Use of a Hybrid Adeno-Associated Viral Vector Transposon System to Deliver the Insulin Gene to Diabetic NOD Mice.

Previously, we used a lentiviral vector to deliver furin-cleavable human insulin (INS-FUR) to the livers in several animal models of diabetes using intervallic infusion in full flow occlusion (FFO), with resultant reversal of diabetes, restoration of glucose tolerance and pancreatic transdifferentiation (PT), due to the expression of beta (β)-cell transcription factors (β-TFs). The present study aimed to determine whether we could similarly reverse diabetes in the non-obese diabetic (NOD) mouse using an adeno-associated viral vector (AAV) to deliver INS-FUR ± the β-TF Pdx1 to the livers of diabetic mice. The traditional AAV8, which provides episomal expression, and the hybrid AAV8/piggyBac that results in transgene integration were used. Diabetic mice that received AAV8-INS-FUR became hypoglycaemic with abnormal intraperitoneal glucose tolerance tests (IPGTTs). Expression of β-TFs was not detected in the livers. Reversal of diabetes was not achieved in mice that received AAV8-INS-FUR and AAV8-Pdx1 and IPGTTs were abnormal. Normoglycaemia and glucose tolerance were achieved in mice that received AAV8/piggyBac-INS-FUR/FFO. Definitive evidence of PT was not observed. This is the first in vivo study using the hybrid AAV8/piggyBac system to treat Type 1 diabetes (T1D). However, further development is required before the system can be used for gene therapy of T1D

Primary Extracranial Meningiomas: An Analysis of 146 Cases

Primary extracranial meningiomas are rare neoplasms, frequently misdiagnosed, resulting in inappropriate clinical management. To date, a large clinicopathologic study has not been reported. One hundred and forty-six cases diagnosed between 1970 and 1999 were retrieved from the files of the Armed Forces Institute of Pathology. Histologic features were reviewed, immunohistochemistry analysis was performed (n = 85), and patient follow-up was obtained (n = 110). The patients included 74 (50.7%) females and 72 (49.3%) males. Tumors of the skin were much more common in males than females (1.7:1). There was an overall mean age at presentation of 42.4 years, with a range of 0.3–88 years. The overall mean age at presentation was significantly younger for skin primaries (36.2 years) than for ear (50.1 years) and nasal cavity (47.1 years) primaries. Symptoms were in general non-specific and reflected the anatomic site of involvement, affecting the following areas in order of frequency: scalp skin (40.4%), ear and temporal bone (26%), and sinonasal tract (24%). The tumors ranged in size from 0.5 up to 8 cm, with a mean size of 2.3 cm. Histologically, the majority of tumors were meningothelial (77.4%), followed by atypical (7.5%), psammomatous (4.1%) and anaplastic (2.7%). Psammoma bodies were present in 45 tumors (30.8%), and bone invasion in 31 (21.2%) of tumors. The vast majority were WHO Grade I tumors (87.7%), followed by Grade II (9.6%) and Grade III (2.7%) tumors. Immunohistochemically, the tumor cells labeled for EMA (76%; 61/80), S-100 protein (19%; 15/78), CK 7 (22%; 12/55), and while there was ki-67 labeling in 27% (21/78), <3% of cells were positive. The differential diagnosis included a number of mesenchymal and epithelial tumors (paraganglioma, schwannoma, carcinoma, melanoma, neuroendocrine adenoma of the middle ear), depending on the anatomic site of involvement. Treatment and follow-up was available in 110 patients: Biopsy, local excision, or wide excision was employed. Follow-up time ranged from 1 month to 32 years, with an average of 14.5 years. Recurrences were noted in 26 (23.6%) patients, who were further managed by additional surgery. At last follow-up, recurrent disease was persistent in 15 patients (mean, 7.7 years): 13 patients were dead (died with disease) and two were alive; the remaining patients were disease free (alive 60, mean 19.0 years, dead 35, mean 9.6 years). There is no statistically significant difference in 5-year survival rates by site: ear and temporal bone: 83.3%; nasal cavity: 81.8%; scalp skin: 78.5%; other sites: 65.5% (P = 0.155). Meningiomas can present in a wide variety of sites, especially within the head and neck region. They behave as slow-growing neoplasms with a good prognosis, with longest survival associated with younger age, and complete resection. Awareness of this diagnosis in an unexpected location will help to avoid potential difficulties associated with the diagnosis and management of these tumors

Physical Analyses of E. coli Heteroduplex Recombination Products In Vivo: On the Prevalence of 5′ and 3′ Patches

