1,2-Di­hydro­spiro­[carbazole-3(4H),2′-[1,3]dioxolane]

In the title compound, C14H15NO2, the hydrogenated six-membered ring of the carbazole unit adopts a half-chair conformation and the dioxolane ring points to one side of the carbazole plane. Neighbouring mol­ecules form edge-to-face inter­actions in which the NH group is directed towards an adjacent carbazole unit, with a shortest H⋯C contact of 2.72 Å. These inter­actions arrange the mol­ecules into one-dimensional herringbone-type motifs, which pack so that the methyl­ene groups of the dioxolane ring lie over the face of a neighbouring carbazole unit with a shortest H⋯C contact of 2.85 Å

Locked nucleoside analogues expand the potential of DNAzymes to cleave structured RNA targets

BACKGROUND: DNAzymes cleave at predetermined sequences within RNA. A prerequisite for cleavage is that the DNAzyme can gain access to its target, and thus the DNAzyme must be capable of unfolding higher-order structures that are present in the RNA substrate. However, in many cases the RNA target sequence is hidden in a region that is too tightly structured to be accessed under physiological conditions by DNAzymes. RESULTS: We investigated how incorporation of LNA (locked nucleic acid) monomers into DNAzymes improves their ability to gain access and cleave at highly-structured RNA targets. The binding arms of DNAzymes were varied in length and were substituted with up to three LNA and α-L-LNA monomers (forming LNAzymes). For one DNAzyme, the overall cleavage reaction proceeded fifty times faster after incorporation of two α-L-LNA monomers per binding arm (k(obs )increased from 0.014 min(-1 )to 0.78 min(-1)). CONCLUSION: The data demonstrate how hydrolytic performance can be enhanced by design of LNAzymes, and indicate that there are optimal lengths for the binding arms and for the number of modified LNA monomers

A genome-wide assessment of conserved SNP alleles reveals a panel of regulatory SNPs relevant to the peripheral nerve

Abstract Background Identifying functional non-coding variation is critical for defining the genetic contributions to human disease. While single-nucleotide polymorphisms (SNPs) within cis-acting transcriptional regulatory elements have been implicated in disease pathogenesis, not all cell types have been assessed and functional validations have been limited. In particular, the cells of the peripheral nervous system have been excluded from genome-wide efforts to link non-coding SNPs to altered gene function. Addressing this gap is essential for defining the genetic architecture of diseases that affect the peripheral nerve. We developed a computational pipeline to identify SNPs that affect regulatory function (rSNPs) and evaluated our predictions on a set of 144 regions in Schwann cells, motor neurons, and muscle cells. Results We identified 28 regions that display regulatory activity in at least one cell type and 13 SNPs that affect regulatory function. We then tailored our pipeline to one peripheral nerve cell type by incorporating SOX10 ChIP-Seq data; SOX10 is essential for Schwann cells. We prioritized 22 putative SOX10 response elements harboring a SNP and rapidly validated two rSNPs. We then selected one of these elements for further characterization to assess the biological relevance of our approach. Deletion of the element from the genome of cultured Schwann cells—followed by differential gene expression studies—revealed Tubb2b as a candidate target gene. Studying the enhancer in developing mouse embryos revealed activity in SOX10-positive cells including the dorsal root ganglia and melanoblasts. Conclusions Our efforts provide insight into the utility of employing strict conservation for rSNP discovery. This strategy, combined with functional analyses, can yield candidate target genes. In support of this, our efforts suggest that investigating the role of Tubb2b in SOX10-positive cells may reveal novel biology within these cell populations.https://deepblue.lib.umich.edu/bitstream/2027.42/143511/1/12864_2018_Article_4692.pd

Probing the O-glycoproteome of gastric cancer cell lines for biomarker discovery

Circulating O-glycoproteins shed from cancer cells represent important serum biomarkers for diagnostic and prognostic purposes. We have recently shown that selective detection of cancer-associated aberrant glycoforms of circulating O-glycoprotein biomarkers can increase specificity of cancer biomarker assays. However, the current knowledge of secreted and circulating O-glycoproteins is limited. Here, we used the COSMC KO "Simple- Cell" (SC) strategy to characterize the O-glycoproteome of two gastric cancer SimpleCell lines (AGS, MKN45) as well as a gastric cell line (KATO III) which naturally expresses at least partially truncated O-glycans. Overall, we identified 499 O-glycoproteins and 1236 O-glycosites in gastric cancer SimpleCells, and a total 47 O-glycoproteins and 73 O-glycosites in the KATO III cell line. We next modified the glycoproteomic strategy to apply it to pools of sera from gastric cancer and healthy individuals to identify circulating O-glycoproteins with the STn glycoform. We identified 37 O-glycoproteins in the pool of cancer sera, and only nine of these were also found in sera from healthy individuals. Two identified candidate O-glycoprotein biomarkers (CD44 and GalNAc-T5) circulating with the STn glycoform were further validated as being expressed in gastric cancer tissue. A proximity ligation assay was used to show that CD44 was expressed with the STn glycoform in gastric cancer tissues. The study provides a discovery strategy for aberrantly glycosylated O-glycoproteins and a set of O-glycoprotein candidates with biomarker potential in gastric cancer.This work was supported by The Danish Research Councils, The Mizutani Foundation, The Danish National Research Foundation (DNRF107) and Fundacão para a Ciência e a Tecnologia (FCT) and COMPETE (Programa Operacional Temático Factores de Competitividade, comparticipado pelo fundo comunitário europeu FEDER) in the framework of the projects: PTDC/BBB-EBI/0786/2012; EXPL/CTM-BIO/0762/2013. Grants were received from FCT (SFRH/BD/73717/2010 to DC), (SFRH/BPD/75871/2011 to AM), (SFRH/BPD/96510/2013 to CG) and (SFRH/BPD/66288/2009 to JAF). IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, and is partially supported by FCT

