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Unlike amantadine (1-adamantanamine), tromantadine (N-1-adamantyl-N-[2-(dimethyl amino)ethoxy]acetamide hydrochloride) inhibits herpes simplex virus type 1 (KOS strain)-induced cytopathic effect and virus replication with limited toxicity to the cells. Vero and HEp-2 cells tolerated up to 2 mg of tromantadine per 2 x 106 cells for 24-, 48-, or 96-h incubation periods with little change in cell morphology. Treatment of the cells with 10 to 50 p.g of tromantadine reduced herpes simplex virus-induced cytopathic effect. Treatment with 100 to 500 pg of tromantadine inhibited herpes simplex virus-induced cytopathic effect and reduced virus production. Complete inhibition of virus production was observed with treatments of 500 jig to 1 mg. The antiherpetic activity of tromantadine was dependent upon the viral inoculum size and the time of addition of the compound with respect to infection. Virion synthesis and viral polypeptide synthesis were inhibited by addition of tromantadine at the time of infection or 4 h postinfection. The results are consistent with tromantadine inhibition of an early event in herpes simplex virus infection, before macromolecular synthesis, and a late event, such as assembly or release of virus.Tromantadine (N-1-adamantyl-N-[2-(dimethylamino)ethoxy]acetamide hydrochloride) (10,11,16,20) is one of relatively few amantadine (1-adamantanamine) derivatives which have antiherpetic activity. Amantadine has been shown to inhibit an early event before viral macromolecular synthesis in influenza A virus replication and is an alternative to vaccination against influenza A virus (15). Tromantadine has been reported to inhibit herpes simplex virus type 1 (HSV-1) and HSV-2 replication and to be effective as a topical antiherpetic drug (1,4,7,9,12,16,20,25). In this paper, we report an improved synthetic scheme for the compound and describe its activity against HSV-1 infection. An indication of the HSV replication step(s) inhibited by tromantadine is discussed. Tromantadine was prepared by addition of N,N-dimethylethanolamine to N-(1-adamantyl)-2-chloroacetamide. First, the lithium alkoxide of N,N-dimethylethanolamine was prepared by the addition of 4.3 ml (0.009 mol) of 2.1 M n-butylithium in hexane to a cold solution of the amino alcohol (0.78 g, 0.0088 mol) in 10 ml of dry THF. A solution of N-(1-adamantyl)-2-chloroacetamide (2.0 g, 0.0088 mol) in 15 ml of dry THF was then added slowly, and the mixture was stirred overnight. The solution was next poured into 50 ml of 10% HCI, and the resulting solution was washed with ether. Addition of NaOH solution liberated the tromantadine, which was extracted into two 100-ml portions of ether. The combined ether extracts were dried over anhydrous Na2SO4, and the ether was evaporated to yield a viscous oil. The oil was dissolved in benzene, and tromantadine was precipitated by 1031 on April 1, 2019 by guest http://aac.asm.org/ Downloaded from

Circular RNAs (circRNAs) have recently gained substantial attention in the cancer research field where most, including the putative oncogene ciRS-7 (CDR1as), have been proposed to function as competitive endogenous RNAs (ceRNAs) by sponging specific microRNAs. Here, we report the first spatially resolved cellular expression patterns of ciRS-7 in colon cancer and show that ciRS-7 is completely absent in the cancer cells, but highly expressed in stromal cells within the tumor microenvironment. Additionally, our data suggest that this generally apply to classical oncogene-driven adenocarcinomas, but not to other cancers, including malignant melanoma. Moreover, we find that correlations between circRNA and mRNA expression, which are commonly interpreted as evidence of a ceRNA function, can be explained by different cancer-to-stromal cell ratios among the studied tumor specimens. Together, these results have wide implications for future circRNA studies and highlight the importance of spatially resolving expression patterns of circRNAs proposed to function as ceRNAs.

BackgroundInsertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined.ResultsWe detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues.ConclusionsWe expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology.

BackgroundDepartures from the standard genetic code in eukaryotic nuclear genomes are known for only a handful of lineages and only a few genetic code variants seem to exist outside the ciliates, the most creative group in this regard. Most frequent code modifications entail reassignment of the UAG and UAA codons, with evidence for at least 13 independent cases of a coordinated change in the meaning of both codons. However, no change affecting each of the two codons separately has been documented, suggesting the existence of underlying evolutionary or mechanistic constraints.ResultsHere, we present the discovery of two new variants of the nuclear genetic code, in which UAG is translated as an amino acid while UAA is kept as a termination codon (along with UGA). The first variant occurs in an organism noticed in a (meta)transcriptome from the heteropteran Lygus hesperus and demonstrated to be a novel insect-dwelling member of Rhizaria (specifically Sainouroidea). This first documented case of a rhizarian with a non-canonical genetic code employs UAG to encode leucine and represents an unprecedented change among nuclear codon reassignments. The second code variant was found in the recently described anaerobic flagellate Iotanema spirale (Metamonada: Fornicata). Analyses of transcriptomic data revealed that I. spirale uses UAG to encode glutamine, similarly to the most common variant of a non-canonical code known from several unrelated eukaryotic groups, including hexamitin diplomonads (also a lineage of fornicates). However, in these organisms, UAA also encodes glutamine, whereas it is the primary termination codon in I. spirale. Along with phylogenetic evidence for distant relationship of I. spirale and hexamitins, this indicates two independent genetic code changes in fornicates.ConclusionsOur study documents, for the first time, that evolutionary changes of the meaning of UAG and UAA codons in nuclear genomes can be decoupled and that the interpretation of the two codons by the cytoplasmic translation apparatus is mechanistically separable. The latter conclusion has interesting implications for possibilities of genetic code engineering in eukaryotes. We also present a newly developed generally applicable phylogeny-informed method for inferring the meaning of reassigned codons.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0353-y) contains supplementary material, which is available to authorized users.