Pulmonary availability of isotretinoin in rats after inhalation of a powder aerosol

Repeated oral administration of chemopreventive retinoids such as isotretinoin over extended periods of time is associated with intolerable systemic toxicity. Here isotretinoin was formulated as a powder aerosol, and its delivery to the lungs of rats was studied with the aim to explore the possibility of minimizing adverse effects associated with its oral administration. Rats received isotretinoin orally (0.5, 1 or 10 mg kg–1) or by inhalation (theoretical dose ~1 or ~10 mg kg–1) in a nose-only inhalation chamber. Isotretinoin was quantitated by high-pressure liquid chromatography in plasma and lung tissue. The ratios of mean area of concentration-vs-time curve (AUC) values in the lungs over mean AUCs in the plasma for isotretinoin following single or repeated aerosol exposure surpassed those determined for the oral route by factors of between two (single low-dose) and five (single high-dose). Similarly, the equivalent ratios for the maximal peak concentrations in lungs and plasma obtained after aerosol exposure consistently exceeded those seen after oral administration, suggesting that lungs were exposed to higher isotretinoin concentrations after aerosol inhalation than after oral administration of similar doses. Repeated high doses of isotretinoin by inhalation resulted in moderate loss of body weight, but microscopic investigation of ten tissues including lung and oesophagus did not detect any significant aerosol-induced damage. The results suggest that administration of isotretinoin via powder aerosol inhalation is probably superior to its application via the oral route in terms of achieving efficacious drug concentrations in the lungs. © 2000 Cancer Research Campaig

Urinary MicroRNA Profiling in the Nephropathy of Type 1 Diabetes

Background: Patients with Type 1 Diabetes (T1D) are particularly vulnerable to development of Diabetic nephropathy (DN) leading to End Stage Renal Disease. Hence a better understanding of the factors affecting kidney disease progression in T1D is urgently needed. In recent years microRNAs have emerged as important post-transcriptional regulators of gene expression in many different health conditions. We hypothesized that urinary microRNA profile of patients will differ in the different stages of diabetic renal disease. Methods and Findings: We studied urine microRNA profiles with qPCR in 40 T1D with >20 year follow up 10 who never developed renal disease (N) matched against 10 patients who went on to develop overt nephropathy (DN), 10 patients with intermittent microalbuminuria (IMA) matched against 10 patients with persistent (PMA) microalbuminuria. A Bayesian procedure was used to normalize and convert raw signals to expression ratios. We applied formal statistical techniques to translate fold changes to profiles of microRNA targets which were then used to make inferences about biological pathways in the Gene Ontology and REACTOME structured vocabularies. A total of 27 microRNAs were found to be present at significantly different levels in different stages of untreated nephropathy. These microRNAs mapped to overlapping pathways pertaining to growth factor signaling and renal fibrosis known to be targeted in diabetic kidney disease. Conclusions: Urinary microRNA profiles differ across the different stages of diabetic nephropathy. Previous work using experimental, clinical chemistry or biopsy samples has demonstrated differential expression of many of these microRNAs in a variety of chronic renal conditions and diabetes. Combining expression ratios of microRNAs with formal inferences about their predicted mRNA targets and associated biological pathways may yield useful markers for early diagnosis and risk stratification of DN in T1D by inferring the alteration of renal molecular processes. © 2013 Argyropoulos et al

miR-122 activates hepatitis C virus translation by a specialized mechanism requiring particular RNA components

In animals, microRNAs (miRNAs) generally repress gene expression by binding to sites in the 3′-untranslated region (UTR) of target mRNAs. miRNAs have also been reported to repress or activate gene expression by binding to 5′-UTR sites, but the extent of such regulation and the factors that govern these different responses are unknown. Liver-specific miR-122 binds to sites in the 5′-UTR of hepatitis C virus (HCV) RNA and positively regulates the viral life cycle, in part by stimulating HCV translation. Here, we characterize the features that allow miR-122 to activate translation via the HCV 5′-UTR. We find that this regulation is a highly specialized process that requires uncapped RNA, the HCV internal ribosome entry site (IRES) and the 3′ region of miR-122. Translation activation does not involve a previously proposed structural transition in the HCV IRES and is mediated by Argonaute proteins. This study provides an important insight into the requirements for the miR-122–HCV interaction, and the broader consequences of miRNAs binding to 5′-UTR sites

