Restoration of tumor suppression in prostate cancer by targeting the E3 ligase E6AP.

Restoration of tumor suppression is an attractive onco-therapeutic approach. It is particularly relevant when a tumor suppressor is excessively degraded by an overactive oncogenic E3 ligase. We previously discovered that the E6-associated protein (E6AP; as classified in the human papilloma virus context) is an E3 ligase that has an important role in the cellular stress response, and it directly targets the tumor-suppressor promyelocytic leukemia protein (PML) for proteasomal degradation. In this study, we have examined the role of the E6AP-PML axis in prostate cancer (PC). We show that knockdown (KD) of E6AP expression attenuates growth of PC cell lines in vitro. We validated this finding in vivo using cell line xenografts, patient-derived xenografts and mouse genetics. We found that KD of E6AP attenuates cancer cell growth by promoting cellular senescence in vivo, which correlates with restoration of tumor suppression by PML. In addition, we show that KD of E6AP sensitizes cells to radiation-induced death. Overall, our findings demonstrate a role for E6AP in the promotion of PC and support E6AP targeting as a novel approach for PC treatment, either alone or in combination with radiation.

Disorders of sexual development in poultry.

Sex in birds is determined genetically, as in mammals. However, in birds, female is the heterogametic sex (ZW), while the male is homogametic (ZZ). Although the exact mechanism of avian sex determination is still unclear, genes on one or both of the sex chromosomes must control sexual differentiation of the embryonic gonads into testes or ovaries, and eventually all other sexually dimorphic features. In this review of disorders of sexual development in poultry, we focus upon the gonads and external dimorphisms. Abnormalities of sexual development in poultry can be broadly divided into 2 types: those due to disturbances in sex hormone production by the gonads, and those due to abnormal inheritance of sex chromosomes. Recent studies on gynandromorphic chickens (half male, half female) point to the importance of genetic over hormonal factors in controlling sexual development in fowl.

Complete genome sequence of Nariva virus, a rodent paramyxovirus.

Nariva virus (NarPV) was isolated from forest rodents (Zygodontomys b. brevicauda) in eastern Trinidad in the early 1960s. Initial classification within the family Paramyxoviridae was based mainly on morphological observations including the structure of nucleocapsids and virion surface projections. Here, we report the characterization of the complete genome sequence of NarPV. The genome is 15,276 nucleotides in length, conforming to the rule-of-six, and has a genome organization typical of most members of the family, with six transcriptional units in the order 3'-N-P-M-F-H-L-5'. The gene junctions contain highly conserved gene start and stop signals and a tri-nucleotide intergenic sequence present in most members of the subfamily Paramyxovirinae. Sequence comparison studies indicate that NarPV is most closely related to Mossman virus, which was isolated from wild rats (Rattus leucopus) in Queensland, Australia, in 1970. This study confirmed the classification of NarPV as a member of the subfamily Paramyxovirinae and established the close genome organization and sequence relationship between the two rodent paramyxoviruses isolated almost a decade apart and from two locations separated by more than 15,000 km.

Manipulation of small RNAs to modify the chicken transcriptome and enhance productivity traits.

In recent years there has been a revolution in our understanding of genes and how they come to control the physical outcomes of development. Central to this has been the understanding of the cellular processes of RNA interference (RNAi), for which the Nobel Prize for Physiology or Medicine was awarded in 2006. Coupled with this has been the recognition that microRNAs are key mediators of this process within cells. RNAi whether mediated exogenously by synthetic oligonucleotides or vector-delivered double stranded RNA or endogenously by microRNAs can have a profound and specific effect on gene expression. Elucidating and understanding these processes in the chicken will provide critical information to enable more precise control over breeding strategies for improvement of traits in production poultry, either by direct or indirect means. It will also provide alternative strategies for the control and prevention of important avian diseases.

Comparison of bovine RNA polymerase III promoters for short hairpin RNA expression.

RNA interference (RNAi) mediated by DNA-based expression of short hairpin RNA (shRNA) is a powerful method of sequence-specific gene knockdown. A number of vectors for expression of shRNA have been developed that feature promoters from RNA polymerase III (pol III)-transcribed genes of mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and for future development of specific therapeutics in the bovine species, we have developed shRNA expression vectors that feature novel bovine RNA pol III promoters. We characterized two bovine U6 small nuclear RNA (snRNA) promoters (bU6-2 and bU6-3) and a bovine 7SK snRNA promoter (b7SK). We compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and suppressing the reporter gene encoding enhanced green fluorescent protein. Results show that the b7SK promoter induced the greatest level of suppression in a range of cell lines. The comparison of these bovine promoters in shRNA expression is an important component for the future development of bovine-specific RNAi-based research.

Group Comparative Study of VRB-2 Agar in the Recovery of Coliforms from Raw Milk, Ice Cream and Cottage Cheese.

The method of Hartman et al. for recovering stressed coliform bacteria was evaluated in a group study. The experimental medium, VRB-2, was more productive than VRB medium by 31% for raw milk, 70% for ice cream and 61% for cottage cheese. Differences in colony counts of coliforms caused by incubating for 48 vs. 24 h and by boiling vs. autoclaving were not statistically significant. Among 40 samples tested in two laboratories, counts obtained at 30 vs. 32 C were not significantly different.

Transmission of rotavirus gastroenteritis from children to a monkey.

A pooled suspension of rotavirus was prepared from the stools of eight children with acute non-bacterial gastroenteritis. The suspension was infused into the duodenum and stomach of an infant monkey (Nemestrina macaque). Biopsy samples of duodenal mucosa were taken at several intervals after inoculation, examined by light and electron microscopy, and assayed for lysosomal activity. Virus-like particles were seen within and around microvilli and intracellularly within vesicles as early as 20 minutes after the infusion. On the fourth and fifth days, large lysosomal bodies containing numerous virus-like particles were found within epithelial cells of the duodenal villi. No such particles were seen in the pre-inoculation sample or at days 16 or 25 after infection. The present study would appear to be the first demonstration of the transmission of this human virus to another species.

Electron-microscopic study of monotreme neuroglia.

Glia cells were examined in the brains of a mature platypus and an immature echidna. In the echidna a few dark, organelle-rich glia cells were encountered. The lighter glia cells had resemblances with the single type of glia cell encountered in the brain of the platypus. These cells were characterised by highly homogeneous areas of nuclear chromatin and light cytoplasm containing dark, finely granular condensations which frequently surrounded Golgi membranes. Microtubules were present within the cytoplasm but neither filaments nor glycogen-like particles were encountered. It was concluded that the cells described conformed to the types of neither astrocytes nor oligodendrocytes as encountered in metatherian and eutherian mammals. Among their functional capacities such cells presumably include, either in the immature or mature forms, the roles of both astrocytes and oligodendrocytes.