Deconvolution of cellular subsets in human tissue based on targeted DNA methylation analysis at individual CpG sites.

BACKGROUND: The complex composition of different cell types within a tissue can be estimated by deconvolution of bulk gene expression profiles or with various single-cell sequencing approaches. Alternatively, DNA methylation (DNAm) profiles have been used to establish an atlas for multiple human tissues and cell types. DNAm is particularly suitable for deconvolution of cell types because each CG dinucleotide (CpG site) has only two states per DNA strand-methylated or non-methylated-and these epigenetic modifications are very consistent during cellular differentiation. So far, deconvolution of DNAm profiles implies complex signatures of many CpGs that are often measured by genome-wide analysis with Illumina BeadChip microarrays. In this study, we investigated if the characterization of cell types in tissue is also feasible with individual cell type-specific CpG sites, which can be addressed by targeted analysis, such as pyrosequencing. RESULTS: We compiled and curated 579 Illumina 450k BeadChip DNAm profiles of 14 different non-malignant human cell types. A training and validation strategy was applied to identify and test for cell type-specific CpGs. We initially focused on estimating the relative amount of fibroblasts using two CpGs that were either hypermethylated or hypomethylated in fibroblasts. The combination of these two DNAm levels into a "FibroScore" correlated with the state of fibrosis and was associated with overall survival in various types of cancer. Furthermore, we identified hypomethylated CpGs for leukocytes, endothelial cells, epithelial cells, hepatocytes, glia, neurons, fibroblasts, and induced pluripotent stem cells. The accuracy of this eight CpG signature was tested in additional BeadChip datasets of defined cell mixtures and the results were comparable to previously published signatures based on several thousand CpGs. Finally, we established and validated pyrosequencing assays for the relevant CpGs that can be utilized for classification and deconvolution of cell types. CONCLUSION: This proof of concept study demonstrates that DNAm analysis at individual CpGs reflects the cellular composition of cellular mixtures and different tissues. Targeted analysis of these genomic regions facilitates robust methods for application in basic research and clinical settings.

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells.

BACKGROUND: The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion. METHODS: In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays. RESULTS: Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs. CONCLUSIONS: Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

Macaques can contribute to greener practices in oil palm plantations when used as biological pest control.

Conversion of tropical forests into oil palm plantations reduces the habitats of many species, including primates, and frequently leads to human-wildlife conflicts. Contrary to the widespread belief that macaques foraging in the forest-oil palm matrix are detrimental crop pests, we show that the impact of macaques on oil palm yield is minor. More importantly, our data suggest that wild macaques have the potential to act as biological pest control by feeding on plantation rats, the major pest for oil palm crops, with each macaque group estimated to reduce rat populations by about 3,000 individuals per year (mitigating annual losses of 112 USD per hectare). If used for rodent control in place of the conventional method of poison, macaques could provide an important ecosystem service and enhance palm oil sustainability.

Climatic differentiation in polyploid apomictic Ranunculus auricomus complex in Europe.

BACKGROUND: Polyploidy and apomixis are important factors influencing plant distributions often resulting in range shifts, expansions and geographical parthenogenesis. We used the Ranunculus auricomus complex as a model to asses if the past and present distribution and climatic preferences were determined by these phenomena. RESULTS: Ecological differentiation among diploids and polyploids was tested by comparing the sets of climatic variables and distribution modelling using 191 novel ploidy estimations and 561 literature data. Significant differences in relative genome size on the diploid level were recorded between the "auricomus" and "cassubicus" groups and several new diploid occurrences were found in Slovenia and Hungary. The current distribution of diploids overlapped with the modelled paleodistribution (22 kyr BP), except Austria and the Carpathians, which are proposed to be colonized later on from refugia in the Balkans. Current and historical presence of diploids from the R. auricomus complex is suggested also for the foothills of the Caucasus. Based on comparisons of the climatic preferences polyploids from the R. auricomus complex occupy slightly drier and colder habitats than the diploids. CONCLUSIONS: The change of reproductive mode and selection due to competition with the diploid ancestors may have facilitated the establishment of polyploids within the R. auricomus complex in environments slightly cooler and drier, than those tolerated by diploid ancestors. Much broader distribution of polyploid apomicts may have been achieved due to faster colonization mediated by uniparental reproductive system.

