Cancer gene prioritization by integrative analysis of mRNA expression and DNA copy number data: a comparative review

A variety of genome-wide profiling techniques are available to probe complementary aspects of genome structure and function. Integrative analysis of heterogeneous data sources can reveal higher-level interactions that cannot be detected based on individual observations. A standard integration task in cancer studies is to identify altered genomic regions that induce changes in the expression of the associated genes based on joint analysis of genome-wide gene expression and copy number profiling measurements. In this review, we provide a comparison among various modeling procedures for integrating genome-wide profiling data of gene copy number and transcriptional alterations and highlight common approaches to genomic data integration. A transparent benchmarking procedure is introduced to quantitatively compare the cancer gene prioritization performance of the alternative methods. The benchmarking algorithms and data sets are available at http://intcomp.r-forge.r-project.orgComment: PDF file including supplementary material. 9 pages. Preprin

MENGA: a new comprehensive tool for the integration of neuroimaging data and the Allen human brain transcriptome atlas

Brain-wide mRNA mappings offer a great potential for neuroscience research as they can provide information about system proteomics. In a previous work we have correlated mRNA maps with the binding patterns of radioligands targeting specific molecular systems and imaged with positron emission tomography (PET) in unrelated control groups. This approach is potentially applicable to any imaging modality as long as an efficient procedure of imaging-genomic matching is provided. In the original work we considered mRNA brain maps of the whole human genome derived from the Allen human brain database (ABA) and we performed the analysis with a specific region-based segmentation with a resolution that was limited by the PET data parcellation. There we identified the need for a platform for imaging-genomic integration that should be usable with any imaging modalities and fully exploit the high resolution mapping of ABA dataset.In this work we present MENGA (Multimodal Environment for Neuroimaging and Genomic Analysis), a software platform that allows the investigation of the correlation patterns between neuroimaging data of any sort (both functional and structural) with mRNA gene expression profiles derived from the ABA database at high resolution.We applied MENGA to six different imaging datasets from three modalities (PET, single photon emission tomography and magnetic resonance imaging) targeting the dopamine and serotonin receptor systems and the myelin molecular structure. We further investigated imaging-genomic correlations in the case of mismatch between selected proteins and imaging targets

A multivariate approach to the integration of multi-omics datasets

Background: To leverage the potential of multi-omics studies, exploratory data analysis methods that provide systematic integration and comparison of multiple layers of omics information are required. We describe multiple co-inertia analysis (MCIA), an exploratory data analysis method that identifies co-relationships between multiple high dimensional datasets. Based on a covariance optimization criterion, MCIA simultaneously projects several datasets into the same dimensional space, transforming diverse sets of features onto the same scale, to extract the most variant from each dataset and facilitate biological interpretation and pathway analysis. Results: We demonstrate integration of multiple layers of information using MCIA, applied to two typical “omics” research scenarios. The integration of transcriptome and proteome profiles of cells in the NCI-60 cancer cell line panel revealed distinct, complementary features, which together increased the coverage and power of pathway analysis. Our analysis highlighted the importance of the leukemia extravasation signaling pathway in leukemia that was not highly ranked in the analysis of any individual dataset. Secondly, we compared transcriptome profiles of high grade serous ovarian tumors that were obtained, on two different microarray platforms and next generation RNA-sequencing, to identify the most informative platform and extract robust biomarkers of molecular subtypes. We discovered that the variance of RNA-sequencing data processed using RPKM had greater variance than that with MapSplice and RSEM. We provided novel markers highly associated to tumor molecular subtype combined from four data platforms. MCIA is implemented and available in the R/Bioconductor “omicade4” package. Conclusion: We believe MCIA is an attractive method for data integration and visualization of several datasets of multi-omics features observed on the same set of individuals. The method is not dependent on feature annotation, and thus it can extract important features even when there are not present across all datasets. MCIA provides simple graphical representations for the identification of relationships between large datasets

