A multi-omics dataset for the analysis of frontotemporal dementia genetic subtypes.

Understanding the molecular mechanisms underlying frontotemporal dementia (FTD) is essential for the development of successful therapies. Systematic studies on human post-mortem brain tissue of patients with genetic subtypes of FTD are currently lacking. The Risk and Modyfing Factors of Frontotemporal Dementia (RiMod-FTD) consortium therefore has generated a multi-omics dataset for genetic subtypes of FTD to identify common and distinct molecular mechanisms disturbed in disease. Here, we present multi-omics datasets generated from the frontal lobe of post-mortem human brain tissue from patients with mutations in MAPT, GRN and C9orf72 and healthy controls. This data resource consists of four datasets generated with different technologies to capture the transcriptome by RNA-seq, small RNA-seq, CAGE-seq, and methylation profiling. We show concrete examples on how to use the resulting data and confirm current knowledge about FTD and identify new processes for further investigation. This extensive multi-omics dataset holds great value to reveal new research avenues for this devastating disease.

iPS Cell-Based Model for MAPT Haplotype as a Risk Factor for Human Tauopathies Identifies No Major Differences in TAU Expression.

The H1 haplotype of the microtubule-associated protein tau (MAPT) gene is a common genetic risk factor for some neurodegenerative diseases such as progressive supranuclear palsy, corticobasal degeneration, and Parkinson's disease. The molecular mechanism causing the increased risk for the named diseases, however, remains unclear. In this paper, we present a valuable tool of eight small molecule neural precursor cell lines (smNPC) homozygous for the MAPT haplotypes (four H1/H1 and four H2/H2 cell lines), which can be used to identify MAPT-dependent phenotypes. The employed differentiation protocol is fast due to overexpression of NEUROGENIN-2 and therefore suitable for high-throughput approaches. A basic characterization of all human cell lines was performed, and their TAU and α-SYNUCLEIN profiles were compared during a differentiation time of 30 days. We could identify higher levels of conformationally altered TAU in cell lines carrying the H2 haplotype. Additionally, we found increased expression levels of α-SYNUCLEIN in H1/H1 cells. With this resource, we aim to fill a gap in neurodegenerative disease modeling with induced pluripotent stem cells (iPSC) for sporadic tauopathies.

Automated Production of Human Induced Pluripotent Stem Cell-Derived Cortical and Dopaminergic Neurons with Integrated Live-Cell Monitoring.

Manual culture and differentiation protocols for human induced pluripotent stem cells (hiPSC) are difficult to standardize, show high variability and are prone to spontaneous differentiation into unwanted cell types. The methods are labor-intensive and are not easily amenable to large-scale experiments. To overcome these limitations, we developed an automated cell culture system coupled to a high-throughput imaging system and implemented protocols for maintaining multiple hiPSC lines in parallel and neuronal differentiation. We describe the automation of a short-term differentiation protocol using Neurogenin-2 (NGN2) over-expression to produce hiPSC-derived cortical neurons within 6‒8 days, and the implementation of a long-term differentiation protocol to generate hiPSC-derived midbrain dopaminergic (mDA) neurons within 65 days. Also, we applied the NGN2 approach to a small molecule-derived neural precursor cells (smNPC) transduced with GFP lentivirus and established a live-cell automated neurite outgrowth assay. We present an automated system with protocols suitable for routine hiPSC culture and differentiation into cortical and dopaminergic neurons. Our platform is suitable for long term hands-free culture and high-content/high-throughput hiPSC-based compound, RNAi and CRISPR/Cas9 screenings to identify novel disease mechanisms and drug targets.

Insecticide resistance in Trialeurodes vaporariorum populations and novel diagnostics for kdr mutations.

BACKGROUND: Neonicotinoids, pyrethroids and ketoenols are currently used for the control of Trialeurodes vaporariorum (Hemiptera: Aleyrodidae). In this study, insecticide resistance status and mechanisms were investigated using classical bioassays and molecular techniques. RESULTS: Dose-response bioassays were performed on 19 Greek populations, among the 35 different whitefly populations used for the whole analysis. Resistance factors scaled up to 207-, 4657- and 59-fold for imidacloprid, bifenthrin and spiromesifen, respectively. Molecular assays were used to investigate the frequency of known resistance mutations. A simple polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed for detecting the pyrethroid-resistant alleles r1 (mutation L925I) and r2 (mutation T929I) of the para-type voltage-gated sodium channel gene (VGSC). Both alleles were present at high frequencies (on average 65% and 33%, respectively) in 14 populations from Greece. The M918 L pyrethroid resistance mutation was not detected in any of the Greek populations. Sequencing and a Taqman allelic discrimination were used to monitor the frequency of the mutation E645K of the acetyl-coenzyme A carboxylase gene (ACC) recently linked to spiromesifen resistance. This mutation was detected in 20 of the 24 populations examined in ∼38% frequency among the 433 individuals tested. However, its association with the spiromesifen resistance phenotype was not confirmed in the Greek populations. Finally, two homologues of the CYP6CM1 Bemisia tabaci P450, the known neonicotinoid metabolizer, were found upregulated in two T. vaporariorum neonicotinoid-resistant populations; they were both functionally expressed in Escherichia coli, but the recombinant proteins encoded did not metabolize those neonicotinoid insecticides tested. CONCLUSION: The development of simple diagnostics and their use alongside classical and molecular techniques for the early detection of resistant populations are of great importance for pest management strategies. The practical implications of our results are discussed in light of whitefly control. © 2017 Society of Chemical Industry.

