Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.     

Mycobacteriosis in zebrafish (Danio rerio) research facilities

The Zebrafish International Resource Center was established to support the zebrafish research community, and includes a diagnostic service. One of the most common diseases that we have diagnosed is mycobacteriosis, which represented 18% of the diagnostic cases submitted from November 1999 to June 2003. We describe here the severity of the disease and associated pathological changes of 24 diagnostic cases from 14 laboratories. Identifications of the bacteria are provided for seven of these cases. For two cases in which culture of the organism was not successful, these identifications were based on ribosomal DNA (rDNA) sequence analysis obtained directly from infected tissues. Biochemical characteristics and rDNA sequence analysis from cultures are reported for the other isolates. Two severe outbreaks from different facilities on different continents were associated with an organism identified as Mycobacterium haemophilum based on rDNA sequence from tissues. Another severe outbreak was associated with an organism most closely related to Mycobacterium peregrinum. These species are recognized pathogens of humans, but this is the first report of them from fish. Bacteria identified as Mycobacterium chelonae or M. abscessus were recovered from fish in cases categorized as moderate disease or as an incidental finding. These findings indicate that species of Mycobacterium previously undescribed from fish (i.e., M. haemophilum and M. peregrinum) may pose significant health problems in zebrafish research facilities, whereas species and strains that are already recognized as common in fish usually cause limited disease on a population basis in zebrafish.     

Alterations in metabolism and gene expression in brain regions during cuprizone-induced demyelination and remyelination

Exposure of mice to the copper chelator, cuprizone, results in CNS demyelination. There is remyelination after removal of the metabolic insult. We present brain regional studies identifying corpus callosum as particularly severely affected; 65% of cerebroside is lost after 6 weeks of exposure. We examined recovery of cerebroside and ability to synthesize cerebroside and cholesterol following removal of the toxicant. The temporal pattern for concentration of myelin basic protein resembled that of cerebroside. We applied Affymetrix GeneChip technology to corpus callosum to identify temporal changes in levels of mRNAs during demyelination and remyelination. Genes coding for myelin structural components were greatly down-regulated during demyelination and up-regulated during remyelination. Genes related to microglia/macrophages appeared in a time-course (peaking at 6 weeks) correlating with phagocytosis of myelin and repair of lesions. mRNAs coding for many cytokines had peak expression at 4 weeks, compatible with intercellular signaling roles. Of interest were other genes with temporal patterns correlating with one of the three above patterns, but of function not obviously related to demyelination/remyelination. The ability to correlate gene expression with known pathophysiological events should help in elucidating further function of such genes as related to demyelination/remyelination.     

MicroRNAs in the miR-106b family regulate p21/CDKN1A and promote cell cycle progression

microRNAs in the miR-106b family are overexpressed in multiple tumor types and are correlated with the expression of genes that regulate the cell cycle. Consistent with these observations, miR-106b family gain of function promotes cell cycle progression, whereas loss of function reverses this phenotype. Microarray profiling uncovers multiple targets of the family, including the cyclin-dependent kinase inhibitor p21/CDKN1A. We show that p21 is a direct target of miR-106b and that its silencing plays a key role in miR-106b-induced cell cycle phenotypes. We also show that miR-106b overrides a doxorubicin-induced DNA damage checkpoint. Thus, miR-106b family members contribute to tumor cell proliferation in part by regulating cell cycle progression and by modulating checkpoint functions.     

Alterations in Gene Expression Associated with Primary Demyelination and Remyelination in the Peripheral Nervous System

Primary demyelination is an important component of a number of human diseases and toxic neuropathies. Animal models of primary demyelination are useful for isolating processes involved in myelin breakdown and remyelination because the complicating events associated with axonal degeneration and regeneration are not present. The tellurium neuropathy model has proven especially useful in this respect. Tellurium specifically blocks synthesis of cholesterol, a major component of PNS myelin. The resulting cholesterol deficit in myelin-producing Schwann cells rapidly leads to synchronous primary demyelination of the sciatic nerve, which is followed by rapid synchronous remyelination when tellurium exposure is discontinued. Known alterations in gene expression for myelin proteins and for other proteins involved in the sequence of events associated with demyelination and subsequent remyelination in the PNS are reviewed, and new data regarding gene expression changes during tellurium neuropathy are presented and discussed.     

