Somatic Mutational Landscape of Splicing Factor Genes and Their Functional Consequences across 33 Cancer Types

Hotspot mutations in splicing factor genes have been recently reported at high frequency in hematological malignancies, suggesting the importance of RNA splicing in cancer. We analyzed whole-exome sequencing data across 33 tumor types in The Cancer Genome Atlas (TCGA), and we identified 119 splicing factor genes with significant non-silent mutation patterns, including mutation over-representation, recurrent loss of function (tumor suppressor-like), or hotspot mutation profile (oncogene-like). Furthermore, RNA sequencing analysis revealed altered splicing events associated with selected splicing factor mutations. In addition, we were able to identify common gene pathway profiles associated with the presence of these mutations. Our analysis suggests that somatic alteration of genes involved in the RNA-splicing process is common in cancer and may represent an underappreciated hallmark of tumorigenesis

Vapor nanobubble is the more reliable photothermal mechanism for inducing endosomal escape of siRNA without disturbing cell homeostasis

Strategies for controlled delivery of therapeutic siRNA into living cells are in high demand as endosomal escape remains the most prominent bottleneck at the intracellular level. Photothermal properties of gold nanoparticles (AuNP) can be used to overcome the endosomal membrane barrier upon laser irradiation by two mechanisms: endosomal rupture by mechanical energy from water vapor nanobubbles (VNBs), or permeabilization of the endosomal membrane by heat diffusion. Here we evaluated how both mechanisms influence cargo release, transfection efficiency, acute cytotoxicity and cell homeostasis. Using a siRNA/AuNP drug delivery system we found that the in vitro release of siRNA from the AuNP carrier occurs equally efficiently by VNB formation or heat generation. Heat-mediated endosomal escape happened more efficiently in cells that had more particles per endosome, resulting in variable siRNA-induced downregulation (20-50%). VNB-mediated endosomal escape did not dependent on the number of AuNP per endosome, yielding high downregulations (50-60%) independent of the cell type. Effects on cell homeostasis by whole transcriptome analysis, showed a quick recover after 24 h or 48 h for either of both photothermal mechanisms. We conclude that VNBs are more consistent to induce efficient endosomal escape and gene silencing independent of the cell type without long lasting effects on cell homeostasis

Medical imaging analysis with artificial neural networks

Given that neural networks have been widely reported in the research community of medical imaging, we provide a focused literature survey on recent neural network developments in computer-aided diagnosis, medical image segmentation and edge detection towards visual content analysis, and medical image registration for its pre-processing and post-processing, with the aims of increasing awareness of how neural networks can be applied to these areas and to provide a foundation for further research and practical development. Representative techniques and algorithms are explained in detail to provide inspiring examples illustrating: (i) how a known neural network with fixed structure and training procedure could be applied to resolve a medical imaging problem; (ii) how medical images could be analysed, processed, and characterised by neural networks; and (iii) how neural networks could be expanded further to resolve problems relevant to medical imaging. In the concluding section, a highlight of comparisons among many neural network applications is included to provide a global view on computational intelligence with neural networks in medical imaging

IVF based approaches towards the treatment and prevention of mitochondrial disease

PhD ThesisMitochondria are strictly maternally inherited, with all paternal mitochondria being destroyed following fertilisation. Women known to be carriers of pathogenic mtDNA mutations are therefore at increased risk of conceiving affected children. These women are currently offered the following options to aid in genetic counselling: oocyte donation, prenatal genetic diagnosis (PND) or preimplantation genetic diagnosis (PGD). One of the aims of this thesis, was to examine the feasibility of PGD for mtDNA inherited disorders, with specific emphasis on answering the following questions: how accurately does the mutation load observed in the biopsied blastomeres reflect the mutation load in the remaining embryo, are those mutation loads initially observed in the biopsied blastomeres maintained throughout preimplantation embryonic development and do mutation loads observed in the inner cell mass reflect those mutation loads observed in the extra-embryonic trophectoderm cells? In my thesis, I have now been able to provide data towards answering each of these questions through the examination of mutation loads in oocytes, embryos and blastocysts obtained from mitochondrial patients undergoing fertility treatment. Techniques, which have been developed in my current laboratory, have facilitated the characterisation of a nuclear transfer technique known as pronuclear transfer (PNT). This is a method to prevent the transmission of mitochondrial DNA disease from mother to child (Craven et al, 2010). As part of the work for my thesis, I have examined the reproducibility of the PNT technique by assessing whether the procedure could be performed by different operators, whilst maintaining levels of efficiency, survival and developmental outcome. Experiments are now being performed to examine the feasibility of PNT in normally fertilised human zygotes, created from donated oocytes. As it is unlikely that egg collection will be possible from two independent donors on the same day, the final purpose of this study was to examine the potential and feasibility of vitrification of eggs or fertilised embryos at both the pronuclear (PN) and Metaphase II (MII) stage for the purpose of the PNT technique. In summary, my studies has examined the reliability of current methods to reduce the likelihood of having a child affected by a mitochondrial DNA disorder and new techniques currently being developed to prevent the transmission of defective mitochondrial DNA, altogether. I hope this will provide fresh hope for patients with mitochondrial DNA disease