Evidence for discrimination between feeding sounds of familiar fish and unfamiliar mammal-eating killer whale ecotypes by long-finned pilot whales

Research funding was provided by the US Office of Naval Research, the DGA/TN (France), the UK Natural Environmental Research Council, and the Ministries of Defence of Norway and The Netherlands. PLT acknowledges funding received from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland). MASTS is funded by the Scottish Funding Council (Grant reference HR09011) and contributing institutions. CC acknowledges statistical support provided by the Multi-study OCean acoustics Human effects Analysis (MOCHA) project funded by the United States Office of Naval Research (Grant N00014-12-1-0204).Killer whales (KW) may be predators or competitors of other cetaceans. Since their foraging behavior and acoustics differ among populations ('ecotypes'), we hypothesized that other cetaceans can eavesdrop on KW sounds and adjust their behavior according to the KW ecotype. We performed playback experiments on long-finned pilot whales (Globicephala melas) in Norway using familiar fish-eating KW sounds (fKW) simulating a sympatric population that might compete for foraging areas, unfamiliar mammal-eating KW sounds (mKW) simulating a potential predator threat, and two control sounds. We assessed behavioral responses using animal-borne multi-sensor tags and surface visual observations. Pilot whales barely changed behavior to a broadband noise (CTRL-), whereas they were attracted and exhibited spyhops to fKW, mKW, and to a repeated-tonal upsweep signal (CTRL+). Whales never stopped nor started feeding in response to fKW, whereas they reduced or stopped foraging to mKW and CTRL+. Moreover, pilot whales joined other subgroups in response to fKW and CTRL+, whereas they tightened individual spacing within group and reduced time at surface in response to mKW. Typical active intimidation behavior displayed to fKW might be an antipredator strategy to a known low-risk ecotype or alternatively a way of securing the habitat exploited by a heterospecific sympatric population. Cessation of feeding and more cohesive approach to mKW playbacks might reflect an antipredator behavior towards an unknown KW ecotype of potentially higher risk. We conclude that pilot whales are able to acoustically discriminate between familiar and unfamiliar KW ecotypes, enabling them to adjust their behavior according to the perceived disturbance type.PostprintPeer reviewe

Long range allostery mediates cooperative adenine nucleotide binding by the Ski2 like RNA helicase Brr2

Brr2 is an essential Ski2 like RNA helicase that exhibits a unique structure among the spliceosomal helicases. Brr2 harbors a catalytically active N terminal helicase cassette and a structurally similar but enzymatically inactive C terminal helicase cassette connected by a linker region. Both cassettes contain a nucleotide binding pocket, but it is unclear whether nucleotide binding in these two pockets is related. Here we use biophysical and computational methods to delineate the functional connectivity between the cassettes and determine whether occupancy of one nucleotide binding site may influence nucleotide binding at the other cassette. Our results show that Brr2 exhibits high specificity for adenine nucleotides, with both cassettes binding ADP tighter than ATP. Adenine nucleotide affinity for the inactive C terminal cassette is more than two orders of magnitude higher than that of the active N terminal cassette, as determined by slow nucleotide release. Mutations at the intercassette surfaces and in the connecting linker diminish the affinity of adenine nucleotides for both cassettes. Moreover, we found that abrogation of nucleotide binding at the C terminal cassette reduces nucleotide binding at the N terminal cassette 70 away. Molecular dynamics simulations identified structural communication lines that likely mediate these long range allosteric effects, predominantly across the intercassette interface. Together, our results reveal intricate networks of intramolecular interactions in the complex Brr2 RNA helicase, which fine tune its nucleotide affinities and which could be exploited to regulate enzymatic activity during splicin

Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers

Genomes are becoming heavily annotated with important features. Analysis of these features often employs oligonucleotides that hybridize at defined locations. When the defined location lies in a poor sequence context, traditional design strategies may fail. Locked Nucleic Acid (LNA) can enhance oligonucleotide affinity and specificity. Though LNA has been used in many applications, formal design rules are still being defined. To further this effort we have investigated the effect of LNA on the performance of sequencing and PCR primers in AT-rich regions, where short primers yield poor sequencing reads or PCR yields. LNA was used in three positional patterns: near the 5′ end (LNA-5′), near the 3′ end (LNA-3′) and distributed throughout (LNA-Even). Quantitative measures of sequencing read length (Phred Q30 count) and real-time PCR signal (cycle threshold, C(T)) were characterized using two-way ANOVA. LNA-5′ increased the average Phred Q30 score by 60% and it was never observed to decrease performance. LNA-5′ generated cycle thresholds in quantitative PCR that were comparable to high-yielding conventional primers. In contrast, LNA-3′ and LNA-Even did not improve read lengths or C(T). ANOVA demonstrated the statistical significance of these results and identified significant interaction between the positional design rule and primer sequence

Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification

Background: Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping. Results: We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 specie