Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose

A Cross-Species Analysis of MicroRNAs in the Developing Avian Face

Higher vertebrates use similar genetic tools to derive very different facial features. This diversity is believed to occur through temporal, spatial and species-specific changes in gene expression within cranial neural crest (NC) cells. These contribute to the facial skeleton and contain species-specific information that drives morphological variation. A few signaling molecules and transcription factors are known to play important roles in these processes, but little is known regarding the role of micro-RNAs (miRNAs). We have identified and compared all miRNAs expressed in cranial NC cells from three avian species (chicken, duck, and quail) before and after species-specific facial distinctions occur. We identified 170 differentially expressed miRNAs. These include thirty-five novel chicken orthologs of previously described miRNAs, and six avian-specific miRNAs. Five of these avian-specific miRNAs are conserved over 120 million years of avian evolution, from ratites to galliforms, and their predicted target mRNAs include many components of Wnt signaling. Previous work indicates that mRNA gene expression in NC cells is relatively static during stages when the beak acquires species-specific morphologies. However, miRNA expression is remarkably dynamic within this timeframe, suggesting that the timing of specific developmental transitions is altered in birds with different beak shapes. We evaluated one miRNA:mRNA target pair and found that the cell cycle regulator p27KIP1 is a likely target of miR-222 in frontonasal NC cells, and that the timing of this interaction correlates with the onset of phenotypic variation. Our comparative genomic approach is the first comprehensive analysis of miRNAs in the developing facial primordial, and in species-specific facial development

In vitro processing and secretion of mutant insulin proteins that cause permanent neonatal diabetes

Permanent neonatal diabetes mellitus is a rare form of insulin-requiring diabetes presenting within the first few weeks or months of life. Mutations in the insulin gene are the second most common cause of this form of diabetes. These mutations are located in critical regions of preproinsulin and are likely to prevent normal processing or folding of the preproinsulin/proinsulin molecule. To characterize these mutations, we transiently expressed proinsulin-GFP fusion proteins in MIN6 mouse insulinoma cells. Our study revealed three groups of mutant proteins: 1) mutations that result in retention of proinsulin in the endoplasmic reticulum (ER) and attenuation of secretion of cotransfected wild-type insulin: C43G, F48C, and C96Y; 2) mutations with partial ER retention, partial recruitment to granules, and attenuation of secretion of wild-type insulin: G32R, G32S, G47V, G90C, and Y108C; and 3) similar to (2) but with no significant attenuation of wild-type insulin secretion: A24D and R89C. The mutant insulin proteins do not prevent targeting of wild-type insulin to secretory granules, but most appear to lead to decreased secretion of wild-type insulin. Each of the mutants triggers the expression of the proapoptotic gene Chop, indicating the presence of ER stress

Rounding up performance measures in German firms: Earnings cosmetics or earnings management on a larger scale?

We use Benford's Law to provide evidence that German firms round up both their net income and earnings per share. We use the introduction of the euro to show that round earnings numbers are likely the result of earnings management. The incentive to round up comes from stakeholders’ left‐digit bias when processing the information in financial statements. Since round numbers are natural benchmarks, stakeholders perceive the performance metrics directly below such thresholds as abnormally lower. However, rounding up is objectionable only if it involves large‐scale earnings management, but not in cases of negligible ‘earnings cosmetics’. Because the difference between the pre‐managed and reported earnings is unobservable, we investigate whether the prevalence of rounding up coincides with specific levels of several earnings characteristics and proxies for audit quality. If the rounding up is cosmetic, then it should occur independently of these characteristics. In contrast, if firms use earnings management on a larger scale, then it might not be possible to simultaneously round up and achieve other objectives of earnings management. Our evidence is in line with substantial earnings management