Navigating the Human Metabolome for Biomarker Identification and Design of Pharmaceutical Molecules

Metabolomics is a rapidly evolving discipline that involves the systematic study of endogenous small molecules that characterize the metabolic pathways of biological systems. The study of metabolism at a global level has the potential to contribute significantly to biomedical research, clinical medical practice, as well as drug discovery. In this paper, we present the most up-to-date metabolite and metabolic pathway resources, and we summarize the statistical, and machine-learning tools used for the analysis of data from clinical metabolomics. Through specific applications on cancer, diabetes, neurological and other diseases, we demonstrate how these tools can facilitate diagnosis and identification of potential biomarkers for use within disease diagnosis. Additionally, we discuss the increasing importance of the integration of metabolomics data in drug discovery. On a case-study based on the Human Metabolome Database (HMDB) and the Chinese Natural Product Database (CNPD), we demonstrate the close relatedness of the two data sets of compounds, and we further illustrate how structural similarity with human metabolites could assist in the design of novel pharmaceuticals and the elucidation of the molecular mechanisms of medicinal plants

Application of “omics” to Prion Biomarker Discovery

The advent of genomics and proteomics has been a catalyst for the discovery of biomarkers able to discriminate biological processes such as the pathogenesis of complex diseases. Prompt detection of prion diseases is particularly desirable given their transmissibility, which is responsible for a number of human health risks stemming from exogenous sources of prion protein. Diagnosis relies on the ability to detect the biomarker PrPSc, a pathological isoform of the host protein PrPC, which is an essential component of the infectious prion. Immunochemical detection of PrPSc is specific and sensitive enough for antemortem testing of brain tissue, however, this is not the case in accessible biological fluids or for the detection of recently identified novel prions with unique biochemical properties. A complementary approach to the detection of PrPSc itself is to identify alternative, “surrogate” gene or protein biomarkers indicative of disease. Biomarkers are also useful to track the progress of disease, especially important in the assessment of therapies, or to identify individuals “at risk”. In this review we provide perspective on current progress and pitfalls in the use of “omics” technologies to screen body fluids and tissues for biomarker discovery in prion diseases

Fishroesomes as carriers with antioxidant and anti-inflammatory bioactivities

The great diversity of marine habitats and organisms renders them a high-value source to find/develop novel drugs and formulations. Therefore, herein, sardine (Sardina pilchardus) roe was used as a lipidic source to produce liposomes. This fish product presents high nutritional value, being its lipidic content associated with important health benefits. Consequently, it can be advantageously used to produce therapeutically active delivery devices. Roe lipids were extracted using the Matyash method. After lipid film hydration and extrusion, sardine roe-derived large unilamellar liposomes (LUVs), designated as fishroesomes, presented a size of â 330 nm and a significant negative surface charge (â - 27 mV). Radical scavenging assays demonstrated that fishroesomes efficiently neutralized peroxyl, hydroxyl and nitric oxide radicals. Moreover, fishroesomes significantly reduced the expression of pro-inflammatory cytokines and chemokines by LPS-stimulated macrophages at non-toxic concentrations for L929 and THP-1 cells. Consequently, the developed liposomes exhibit unique properties as bioactive drug carriers for inflammatory diseases treatment.This work was supported by FCT/MCTES (Portuguese Foundation for Science and Technology / Ministry of Science, Technology and Higher Education) or FSE/POCH (European Social Fund through the Operational Program of Human Capital) (grant numbers PD/169/2013, PD/ BD/113795/2015, PD/BD/135246/2017, PTDC/BTM-SAL/28882/ 2017), and the NORTE 2020 Structured Project, co-funded by Norte2020 (NORTE-01-0145-FEDER-000021). Authors also thank the local fisherman for the donation of the samples