Faculty Bibliography

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3 degrees C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.

High cholesterol and diabetes are major risk factors for atherosclerosis. Regression of atherosclerosis is mediated in part by the Liver X Receptor (LXR) through the induction of genes involved in cholesterol transport and efflux. In the context of diabetes, regression of atherosclerosis is impaired. We proposed that changes in glucose levels modulate LXR-dependent gene expression. Using a mouse macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages (BMDMs) cultured in normal or diabetes relevant high glucose conditions we found that high glucose inhibits the LXR-dependent expression of ATP-binding cassette transporter A1 (ABCA1), but not ABCG1. To probe for this mechanism, we surveyed the expression of a host of chromatin-modifying enzymes and found that Protein Arginine Methyltransferase 2 (PRMT2) was reduced in high compared to normal glucose conditions. Importantly, ABCA1 expression and ABCA1-mediated cholesterol efflux were reduced in Prmt2-/- compared to wild type BMDMs. Monocytes from diabetic mice also showed decreased expression of Prmt2 compared to non-diabetic counterparts. Thus, PRMT2 represents a glucose-sensitive factor that plays a role in LXR-mediated ABCA1-dependent cholesterol efflux and lends insight to the presence of increased atherosclerosis in diabetic patients.

The induction and maintenance of pluripotency requires the expression of several core factors at appropriate levels (Oct4, Sox2, Klf4, Prdm14). A subset of these proteins (Oct4, Sox2, Prdm14) also plays crucial roles for the establishment of primordial germ cells (PGCs). Here we demonstrate that the Mad2l2 (MAD2B, Rev7) gene product is not only required by PGCs, but also by pluripotent embryonic stem cells (ESCs), depending on the growth conditions. Mad2l2(-/-) ESCs were unstable in LIF/serum medium, and differentiated into primitive endoderm. However, they could be stably propagated using small molecule inhibitors of MAPK signaling. Several components of the MAPK cascade were up- or downregulated even in undifferentiated Mad2l2(-/-) ESCs. Global levels of repressive histone H3 variants were increased in mutant ESCs, and the epigenetic signatures on pluripotency-, primitive endoderm-, and MAPK-related loci differed. Thus, H3K9me2 repressed the Nanog promoter, while the promoter of Gata4 lost H3K27me3 and became de-repressed in LIF/serum condition. Promoters associated with genes involved in MAPK signaling also showed misregulation of these histone marks. Such epigenetic modifications could be indirect consequences of mutating Mad2l2. However, our previous observations suggested the histone methyltransferases as direct (G9a) or indirect (Ezh2) targets of Mad2l2. In effect, the intricate balance necessary for pluripotency becomes perturbed in the absence of Mad2l2.

Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

Genetically encoded calcium indicators (GECIs) permit imaging intracellular calcium transients. Among GECIs, the GFP-based GCaMPs are the most widely used because of their high sensitivity and rapid response to changes in intracellular calcium concentrations. Here we report that the fluorescence of GCaMPs-including GCaMP3, GCaMP5 and GCaMP6-can be converted from green to red following exposure to blue-green light (450-500 nm). This photoconversion occurs in both insect and mammalian cells and is enhanced in a low oxygen environment. The red fluorescent GCaMPs retained calcium responsiveness, albeit with reduced sensitivity. We identified several amino acid residues in GCaMP important for photoconversion and generated a GCaMP variant with increased photoconversion efficiency in cell culture. This light-induced spectral shift allows the ready labeling of specific, targeted sets of GCaMP-expressing cells for functional imaging in the red channel. Together, these findings indicate the potential for greater utility of existing GCaMP reagents, including transgenic animals.

Primary Cilia (PC) are a very likely place for signal integration where multiple signaling pathways converge. Two major signaling pathways clearly shown to signal through the PC, Sonic Hedgehog (Shh) and PDGF-Ralpha, are particularly important for the proliferation and differentiation of oligodendrocytes, suggesting that their interaction occurs in or around this organelle. We identified PC in rat oligodendrocyte precursor cells (OPCs) and found that, while easily detectable in early OPCs, PC are lost as these cells progress to terminal differentiation. We confirmed the interaction between these pathways, as cyclopamine inhibition of Hedgehog function impairs both PDGF-mediated OPC proliferation and Shh-dependent cell branching. However, we failed to detect PDGF-Ralpha localization into the PC. Remarkably, ciliobrevin-mediated disruption of PC and reduction of OPC process extension was counteracted by recombinant Shh treatment, while PDGF had no effect. Therefore, while PDGF-Ralpha-dependent OPC proliferation and survival most probably does not initiate at the PC, still the integrity of this organelle and cilium-centered pathway is necessary for OPC survival and differentiation.

We report an 83-year-old immune-competent female with unilateral endophthalmitis extraordinarily caused by cytomegalovirus (CMV). Since she was suspected of suffering possible bacterial endophthalmitis, she was referred to our hospital. At the first visit, hypopyon in the anterior chamber and the opacity of vitreous body were observed in the left eye. The best-corrected visual acuity (BCVA) of the left eye was counting fingers and the intraocular pressure (IOP) was 20 mmHg. Bacterial and fungus culture of the aqueous humor revealed no infection. However, the density of corneal endothelial cell was less than the measurable range and CMV was detected by PCR of the aqueous humor. She was immune-competent and the data indicated neither systemic infections nor diseases. Systemic valganciclovir and corticosteroid were administered. After that, hypopyon in the anterior chamber and the opacity of vitreous body of the left eye were improved, and the BCVA of the left eye was 20/200 one year after the first visit. However, the inflammation of the anterior chamber recurred accompanied by elevated IOP after the discontinuance of administering valganciclovir. CMV-induced uveitis accompanied with hypopyon is quite rare. Therefore, it can be easily misdiagnosed as bacterial endophthalmitis.