry is a hallmark characteristic of all eukaryotic cells. The balance of phospholipid composition within the cytoplasmic inner leaflet and the extracellular outer leaflet of the plasma membrane (PM) maintains cellular function and vitality. The proper exposure of particular phospholipids is necessary to maintain cellular signalling, controlled apoptosis, and vesicle transportation among other roles. Phospholipid asymmetry is coordinated by P4-type phospholipid transferases (flippases or ATPases). ATP11A, ATP11B, and ATP11C belong to class VI of the P4-flippase family (vertebrates) and are responsible for the movement of phosphatidylserine (PS) from the outer leaflet to the inner leaflet of the PM. To date, there is a lack of knowledge of the tissue specific expression of these three flippases on a whole-organism level in a vertebrate system. Here we have determined the spatial-temporal expression profiles of each gene in a zebrafish model using in situ hybridization and performed comparative phylogenetic analyses with other vertebrates. Our data reveals sequence similarity between vertebrate flippases and specific synteny of zebrafish and human chromosomes. Both atp11b and atp11c are maternally expressed in zebrafish, while zygotic expression analysis demonstrates tissue and temporal specificity for all three genes. atp11a is expressed in the neural crest cells as well as in the developing eye and ear, while atp11b is expressed early in the ventricular epithelial lining and later in the ear. atp11c is expressed in the anterior most rhombomeres of the hindbrain, pharyngeal arches, and liver. Our expression data suggests that each of the three flippases are integral for the development of specific tissues, and aberrant function of either could lead to visual, hearing, neural, or liver dysfunction.

Loss of foxc1 in zebrafish reduces optic nerve size and cell number in the retinal ganglion cell layer.

Mutation of FOXC1 causes Axenfeld-Rieger Syndrome (ARS) with early onset or congenital glaucoma. We assessed retinal ganglion cell (RGC) number in zebrafish due to CRISPR-mediated mutation and antisense inhibition of two-forkhead box transcription factors, foxc1a and foxc1b. These genes represent duplicated homologues of human FOXC1. Using a CRISPR induced null mutation in foxc1b, in combination with antisense inhibition of foxc1a, we demonstrate reduced cell number in the retinal ganglion cell layer of developing zebrafish eyes. As early as 5 days post fertilization (dpf), fewer RGCs are found in foxc1b homozygous mutants injected with foxc1a morpholinos, and a thinner optic nerve results. Our data illustrates that foxc1 is required for the expression of atonal homolog 7 (atoh7), a gene that is necessary for RGC differentiation. As markers of differentiated RGCs (pou4f2) are downregulated in foxc1b-/- mutants injected with foxc1a morpholinos and no cell death is observed, our results are consistent with defects in the differentiation of RGCs leading to reduced cell number, as opposed to increased cell death of RGCs or off targets effects of morpholino injection. Our zebrafish model demonstrates that aberrant regulation of RGC number could act in concert with other known glaucoma risk factors to influence the development of congenital and early onset glaucoma due to FOXC1 mutation.

The Performance of Direct Disk Diffusion for Community Acquired Bacteremia due to Gram-Negative Bacilli and Its Impact on Physician Treatment Decisions.

Background. Direct disk diffusion susceptibility testing provides faster results than standard microtitre susceptibility. The direct result may impact patient outcome in sepsis if it is accurate and if physicians use the information to promptly and appropriately change antibiotic treatment. Objective. To compare the performance of direct disk diffusion with standard susceptibility and to consider physician decisions in response to these early results, for community acquired bacteremia with Gram-negative Bacilli. Methods. Retrospective observational study of all positive blood cultures with Gram-negative Bacilli, collected over one year. Physician antibiotic treatment decisions were assessed by an infectious diseases physician based on information available to the physician at the time of the decision. Results. 89 bottles growing Gram-negative Bacilli were included in the analysis. Direct disk diffusion agreement with standard susceptibility varied widely. In 47 cases (52.8%), the physician should have changed to a narrower spectrum but did not, in 18 cases (20.2%), the physician correctly narrowed from appropriate broad coverage, and in 8 cases (9.0%), the empiric therapy was correct. Discussion. Because inoculum is not standardized, direct susceptibility results do not agree with standard susceptibility results for all drugs. Physicians do not act on direct susceptibility results. Conclusion. Direct susceptibility should be discontinued in clinical microbiology laboratories.