BRCA Mutations-The Achilles Heel of Breast, Ovarian and Other Epithelial Cancers.

Two related tumor suppressor genes, BRCA1 and BRCA2, attract a lot of attention from both fundamental and clinical points of view. Oncogenic hereditary mutations in these genes are firmly linked to the early onset of breast and ovarian cancers. However, the molecular mechanisms that drive extensive mutagenesis in these genes are not known. In this review, we hypothesize that one of the potential mechanisms behind this phenomenon can be mediated by Alu mobile genomic elements. Linking mutations in the BRCA1 and BRCA2 genes to the general mechanisms of genome stability and DNA repair is critical to ensure the rationalized choice of anti-cancer therapy. Accordingly, we review the literature available on the mechanisms of DNA damage repair where these proteins are involved, and how the inactivating mutations in these genes (BRCAness) can be exploited in anti-cancer therapy. We also discuss a hypothesis explaining why breast and ovarian epithelial tissues are preferentially susceptible to mutations in BRCA genes. Finally, we discuss prospective novel therapeutic approaches for treating BRCAness cancers.

Dual Role of p73 in Cancer Microenvironment and DNA Damage Response.

Understanding the mechanisms that regulate cancer progression is pivotal for the development of new therapies. Although p53 is mutated in half of human cancers, its family member p73 is not. At the same time, isoforms of p73 are often overexpressed in cancers and p73 can overtake many p53 functions to kill abnormal cells. According to the latest studies, while p73 represses epithelial-mesenchymal transition and metastasis, it can also promote tumour growth by modulating crosstalk between cancer and immune cells in the tumor microenvironment, M2 macrophage polarisation, Th2 T-cell differentiation, and angiogenesis. Thus, p73 likely plays a dual role as a tumor suppressor by regulating apoptosis in response to genotoxic stress or as an oncoprotein by promoting the immunosuppressive environment and immune cell differentiation.

The p53 family member p73 in the regulation of cell stress response.

During oncogenesis, cells become unrestrictedly proliferative thereby altering the tissue homeostasis and resulting in subsequent hyperplasia. This process is paralleled by resumption of cell cycle, aberrant DNA repair and blunting the apoptotic program in response to DNA damage. In most human cancers these processes are associated with malfunctioning of tumor suppressor p53. Intriguingly, in some cases two other members of the p53 family of proteins, transcription factors p63 and p73, can compensate for loss of p53. Although both p63 and p73 can bind the same DNA sequences as p53 and their transcriptionally active isoforms are able to regulate the expression of p53-dependent genes, the strongest overlap with p53 functions was detected for p73. Surprisingly, unlike p53, the p73 is rarely lost or mutated in cancers. On the contrary, its inactive isoforms are often overexpressed in cancer. In this review, we discuss several lines of evidence that cancer cells develop various mechanisms to repress p73-mediated cell death. Moreover, p73 isoforms may promote cancer growth by enhancing an anti-oxidative response, the Warburg effect and by repressing senescence. Thus, we speculate that the role of p73 in tumorigenesis can be ambivalent and hence, requires new therapeutic strategies that would specifically repress the oncogenic functions of p73, while keeping its tumor suppressive properties intact.

Viral Membrane Fusion Proteins and RNA Sorting Mechanisms for the Molecular Delivery by Exosomes.

The advancement of precision medicine critically depends on the robustness and specificity of the carriers used for the targeted delivery of effector molecules in the human body. Numerous nanocarriers have been explored in vivo, to ensure the precise delivery of molecular cargos via tissue-specific targeting, including the endocrine part of the pancreas, thyroid, and adrenal glands. However, even after reaching the target organ, the cargo-carrying vehicle needs to enter the cell and then escape lysosomal destruction. Most artificial nanocarriers suffer from intrinsic limitations that prevent them from completing the specific delivery of the cargo. In this respect, extracellular vesicles (EVs) seem to be the natural tool for payload delivery due to their versatility and low toxicity. However, EV-mediated delivery is not selective and is usually short-ranged. By inserting the viral membrane fusion proteins into exosomes, it is possible to increase the efficiency of membrane recognition and also ease the process of membrane fusion. This review describes the molecular details of the viral-assisted interaction between the target cell and EVs. We also discuss the question of the usability of viral fusion proteins in developing extracellular vesicle-based nanocarriers with a higher efficacy of payload delivery. Finally, this review specifically highlights the role of Gag and RNA binding proteins in RNA sorting into EVs.

Evaluation of the potential defensive strategy against Influenza A in cell line models.

Background: Influenza virus can cause both seasonal infections and unpredictable pandemics. Rapidly evolving avian H5N1 and  H7N9 viruses have a potential pandemic threat for humans. Since avian Influenza can be transmitted by domestic birds, serving as a key link between wild birds and humans, an effective measure to control the influenza transmission would be eradication of the infection in poultry. It is known that the virus penetrates into the cell through binding with the terminal oligosaccharides - sialic acids (SA) - on the cell surfaces. Removal of SA might be a potential antiviral strategy. An approach to developing chicken lines that are resistant to influenza viruses could be the creation of genetically modified birds. Thus it is necessary to select a gene that provides defense to influenza. Here we have expressed in cells a range of exogenous sialidases and estimated their activity and specificity towards SA residues. Methods: Several bacterial, viral and human sialidases were tested. We adopted bacterial sialidases from Salmonella and Actinomyces for expression on the cell surface by fusing catalytic domains with transmembrane domains. We also selected Influenza A/PuertoRico/8/34/H1N1 neuraminidase and human membrane sialidase ( hNeu3) genes. Lectin binding assay was used for estimation of a α (2,3)-sialylation level by fluorescent microscopy and FACS.   Results: We compared sialidases from bacteria, Influenza virus and human. Sialidases from Salmonella and Influenza A neuraminidase effectively cleaved α (2-3)-SA receptors. Viral neuraminidase demonstrated a higher activity. Sialidases from Actinomyces and hNeu3 did not show any activity against α (2-3) SA under physiological conditions. Conclusion: Our results demonstrated that sialidases with different specificity and activity can be selected as genes providing antiviral defence. Combining chosen sialidases with different activity together with tissue-specific promoters would provide an optimal level of desialylation. Tissue specific expression of the sialidases could protect domestic birds from infection.