Insecticide resistance status of Myzus persicae in Greece: long-term surveys and new diagnostics for resistance mechanisms.

BACKGROUND: Myzus persicae nicotianae is an important pest in Greece, controlled mainly by neonicotinoids. Monitoring of the aphid populations for resistance mechanisms is essential for effective control. RESULTS: Two new RFLP-based diagnostics for the detection of the M918T (super-kdr pyrethroid resistance) and nAChR R81T (neonicotinoid resistance) mutations were applied, along with other established assays, on 131 nicotianae multilocus genotypes (MLGs) collected from tobacco and peach in Greece in 2012-2013. Furthermore, we present resistance data from aphid clones (>500, mainly nicotianae) collected in 2006-2007. About half of the clones tested with a diagnostic dose of imidacloprid were tolerant. The R81T mutation was not found in the 131 MLGs and 152 clones examined. Over half (58.6%) of a subset of 29 clones showed a 9-36-fold overexpression of CYP6CY3. M918T was found at low to moderate frequencies. The kdr and MACE mechanisms and carboxylesterase-based resistance were found at high frequency in all years. CONCLUSION: The aphid retains costly resistance mechanisms even in the absence of pressure from certain insecticides, which could be attributed to factors related to climate and genetic properties of the populations. The indication of build-up of resistance/tolerance to neonicotinoids, related to CYP6CY3 overexpression, is a matter of concern. © 2015 Society of Chemical Industry.

Comparative analysis of internal ribosomal entry sites as molecular tools for bicistronic expression.

Internal ribosomal entry sites (IRESes) are sequences that drive cap-independent translation. They are found in some viral and cellular transcripts and they have been extensively used in both basic and applied research for the translation of two or more polypeptides from a single mRNA molecule in eukaryotic cells. Although the most widely used IRES comes from the encephalomyocarditis virus (EMCV), several other viral and cellular IRES elements have been identified and successfully used, including those of the human VCIP gene and the mouse Gtx gene. In this report we have compared the EMCV IRES with the VCIP and the Gtx IRESes, and we provide evidence that by using the EMCV IRES much higher levels of second cistron expression can be achieved.

Effect of an all-trans-retinoic acid conjugate with spermine on viability of human prostate cancer and endothelial cells in vitro and angiogenesis in vivo.

Retinoids constitute a family of organic compounds that are being used for the treatment of various diseases, ranging from acne vulgaris to acute promyelocytic leukemia. Their use however is limited due to serious adverse effects and there is a great need for analogues with better safety profile. In the present work, the effect of N(1),N(12)-bis(all-trans-retinoyl)spermine (RASP), a conjugate of all-trans-retinoic acid (atRA) with spermine, on angiogenesis in vivo and viability of human endothelial and prostate cancer cells in vitro were studied. Both atRA and RASP dose-dependently inhibited angiogenesis in the chicken embryo chorioallantoic membrane model. RASP was more effective and could be used in a wider dose range due to lower toxicity compared with atRA. Both retinoids decreased the number of human umbilical vein endothelial and prostate cancer LNCaP and PC3 cells in a concentration-dependent manner. RASP was more effective and potent compared with atRA, spermine, their combination, or conjugates of spermine with other acidic retinoids and/or psoralens in prostate cancer cells. The inhibitory effect of both atRA and RASP seems to be related to an increase of the tumour repressing gene retinoic acid receptor beta mRNA, was mediated by retinoic acid receptor alpha, and was proportional to endogenous retinoic acid receptor beta expression. These data suggest that RASP is more effective than atRA in decreasing angiogenesis and prostate cancer cell growth and identify retinoic acid receptor alpha as the receptor through which it causes retinoic acid receptor beta up-regulation and decrease of prostate cancer cell growth.

Effect of conjugates of all-trans-retinoic acid and shorter polyene chain analogues with amino acids on prostate cancer cell growth.

In the present work, a series of conjugates of amino acids with all-trans-retinoic acid (ATRA) and shorter polyene chain analogues were rationally designed, synthesized by coupling the succinimidyl active esters of the acidic retinoids with appropriately protected amino acids or peptides followed by deprotection, and examined for their possible effect on viability of human prostate cancer LNCaP cells. In contrast to ATRA, all conjugates bearing amino acids with polar side chains showed no inhibitory effect on LNCaP cell proliferation, while conjugates with alpha-amino acids with lipophilic side chain, such as 7, or linear amino acids, such as 9, significantly decreased prostate cancer LNCaP cell number. Interestingly, while the effect of ATRA was RARalpha-dependent, the effect of its active analogues was not inhibited by a selective RARalpha antagonist. Cell cycle analysis showed no effect on cell cycle, while quantitative analysis by annexin V-propidium iodide staining revealed that neither ATRA nor its analogues affected LNCaP cell apoptosis or necrosis. These results demonstrate that compounds 7 and 9 are potentially useful agents that warrant further preclinical development for treatment of prostate cancer.