Design, synthesis, and qualitative structure-activity evaluations of novel ß-secretase inhibitors as potential Alzheimer's drug leads.

We have identified highly selective imidazopyridines armed with benzimidazol and/or arylimidazole as potent ß-secretase inhibitors. The most effective and selective analogues demonstrated low nanomolar potency for the BACE1 enzyme as measured by FRET and cell-based (ELISA) assays and exhibited comparable affinity (KI) and high ligand efficiency (LE). In addition, these motifs were highly selective (>200) against the structurally related aspartyl protease BACE2. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on the previously reported hydroxyethylene transition state inhibitor derived from isophthalic acid I. Of the most potent compounds, 34 displayed an IC50 for BACE1 of 18 nM and exhibited cellular activity with an EC50 of 37 nM in the cell-based ELISA assay, as well as high affinity (KI=17 nM) and ligand efficiency (LE=1.7 kJ/mol). Compound 34 was found to be 204-fold more selective for BACE1 compared to the closely related aspartyl protease BACE2.

Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease.

SARS coronavirus main protease (SARS-CoV M(pro)) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV M(pro). Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV M(pro) requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We determined four crystal structures of SARS-CoV M(pro) in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV M(pro), with K(i) values in the µM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2=Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of SARS-CoV M(pro) in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV M(pro), with K(i)=2.24±0.58 µM. These results show that the stringent substrate specificity of the SARS-CoV M(pro) with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the observed cleavage specificity of the target protease.

Comparison of prognostic usefulness (three years) of computed tomographic angiography versus 64-slice computed tomographic calcium scanner in subjects without significant coronary artery disease.

Coronary computerized tomographic angiography (CTA) has been used as a noninvasive method for ruling out high-grade stenoses. Even in the absence of such stenoses, analysis of coronary atherosclerosis may provide for important prognostic information, and this may be superior to exclusive coronary artery calcium scoring. We tested this hypothesis in patients undergoing CTA for clinical indications who had no stenoses requiring revascularization. From December 2004 to December 2006, 706 consecutive patients who underwent CTA but had no high-grade stenoses were included (58% men, mean age 59 ± 11 years). CTA and coronary artery calcium scoring (Agatston method) were performed using a 64-slice CT scanner with a gantry rotation time of 330 ms. CT angiograms were categorized as completely normal (group 1), showing minor plaque (group 2), or showing intermediate stenoses (group 3). Follow-up information was obtained in 670 patients (95%) over a mean of 3.2 years. There were 31 major adverse events (5%), namely 9 deaths (all noncoronary), 2 myocardial infarctions, 5 strokes, 13 coronary revascularization procedures (percutaneous or surgical > 6 months after CTA), and 2 peripheral percutaneous interventions. Coronary status as defined by CTA was predictive of major events after adjustment for age and gender. In group 1, the probability of event-free survival at 3 years was 100%; in group 2, it was 96%; and in group 3, it was 91%. Compared to group 1, the risk in group 2 was increased 2.3-fold, and in group 3, it was increased 5.6-fold after adjusting for age and gender. However, after addition of the coronary artery calcium score to the regression analysis, CT angiographic status no longer appeared to be predictive. In conclusion, the risk of a major adverse cardiovascular event or death increased in a graded manner with degree of coronary atherosclerosis as defined by CTA even in the absence of high-grade coronary stenoses. However, in the absence of high-grade stenoses, we were unable to demonstrate a superior prognostic value of CTA compared to coronary artery calcium.