Successful CRISPR/Cas9 mediated homologous recombination in a chicken cell line.

Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3'-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene ( neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

Manual annotation of protein interactions.

Protein interactions are the basic building blocks for assembly of pathways and networks. Almost any biologically meaningful functionality (for instance, linear signaling pathways, chains of metabolic reactions, transcription factor dimmers, protein complexes of transcriptosome, gene-disease associations) can be represented as a combination of binary relationships between "network objects" (genes, proteins, RNA species, bioactive compounds). Naturally, the assembled pathways and networks are only as good as their "weakest" link (i.e., a wrongly assigned interaction), and the errors multiply in multi-step pathways. Therefore, the utility of "systems biology" is fundamentally dependent on quality and relevance of protein interactions. The second important parameter is the sheer number of interactions assembled in the database. One needs a "critical mass" of species-specific interactions in order to build cohesive networks for a gene list, not a constellation of non-connected proteins and protein pairs. The third issue is semantic consistency between interactions of different types. Transient physical signal transduction interactions, reactions of endogenous metabolism, transcription factor-promoter binding, and kinetic drug-target interactions are all very different in nature. Yet, they have to fit well into one database format and be consistent in order to be useful in reconstruction of cellular processes.High-quality protein interactions are available in peer-reviewed "small experiment" literature and, to a much smaller extent, patents. However, it is very challenging to find the interactions, annotate with searchable (and computable) parameters, catalogue in the database format in computer readable form, and assemble into a database. There are hundreds of thousands of mammalian interactions scattered in tens of thousands of papers in a few thousands of scientific journals. There are no widely used standards for reporting the interactions in scientific texts and, therefore, text-mining tools have only limited applicability. In order to generate a meaningful database of protein interactions, one needs a well-developed technology of manual curation, equipped with computational solutions, managerial procedures, quality control, and users' feedback. Here we describe our ever-evolving annotation approach, the important annotation issues and our solutions, and the mammalian protein interactions database MetaBase which we have been working on for over 8 years.

Network analysis of human glaucomatous optic nerve head astrocytes.

BACKGROUND: Astrocyte activation is a characteristic response to injury in the central nervous system, and can be either neurotoxic or neuroprotective, while the regulation of both roles remains elusive. METHODS: To decipher the regulatory elements controlling astrocyte-mediated neurotoxicity in glaucoma, we conducted a systems-level functional analysis of gene expression, proteomic and genetic data associated with reactive optic nerve head astrocytes (ONHAs). RESULTS: Our reconstruction of the molecular interactions affected by glaucoma revealed multi-domain biological networks controlling activation of ONHAs at the level of intercellular stimuli, intracellular signaling and core effectors. The analysis revealed that synergistic action of the transcription factors AP-1, vitamin D receptor and Nuclear Factor-kappaB in cross-activation of multiple pathways, including inflammatory cytokines, complement, clusterin, ephrins, and multiple metabolic pathways. We found that the products of over two thirds of genes linked to glaucoma by genetic analysis can be functionally interconnected into one epistatic network via experimentally-validated interactions. Finally, we built and analyzed an integrative disease pathology network from a combined set of genes revealed in genetic studies, genes differentially expressed in glaucoma and closely connected genes/proteins in the interactome. CONCLUSION: Our results suggest several key biological network modules that are involved in regulating neurotoxicity of reactive astrocytes in glaucoma, and comprise potential targets for cell-based therapy.

Polypurine (A)-rich sequences promote cross-kingdom conservation of internal ribosome entry.

The internal ribosome entry sites (IRES), IRES(CP,148)(CR) and IRES(MP,75)(CR), precede the coat protein (CP) and movement protein (MP) genes of crucifer-infecting tobamovirus (crTMV), respectively. In the present work, we analyzed the activity of these elements in transgenic plants and other organisms. Comparison of the relative activities of the crTMV IRES elements and the IRES from an animal virus--encephalomyocarditis virus--in plant, yeast, and HeLa cells identified the 148-nt IRES(CP,148)(CR) as the strongest element that also displayed IRES activity across all kingdoms. Deletion analysis suggested that the polypurine (A)-rich sequences (PARSs) contained in IRES(CP,148)(CR) are responsible for these features. On the basis of those findings, we designed artificial PARS-containing elements and showed that they, too, promote internal translation from dicistronic transcripts in vitro, in tobacco protoplasts and in HeLa cells. The maximum IRES activity was obtained from multiple copies of either (A)(4)G(A)(2)(G)(2) or G(A)(2-5) as contained in IRES(CP,148)(CR). Remarkably, even homopolymeric poly(A) was moderately active, whereas a poly(G) homopolymer was not active. Furthermore, a database search for existing PARS sequences in 5'-untranslated regions (5'UTR) of genes in tobacco genome allowed the easy identification of a number of IRES candidates, in particular in the 5'UTR of the gene encoding Nicotiana tabacum heat-shock factor 1 (NtHSF1). Consistent with our prediction, the 5'UTR of NtHSF1 turned out to be an IRES element active in vitro, in plant protoplasts and HeLa cells. We predict that PARS elements, when found in other mRNAs, will show a similar activity.