Effect of Selenium Yeast Supplementation on Naturally Acquired Parasitic Infection in Ewes

Gastrointestinal parasites cause substantial economic losses in pasture-based sheep production systems. Supranutritional organic selenium (Se) supplementation may be beneficial because it improves immune responses to pathogens. To evaluate the effect of Se-yeast supplementation on gastrointestinal parasite load, 30 ewes per treatment group were drenched weekly with no Se, 4.9 mg Se/week as Se yeast (maximum FDA-allowed concentration), or supranutritional concentrations of Se yeast (14.7 and 24.5 mg Se/week) starting early fall for 85 weeks. Fecal samples were collected at weeks 63, 66, 78, and 84 and counted for total trichostrongyle-type eggs and Haemonchus contortus eggs (in samples with ≥200 trichostrongyle eggs/g feces). During breeding season (fall), ewes were kept on pasture; ewes receiving 24.5 mg Se/week had lower fecal trichostrongyle egg counts (93?±?40 eggs/g feces) compared with ewes receiving no Se (537?±?257 eggs/g feces; P?=?0.007) or ewes receiving 4.9 mg Se/week as Se yeast (398?±?208 eggs/g feces; P?=?0.03). In winter, fecal trichostrongyle egg counts decreased, and group differences were not apparent. During lambing season (spring), ewes were kept in the barn and fecal trichostrongyle egg counts increased, although no group differences were observed. However, none of the ewes receiving supranutritional Se yeast, and with trichostrongyle egg counts ≥200 eggs/g of feces, but four of the ewes receiving lower Se dosages had H. contortus egg counts ≥1,000 eggs/g feces (P?=?0.04). Our results suggest that supranutritional Se-yeast supplementation may enhance resistance to naturally occurring H. contortus gastrointestinal parasitism in sheep.     

Shifts in Geographic Distribution and Antimicrobial Resistance during a Prolonged Typhoid Fever Outbreak — Bundibugyo and Kasese Districts,Uganda, 2009–2011

Background

Salmonella enterica serovar Typhi is transmitted by fecally contaminated food and water and causes approximately 22 million typhoid fever infections worldwide each year. Most cases occur in developing countries, where approximately 4% of patients develop intestinal perforation (IP). In Kasese District, Uganda, a typhoid fever outbreak notable for a high IP rate began in 2008. We report that this outbreak continued through 2011, when it spread to the neighboring district of Bundibugyo.

Methodology/Principal Findings

A suspected typhoid fever case was defined as IP or symptoms of fever, abdominal pain, and ≥1 of the following: gastrointestinal disruptions, body weakness, joint pain, headache, clinically suspected IP, or non-responsiveness to antimalarial medications. Cases were identified retrospectively via medical record reviews and prospectively through laboratory-enhanced case finding. Among Kasese residents, 709 cases were identified from August 1, 2009–December 31, 2011; of these, 149 were identified during the prospective period beginning November 1, 2011. Among Bundibugyo residents, 333 cases were identified from January 1–December 31, 2011, including 128 cases identified during the prospective period beginning October 28, 2011. IP was reported for 507 (82%) and 59 (20%) of Kasese and Bundibugyo cases, respectively. Blood and stool cultures performed for 154 patients during the prospective period yielded isolates from 24 (16%) patients. Three pulsed-field gel electrophoresis pattern combinations, including one observed in a Kasese isolate in 2009, were shared among Kasese and Bundibugyo isolates. Antimicrobial susceptibility was assessed for 18 isolates; among these 15 (83%) were multidrug-resistant (MDR), compared to 5% of 2009 isolates.

Conclusions/Significance

Molecular and epidemiological evidence suggest that during a prolonged outbreak, typhoid spread from Kasese to Bundibugyo. MDR strains became prevalent. Lasting interventions, such as typhoid vaccination and improvements in drinking water infrastructure, should be considered to minimize the risk of prolonged outbreaks in the future.     

A Distinct Smoothened Mutation Causes Severe Cerebellar Developmental Defects and Medulloblastoma in a Novel Transgenic Mouse Model

Deregulated developmental processes in the cerebellum cause medulloblastoma, the most common pediatric brain malignancy. About 25 to 30% of cases are caused by mutations increasing the activity of the Sonic hedgehog (Shh) pathway, a critical mitogen in cerebellar development. The proto-oncogene Smoothened (Smo) is a key transducer of the Shh pathway. Activating mutations in Smo that lead to constitutive activity of the Shh pathway have been identified in human medulloblastoma. To understand the developmental and oncogenic effects of two closely positioned point mutations in Smo, we characterized NeuroD2-SmoA2 mice and compared them to NeuroD2-SmoA1 mice. While both SmoA1 and SmoA2 transgenes cause medulloblastoma with similar frequencies and timing, SmoA2 mice have severe aberrations in cerebellar development, whereas SmoA1 mice are largely normal during development. Intriguingly, neurologic function, as measured by specific tests, is normal in the SmoA2 mice despite extensive cerebellar dysplasia. We demonstrate how two nearly contiguous point mutations in the same domain of the encoded Smo protein can produce striking phenotypic differences in cerebellar development and organization in mice.