RDR2 Partially Antagonizes the Production of RDR6-Dependent siRNA in Sense Transgene-Mediated PTGS

Background: RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and SUPPRESSOR of GENE SILENCING 3 (SGS3) are required for DNA methylation and post-transcriptional gene silencing (PTGS) mediated by 21-nt siRNAs produced by sense transgenes (S-PTGS). In contrast, RDR2, but not RDR6, is required for DNA methylation and TGS mediated by 24-nt siRNAs, and for cellto-cell spreading of IR-PTGS mediated by 21-nt siRNAs produced by inverted repeat transgenes under the control of a phloem-specific promoter. Principal Findings: In this study, we examined the role of RDR2 and RDR6 in S-PTGS. Unlike RDR6, RDR2 is not required for DNA methylation of transgenes subjected to S-PTGS. RDR6 is essential for the production of siRNAs by transgenes subjected to S-PTGS, but RDR2 also contributes to the production of transgene siRNAs when RDR6 is present because rdr2 mutations reduce transgene siRNA accumulation. However, the siRNAs produced via RDR2 likely are counteractive in wildtype plants because impairement of RDR2 increases S-PTGS efficiency at a transgenic locus that triggers limited silencing, and accelerates S-PTGS at a transgenic locus that triggers efficient silencing. Conclusions/Significance: These results suggest that RDR2 and RDR6 compete for RNA substrates produced by transgenes subjected to S-PTGS. RDR2 partially antagonizes RDR6 because RDR2 action likely results in the production of counteractiv

Tamoxifen-elicited uterotrophy: cross-species and cross-ligand analysis of the gene expression program

<p>Abstract</p> <p>Background</p> <p>Tamoxifen (TAM) is a well characterized breast cancer drug and selective estrogen receptor modulator (SERM) which also has been associated with a small increase in risk for uterine cancers. TAM's partial agonist activation of estrogen receptor has been characterized for specific gene promoters but not at the genomic level <it>in vivo</it>.Furthermore, reducing uncertainties associated with cross-species extrapolations of pharmaco- and toxicogenomic data remains a formidable challenge.</p> <p>Results</p> <p>A comparative ligand and species analysis approach was conducted to systematically assess the physiological, morphological and uterine gene expression alterations elicited across time by TAM and ethynylestradiol (EE) in immature ovariectomized Sprague-Dawley rats and C57BL/6 mice. Differential gene expression was evaluated using custom cDNA microarrays, and the data was compared to identify conserved and divergent responses. 902 genes were differentially regulated in all four studies, 398 of which exhibit identical temporal expression patterns.</p> <p>Conclusion</p> <p>Comparative analysis of EE and TAM differentially expressed gene lists suggest TAM regulates no unique uterine genes that are conserved in the rat and mouse. This demonstrates that the partial agonist activities of TAM extend to molecular targets in regulating only a subset of EE-responsive genes. Ligand-conserved, species-divergent expression of carbonic anhydrase 2 was observed in the microarray data and confirmed by real time PCR. The identification of comparable temporal phenotypic responses linked to related gene expression profiles demonstrates that systematic comparative genomic assessments can elucidate important conserved and divergent mechanisms in rodent estrogen signalling during uterine proliferation.</p

Implications of a RAD54L polymorphism (2290C/T) in human meningiomas as a risk factor and/or a genetic marker

