siRNA-Like Double-Stranded RNAs Are Specifically Protected Against Degradation in Human Cell Extract

RNA interference (RNAi) is a set of intracellular pathways in eukaryotes that controls both exogenous and endogenous gene expression. The power of RNAi to knock down (silence) any gene of interest by the introduction of synthetic small-interfering (si)RNAs has afforded powerful insight into biological function through reverse genetic approaches and has borne a new field of gene therapeutics. A number of questions are outstanding concerning the potency of siRNAs, necessitating an understanding of how short double-stranded RNAs are processed by the cell. Recent work suggests unmodified siRNAs are protected in the intracellular environment, although the mechanism of protection still remains unclear. We have developed a set of doubly-fluorophore labeled RNAs (more precisely, RNA/DNA chimeras) to probe in real-time the stability of siRNAs and related molecules by fluorescence resonance energy transfer (FRET). We find that these RNA probes are substrates for relevant cellular degradative processes, including the RNase H1 mediated degradation of an DNA/RNA hybrid and Dicer-mediated cleavage of a 24-nucleotide (per strand) double-stranded RNA. In addition, we find that 21- and 24-nucleotide double-stranded RNAs are relatively protected in human cytosolic cell extract, but less so in blood serum, whereas an 18-nucleotide double-stranded RNA is less protected in both fluids. These results suggest that RNAi effector RNAs are specifically protected in the cellular environment and may provide an explanation for recent results showing that unmodified siRNAs in cells persist intact for extended periods of time

Basonuclin-2 Requirements for Zebrafish Adult Pigment Pattern Development and Female Fertility

Relatively little is known about the generation of adult form. One complex adult trait that is particularly amenable to genetic and experimental analysis is the zebrafish pigment pattern, which undergoes extensive remodeling during post-embryonic development to form adult stripes. These stripes result from the arrangement of three classes of neural crest-derived pigment cells, or chromatophores: melanophores, xanthophores, and iridophores. Here, we analyze the zebrafish bonaparte mutant, which has a normal early pigment pattern but exhibits a severe disruption to the adult stripe pattern. We show that the bonaparte mutant phenotype arises from mutations in basonuclin-2 (bnc2), encoding a highly conserved, nuclear-localized zinc finger protein of unknown function. We show that bnc2 acts non-autonomously to the melanophore lineage and is expressed by hypodermal cells adjacent to chromatophores during adult pigment pattern formation. In bonaparte (bnc2) mutants, all three types of chromatophores differentiate but then are lost by extrusion through the skin. We further show that while bnc2 promotes the development of two genetically distinct populations of melanophores in the body stripes, chromatophores of the fins and scales remain unaffected in bonaparte mutants, though a requirement of fin chromatophores for bnc2 is revealed in the absence of kit and colony stimulating factor-1 receptor activity. Finally, we find that bonaparte (bnc2) mutants exhibit dysmorphic ovaries correlating with infertility and bnc2 is expressed in somatic ovarian cells, whereas the related gene, bnc1, is expressed within oocytes; and we find that both bnc2 and bnc1 are expressed abundantly within the central nervous system. These findings identify bnc2 as an important mediator of adult pigment pattern formation and identify bonaparte mutants as an animal model for dissecting bnc2 functions

The pathological basis of resection margin for hepatocellular carcinoma

At the time of hepatectomy for primary hepatocellular carcinoma (HCC), the necessary amount of surrounding nontumorous liver to be sacrificed, or the resection margin, which will ensure a complete histologic disease clearance is uncertain. Twenty-three resected liver specimens were studied by serial section followed by histological examination prospectively. All histologic diseases identified were assumed to spread radially in all directions from a unicentric lesion. Histologic disease from capsular and liver invasion was found confined to the immediate surrounding liver parenchyma. Eleven of the 14 large (≥5 cm) tumors and all six small (<5 cm) HCCs had either microsatellites and/or histologic venous permeation found beyond 1 cm from the lesion. A large tumor, multinodular lesions, macroscopic venous thrombi, liver invasion, and nonencapsulation were associated with an extensive spread of histologic disease. In the presence of either microsatellites or histologic venous permeation, no distance could ensure a complete disease clearance.link_to_subscribed_fulltex