Modelling leukemia in the mouse: novel strategies in genome engineering

The work of this thesis presents novel genome engineering strategies for assembling complex targeting vectors for functional studies in the mouse. With the objective of establishing a conditional mouse model of the human acute leukemia associated with the t(4;ll)(q21;q23) translocation, an approach based on the Cre-loxP technology was chosen, and two mouse lines and one ES cell line were engineered starting from BACs. For the Af4 mouse line, a combinatorial mutant allele was designed, which supports Cre-driven interchromosomal translocation, can result in a knock-out or hypomorphic allele (also amenable to conditional gene repair strategies) and can report endogenous expression of the gene through |3-galactosidase staining. The work utilised several variations of the ET cloning methodology and served to establish their feasibility for BAG engineering as a novel approach to the generation of complex mouse knock-in/knock-out targeting vectors. Particularly noteworthy was the application of ET technology to the direct subcloning of very large BAG fragments. The Mll targeting construct constitutes the first example of a BAG based, very large knock-in vector (about 70 kb) which was successfully used to target the endogenous Mll locus by homologous recombination in mouse ES cells. The construct was engineered via ET recombination approaches. The major novelty consists in the targeting design which can simultaneously mutate two sites of the gene located very far from each other. Combined with the power of site specific recombinases (SSRs) technology, this opens the way to establishing complex, versatile mouse lines with only one round of ES cell targeting. For the second mouse line, a BAG transgenesis approach was chosen to place Cre recombinase under the control of the Ikaros gene, in a new configuration which can yield either constitutive or regulated Cre activity. Several BAG modifications were applied, and the usefulness of Tnpl recombinase in BAG engineering was established

Transcutaneous immunisation with antigens derived from tetanus toxin

Transcutaneous immunisation (TCI) is a novel needle-free method of vaccine delivery, which involves the application of soluble antigens onto the surface of intact skin. In this thesis, the immunogenicity and neutralising potency of tetanus neurotoxin (TeNT) fragments were compared to that of tetanus toxoid (TTxd) following TCI. In addition, to understand the mechanisms of induction of immune response by TCI, in vitro and in vivo interaction of tetanus proteins with skin immune cells was also investigated. The 50kDa recombinant carboxyl-terminal fragment of tetanus toxin (HCWT) and a 50kDa HC mutant (HCM115) which is devoid of neuronal binding properties, were expressed and purified by affinity chromatography, and excess endotoxin removed by size exclusion chromatography. Mice immunized with HCWT, in the absence of adjuvant, induced the highest anti-toxoid and anti-HCWT antibody titers, with significant increases in the toxin neutralising antibody response when compared with TTxd. In vitro studies demonstrated that both HC fragments and TTxd were capable of up regulating the surface expression of activation marker ICAM-1 on murine bone-marrow derived dendritic cells (DC), but not on the human keratinocyte cell line HaCaT. Real-time reverse transcriptase-PCR (Realtime RT-PCR) analysis showed that TNF-α expression was up regulated in vivo as early as 10 minutes following TCI, and this was caused by shaving rather than by tetanus proteins themselves. Immunohistochemistry staining to monitor the translocation of HCWT fragment and TTxd through the skin following TCI showed that HCWT protein could be detected in both the epidermis and dermis within four hours post-application. However, TTxd translocation appeared to be much slower in comparison. Collectively, the results presented in this thesis suggest that TCI may provide an opportunity for effective delivery of toxin-like antigens, which harbor protective epitopes and that traditional toxoid proteins may not be optimal antigens for skin immunisation

Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture

ヒトのiPS細胞から腱の細胞を作製する --アキレス腱断裂のラットに移植し、機能回復を確認--. 京都大学プレスリリース. 2021-08-31.Repairing tendon injuries with stem cells. 京都大学プレスリリース. 2021-08-31.Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury

N-Acetylcysteine prevents amyloid-β secretion in neurons derived from human pluripotent stem cells with trisomy 21

ダウン症患者さん由来の神経細胞からのアミロイドβ分泌は抗酸化剤で抑止される. 京都大学プレスリリース. 2021-08-31.Stopping dementia in Down syndrome patients. 京都大学プレスリリース. 2021-08-31.Down syndrome (DS) is caused by the trisomy of chromosome 21. Among the many disabilities found in individuals with DS is an increased risk of early-onset Alzheimer's disease (AD). Although higher oxidative stress and an upregulation of amyloid β (Aβ) peptides from an extra copy of the APP gene are attributed to the AD susceptibility, the relationship between the two factors is unclear. To address this issue, we established an in vitro cellular model using neurons differentiated from DS patient-derived induced pluripotent stem cells (iPSCs) and isogenic euploid iPSCs. Neurons differentiated from DS patient-derived iPSCs secreted more Aβ compared to those differentiated from the euploid iPSCs. Treatment of the neurons with an antioxidant, N-acetylcysteine, significantly suppressed the Aβ secretion. These findings suggest that oxidative stress has an important role in controlling the Aβ level in neurons differentiated from DS patient-derived iPSCs and that N-acetylcysteine can be a potential therapeutic option to ameliorate the Aβ secretion

Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle.

Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes