Pausing during Reverse Transcription Increases the Rate of Retroviral Recombination

Retroviruses package two copies of genomic RNA into viral particles. During the minus-sense DNA synthesis step of reverse transcription, the nascent DNA can transfer multiple times between the two copies of the genome, resulting in recombination. The mechanism for this process is similar to the process of obligate strand transfers mediated by the repeat and primer binding site sequences. The location at which the DNA 3′ terminus completely transfers to the second RNA strand defines the point of crossover. Previous work in vitro demonstrated that reverse transcriptase pausing has a significant impact on the location of the crossover, with a proportion of complete transfer events occurring very close to pause sites. The role of pausing in vivo, however, is not clearly understood. By employing a murine leukemia virus-based single-cycle infection assay, strong pausing was shown to increase the probability of recombination, as reflected in the reconstitution of green fluorescent protein expression. The infection assay results were directly correlated with the presence of strong pause sites in reverse transcriptase primer extension assays in vitro. Conversely, when pausing was diminished in vitro, without changing the sequence of the RNA template involved in recombination, there was a significant reduction in recombination in vivo. Together, these data demonstrate that reverse transcriptase pausing, as observed in vitro, directly correlates with recombination during minus-sense DNA synthesis in vivo

microRNA-451a regulates colorectal cancer proliferation in response to radiation

Abstract Background Colorectal cancer (CRC) is a leading cause of cancer-related death. The biologic response of CRC to standard of care adjuvant therapies such as chemotherapy and radiation are poorly understood. MicroRNAs (miRs) have been shown to affect CRC progression and metastasis. Therefore, we hypothesized that specific miRs modulate CRC response to chemoradiation. Methods In this study, we used miR expression profiling and discovered a set of microRNAs upregulated rapidly in response to either a single 2 Gy dose fraction or a 10 Gy dose of γ-radiation in mouse colorectal carcinoma models. We used gain and loss-of-function studies in 2D and 3Dcell proliferation assays and colony formation assays to understand the role of the top miR candidate from our profiling. We used Student’s T-tests for simple comparisons and two-factor ANOVA for evaluating significance. Results The most upregulated candidate at early time points in our signature, miR-451a inhibited tumor cell proliferation and attenuated surviving fraction in longer-term cultures. Conversely, inhibition of miR-451a increased proliferation, tumorsphere formation, and surviving fraction of tumor cells. Using a bioinformatics approach, we identified four genes, CAB39, EMSY, MEX3C, and EREG, as targets of miR-451a. Transfection of miR-451a decreased both mRNA and protein levels of these targets. Importantly, we found miR-451a expression was high and CAB39, EMSY levels were low in a small subset of rectal cancer patients who had a partial response to chemoradiation when compared to patients that had no response. Finally, analysis of a TCGA colorectal cancer dataset revealed that CAB39 and EMSY are upregulated at the protein level in a significant number of CRC patients. Higher levels of CAB39 and EMSY correlated with poorer overall survival. Conclusions Taken together, our data indicates miR-451a is induced by radiation and may influence colorectal carcinoma proliferation via CAB39 and EMSY pathways

Differential intrahepatic phospholipid zonation in simple steatosis and nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) occurs frequently in a setting of obesity, dyslipidemia and insulin resistance, but the etiology of the disease, particularly the events favoring progression to nonalcoholic steatohepatitis (NASH) as opposed to simple steatosis (SS), are not fully understood. Based on known zonation patterns in protein, glucose and lipid metabolism, coupled with evidence that phosphatidylcholine may play a role in NASH pathogenesis, we hypothesized that phospholipid zonation exists in liver and that specific phospholipid abundance and distribution may be associated with histologic disease. A survey of normal hepatic protein expression profiles in the Human Protein Atlas revealed pronounced zonation of enzymes involved in lipid utilization and storage, particularly those facilitating phosphatidylcholine (PC) metabolism. Immunohistochemistry of obese normal, SS and NASH liver specimens with anti-phosphatidylethanomine N-methyltransferase (PEMT) antibodies showed a progressive decrease in the zonal distribution of this PC biosynthetic enzyme. Phospholipid quantitation by liquid chromatography mass spectrometry (LC-MS) in hepatic extracts of Class III obese patients with increasing NAFLD severity revealed that most PC species with 32, 34 and 36 carbons as well as total PC abundance was decreased with SS and NASH. Matrix assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) imaging revealed strong zonal distributions for 32, 34 and 36 carbon PCs in controls (minimal histologic findings) and SS that was lost in NASH specimens. Specific lipid species such as PC 34:1 and PC 36:2 best illustrated this phenomenon. These findings suggest that phospholipid zonation may be associated with the presence of an intrahepatic proinflammatory phenotype and thus have broad implications in the etiopathogenesis of NASH

Additional file 1: of microRNA-451a regulates colorectal cancer proliferation in response to radiation

Figure S1 Affymetrix .cel files were uploaded to Partek Genomics Suite 6.6 and normalized using the default parameters of the RMA subroutine (background correction, quantile normalization, median polish summarization) as a single set. All probesets on each array were included in normalization. Following normalization, the log2 transformed signal data set was filtered to exclude all non-human probesets and control probesets. These miRNAs are qualitatively identified as being differentially expressed in both tumor types within a time point in the same direction. (XLSX 1528 kb

Additional file 2: of microRNA-451a regulates colorectal cancer proliferation in response to radiation

Figure S1 Responses of CT26 mouse and HCT-116 human colorectal carcinoma cells to radiation. Figure S2 miR-451a levels in HCT-116 and CT26 cells at different doses of radiation. Figure S3 miR-451a levels in non-transformed primary cells. Figure S4 miR-451a levels in HCT-116 in survival fraction studies. Figure S5 Ectopic expression of miR-451a inhibits proliferation and clonogenic survival of CT26 cells. Figure S6 Inhibition of miR-451a does not affect proliferation of endothelial cells in response radiation. Figure S7 Ectopic expression of miR-451a inhibits of HCT-116 cells in combination with radiation and 5-FU.Figure S8 miR binding site predictions for miR-451a on target mRNAs. Figure S9 Regulation of miR-451a and target genes in human colorectal cancer. (PPTX 1174 kb

Multiple phospholipid distribution patterns in human liver as revealed by MALDI IMS.

<p>Representative photomicrographs of H&E sections (left panels, <b>A–D</b>) are marked to indicate locations of zone 1 (Z1) or zone 3 (Z3) hepatocytes. MALDI IMS images (right panels, <b>A–D</b>) of <i>m/z</i> 820.57, corresponding to PC 36∶4 [M+K]⁺, in (<b>A</b>) obese normal and (<b>B</b>) SS specimens, <i>m/z</i> 758.56, corresponding to PC 34∶2 [M+H]⁺ in SS specimens (<b>C</b>), and <i>m/z</i> 772.52, corresponding to PC 32∶0 [M+K]⁺ in (<b>D</b>) NASH specimens reveal distinct phospholipid patterning. An ion intensity color scale applicable to all images is shown at the top of figure. Scale bar = 500 µm.</p

Schematic representation of major phosphatidylcholine (PC) biosynthetic pathways in human liver.

<p>Dietary choline is converted to phosphocholine by the actions of choline kinase (CHK), and then reacted with cytidine triphosphate (CTP) to form cytidine diphosphocholine (CDP-choline). Choline/ethanolaminephosphotransferase (CEPT) catalyzes the final step in PC biosynthesis by reacting <i>sn-</i>1,2-diacylglycerols (DAGs) with CDP-choline to form PCs. The second major pathway for PC biosynthesis involves three sequential methylation reactions of phosphatidylethanolamines (PE) with S-adenosylmethionine (SAM) by the actions of phosphatidylethanolamine <i>N-</i>methyltransferase (PEMT). The acyl distribution and composition of fatty acids (FAs) within each PC is continually modified by the actions of multiple phospholipases (primarily A1 and A2 isoforms) and lysophosphatidylcholine acyltransferases (LPCAT1-4). The PCs synthesized by either pathway are readily converted into TAG.</p

Phosphatidylethanolamine methyltransferase (PEMT) localization differs with progression of NAFLD.

<p>Monoclonal antibodies were used to localize PEMT in liver specimens of bariatric subjects sampled intra-operatively at the time of bariatric surgery. (<b>A</b>) The <i>in situ</i> localization of PEMT in an obese normal control subjects was primarily in hepatocytes surrounding the central vein (zone 3; black arrows). (<b>B</b>) PEMT in a SS specimen was localized throughout the liver acinus in zones 1–3. (<b>C</b>) PEMT in NASH specimen accumulated at sites of inflammation (white arrows), as well as in zones 1–3.</p