BACKGROUND: RAD54L (OMIM 603615, Locus Link 8438) has been proposed as a candidate oncosupressor in tumours bearing a non-random deletion of 1p32, such as breast or colon carcinomas, lymphomas and meningiomas. In a search for RAD54L mutations in 29 menigiomas with allelic deletions in 1p, the only genetic change observed was a silent C/T transition at nucleotide 2290 in exon 18. In this communication the possible association of the 2290C/T polymorphism with the risk of meningiomas was examined. In addition, the usefulness of this polymorphism as a genetic marker within the meningioma consensus deletion region in 1p32 was also verified. The present study comprises 287 blood control samples and 70 meningiomas from Spain and Ecuador. Matched blood samples were only available from Spanish patients. RESULTS: The frequency of the rare allele-T and heterozygotes for the 2290C/T polymorphism in the blood of Spanish meningioma patients and in the Ecuadorian meningioma tumours was higher than in the control population (P < 0.05). Four other rare variants (2290C/G, 2299C/G, 2313G/A, 2344A/G) were found within 50 bp at the 3' end of RAD54L. Frequent loss of heterozygosity for the 2290C/T SNP in meningiomas allowed to further narrow the 1p32 consensus region of deletion in meningiomas to either 2.08 Mbp – within D1S2713 (44.35 Mbp) and RAD54L (46.43 Mbp) – or to 1.47 Mbp – within RAD54L and D1S2134 (47.90 Mbp) – according to recent gene mapping results. CONCLUSION: The statistical analysis of genotypes at the 2290C/T polymorphism suggest an association between the rare T allele and the development of meningeal tumours. This polymorphism can be used as a genetic marker inside the consensus deletion region at 1p32 in meningiomas

Identification of a novel Drosophila gene, beltless, using injectable embryonic and adult RNA interference (RNAi)

BACKGROUND: RNA interference (RNAi) is a process triggered by a double-stranded RNA that leads to targeted down-regulation/silencing of gene expression and can be used for functional genomics; i.e. loss-of-function studies. Here we report on the use of RNAi in the identification of a developmentally important novel Drosophila (fruit fly) gene (corresponding to a putative gene CG5652/GM06434), that we named beltless based on an embryonic loss-of-function phenotype. RESULTS: Beltless mRNA is expressed in all developmental stages except in 0–6 h embryos. In situ RT-PCR localized beltless mRNA in the ventral cord and brain of late stage embryos and in the nervous system, ovaries, and the accessory glands of adult flies. RNAi was induced by injection of short (22 bp) beltless double-stranded RNAs into embryos or into adult flies. Embryonic RNAi altered cuticular phenotypes ranging from partially-formed to missing denticle belts (thus beltless) of the abdominal segments A2–A4. Embryonic beltless RNAi was lethal. Adult RNAi resulted in the shrinkage of the ovaries by half and reduced the number of eggs laid. We also examined Df(1)RK4 flies in which deletion removes 16 genes, including beltless. In some embryos, we observed cuticular abnormalities similar to our findings with beltless RNAi. After differentiating Df(1)RK4 embryos into those with visible denticle belts and those missing denticle belts, we assayed the presence of beltless mRNA; no beltless mRNA was detectable in embryos with missing denticle belts. CONCLUSIONS: We have identified a developmentally important novel Drosophila gene, beltless, which has been characterized in loss-of-function studies using RNA interference. The putative beltless protein shares homologies with the C. elegans nose resistant to fluoxetine (NRF) NRF-6 gene, as well as with several uncharacterized C. elegans and Drosophila melanogaster genes, some with prominent acyltransferase domains. Future studies should elucidate the role and mechanism of action of beltless during Drosophila development and in adults, including in the adult nervous system

AGO1 and AGO2 Act Redundantly in miR408-Mediated Plantacyanin Regulation

Background: In Arabidopsis, AGO1 and AGO2 associate with small RNAs that exhibit a Uridine and an Adenosine at their 59 end, respectively. Because most plant miRNAs have a 59U, AGO1 plays many essential roles in miRNA-mediated regulation of development and stress responses. In contrast, AGO2 has only been implicated in antibacterial defense in association with miR393*, which has a 59A. AGO2 also participates in antiviral defense in association with viral siRNAs. Principal Findings: This study reveals that miR408, which has a 59A, regulates its target Plantacyanin through either AGO1 or AGO2. Indeed, neither ago1 nor ago2 single mutations abolish miR408-mediated regulation of Plantacyanin. Only an ago1 ago2 double mutant appears compromised in miR408-mediated regulation of Plantacyanin, suggesting that AGO1 and AGO2 have redundant roles in this regulation. Moreover, the nature of the 59 nucleotide of miR408 does not appear essential for its regulatory role because both a wildtype 59A-MIR408 and a mutant 59U-MIR408 gene complement a mir408 mutant. Conclusions/Significance: These results suggest that miR408 associates with both AGO1 and AGO2 based on criteria that differ from the 59 end rule, reminiscent of miR390-AGO7 and miR165/166-AGO10 associations, which are not based on the nature of the 59 nucleotide