BACKGROUND: Homologous recombination in Escherichia coli creates patches (non-crossovers) or splices (half crossovers), each of which may have associated heteroduplex DNA. Heteroduplex patches have recombinant DNA in one strand of the duplex, with parental flanking markers. Which DNA strand is exchanged in heteroduplex patches reflects the molecular mechanism of recombination. Several models for the mechanism of E. coli RecBCD-mediated recombinational double-strand-end (DSE) repair specify that only the 3'-ending strand invades the homologous DNA, forming heteroduplex in that strand. There is, however, in vivo evidence that patches are found in both strands. METHODOLOGY/PRINCIPLE FINDINGS: This paper re-examines heteroduplex-patch-strand polarity using phage lambda and the lambdadv plasmid as DNA substrates recombined via the E. coli RecBCD system in vivo. These DNAs are mutant for lambda recombination functions, including orf and rap, which were functional in previous studies. Heteroduplexes are isolated, separated on polyacrylamide gels, and quantified using Southern blots for heteroduplex analysis. This method reveals that heteroduplexes are still found in either 5' or 3' DNA strands in approximately equal amounts, even in the absence of orf and rap. Also observed is an independence of the RuvC Holliday-junction endonuclease on patch formation, and a slight but statistically significant alteration of patch polarity by recD mutation. CONCLUSIONS/SIGNIFICANCE: These results indicate that orf and rap did not contribute to the presence of patches, and imply that patches occurring in both DNA strands reflects the molecular mechanism of recombination in E. coli. Most importantly, the lack of a requirement for RuvC implies that endonucleolytic resolution of Holliday junctions is not necessary for heteroduplex-patch formation, contrary to predictions of all of the major previous models. This implies that patches are not an alternative resolution of the same intermediate that produces splices, and do not bear on models for splice formation. We consider two mechanisms that use DNA replication instead of endonucleolytic resolution for formation of heteroduplex patches in either DNA strand: synthesis-dependent-strand annealing and a strand-assimilation mechanism

Comparative Genomic Analysis of Drosophila melanogaster and Vector Mosquito Developmental Genes

Genome sequencing projects have presented the opportunity for analysis of developmental genes in three vector mosquito species: Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. A comparative genomic analysis of developmental genes in Drosophila melanogaster and these three important vectors of human disease was performed in this investigation. While the study was comprehensive, special emphasis centered on genes that 1) are components of developmental signaling pathways, 2) regulate fundamental developmental processes, 3) are critical for the development of tissues of vector importance, 4) function in developmental processes known to have diverged within insects, and 5) encode microRNAs (miRNAs) that regulate developmental transcripts in Drosophila. While most fruit fly developmental genes are conserved in the three vector mosquito species, several genes known to be critical for Drosophila development were not identified in one or more mosquito genomes. In other cases, mosquito lineage-specific gene gains with respect to D. melanogaster were noted. Sequence analyses also revealed that numerous repetitive sequences are a common structural feature of Drosophila and mosquito developmental genes. Finally, analysis of predicted miRNA binding sites in fruit fly and mosquito developmental genes suggests that the repertoire of developmental genes targeted by miRNAs is species-specific. The results of this study provide insight into the evolution of developmental genes and processes in dipterans and other arthropods, serve as a resource for those pursuing analysis of mosquito development, and will promote the design and refinement of functional analysis experiments

Frequent Loss of Heterozygosity Targeting the Inactive X Chromosome in Melanoma

After previous preliminary observations of paradoxical deletion events affecting the inactive X chromosome in melanoma, we have surveyed the X chromosome for deletions using 23 polymorphic microsatellite markers in 28 informative (female XX) metastatic melanomas. Ten tumors (36%) showed at least one loss of heterozygosity (LOH) event, and in two cases an entire chromosome showed LOH at all informative loci. Four distinct X chromosome smallest regions of overlap can be resolved. An 18.6-Mb region on the p arm involving 9 of 28 (32%) samples lies between the markers DXS1061 and DXS1068. An equally frequently deleted smallest region of overlap straddled the centromere, bounded by DX1204 on the p arm and DXS983 14.6 Mb away in Xq11-12. One tumor potentially defines this region more tightly to a 10.6-Mb smallest region of overlap bounded by DXS1190 and DXS981 that contains the androgen receptor (AR) gene. A 6.2-Mb deleted region can be defined between the markers DXS8051 and DXS9902 in 8 of 28 (28%) tumors. An additional, less frequently deleted region of 25.7 Mb was found on distal Xq between the markers DXS1212 and DXS1193 in 5 of 28 (18%) tumors. X inactivation analysis of five tumors with LOH, using the AR exon I CAG repeat, showed that in each case, the inactive, hypermethylated allele was the one deleted. Analysis of copy number in this region by quantitative PCR showed restoration to disomy and, in one case, trisomy at AR