Use of in silico approaches, synthesis and profiling of Pan-filovirus GP-1,2 preprotein specific antibodies.

Intermolecular interactions of protein-protein complexes play a principal role in the process of discovering new substances used in the diagnosis and treatment of many diseases. Among such complexes of proteins, we have to mention antibodies; they interact with specific antigens of two genera of single-stranded RNA viruses belonging to the family Filoviridae-Ebolavirus and Marburgvirus; both cause rare but fatal viral hemorrhagic fever in Africa, with pandemic potential. In this research, we conduct studies aimed at the design and evaluation of antibodies targeting the filovirus glycoprotein precursor GP-1,2 to develop potential targets for the pan-filovirus easy-to-use rapid diagnostic tests. The in silico research using the available 3D structure of the natural antibody-antigen complex was carried out to determine the stability of individual protein segments in the process of its formation and maintenance. The computed free binding energy of the complex and its decomposition for all amino acids allowed us to define the residues that play an essential role in the structure and indicated the spots where potential antibodies can be improved. Following that, the study involved targeting six epitopes of the filovirus GP1,2 with two polyclonal antibodies (pABs) and 14 monoclonal antibodies (mABs). The evaluation conducted using Enzyme Immunoassays tested 62 different sandwich combinations of monoclonal antibodies (mAbs), identifying 10 combinations that successfully captured the recombinant GP1,2 (rGP). Among these combinations, the sandwich option (3G2G12* - (rGP) - 2D8F11) exhibited the highest propensity for capturing the rGP antigen.

Novel combinatorial therapy of oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with anti PD-1 exhibits enhanced anti-cancer efficacy through promotion of intratumoral T-cell infiltration and modulation of tumour microenvironment in mesothelioma mouse model.

Introduction: Malignant mesothelioma is a rare and aggressive form of cancer. Despite improvements in cancer treatment, there are still no curative treatment modalities for advanced stage of the malignancy. The aim of this study was to evaluate the anti-tumor efficacy of a novel combinatorial therapy combining AdV5/3-D24-ICOSL-CD40L, an oncolytic vector, with an anti-PD-1 monoclonal antibody. Methods: The efficacy of the vector was confirmed in vitro in three mesothelioma cell lines - H226, Mero-82, and MSTO-211H, and subsequently the antineoplastic properties in combination with anti-PD-1 was evaluated in xenograft H226 mesothelioma BALB/c and humanized NSG mouse models. Results and discussion: Anticancer efficacy was attributed to reduced tumour volume and increased infiltration of tumour infiltrating lymphocytes, including activated cytotoxic T-cells (GrB+CD8+). Additionally, a correlation between tumour volume and activated CD8+ tumour infiltrating lymphocytes was observed. These findings were confirmed by transcriptomic analysis carried out on resected human tumour tissue, which also revealed upregulation of CD83 and CRTAM, as well as several chemokines (CXCL3, CXCL9, CXCL11) in the tumour microenvironment. Furthermore, according to observations, the combinatorial therapy had the strongest effect on reducing mesothelin and MUC16 levels. Gene set enrichment analysis suggested that the combinatorial therapy induced changes to the expression of genes belonging to the "adaptive immune response" gene ontology category. Combinatorial therapy with oncolytic adenovirus with checkpoint inhibitors may improve anticancer efficacy and survival by targeted cancer cell destruction and triggering of immunogenic cell death. Obtained results support further assessment of the AdV5/3-D24-ICOSL-CD40L in combination with checkpoint inhibitors as a novel therapeutic perspective for mesothelioma treatment.

In vitro immune evaluation of adenoviral vector-based platform for infectious diseases.

New prophylactic vaccine platforms are imperative to combat respiratory infections. The efficacy of T and B memory cell-mediated protection, generated through the adenoviral vector, was tested to assess the effectiveness of the new adenoviral-based platforms for infectious diseases. A combination of adenovirus AdV1 (adjuvant), armed with costimulatory ligands (ICOSL and CD40L), and rRBD (antigen: recombinant nonglycosylated spike protein rRBD) was used to promote the differentiation of T and B lymphocytes. Adenovirus AdV2 (adjuvant), without ligands, in combination with rRBD, served as a control. In vitro T-cell responses to the AdV1+rRBD combination revealed that CD8+ platform-specific T-cells increased (37.2 ± 0.7% vs. 23.1 ± 2.1%), and T-cells acted against SARS-CoV-2 via CD8+TEMRA (50.0 ± 1.3% vs. 36.0 ± 3.2%). Memory B cells were induced after treatment with either AdV1+rRBD (84.1 ± 0.8% vs. 82.3 ± 0.4%) or rRBD (94.6 ± 0.3% vs. 82.3 ± 0.4%). Class-switching from IgM and IgD to isotype IgG following induction with rRBD+Ab was observed. RNA-seq profiling identified gene expression patterns related to T helper cell differentiation that protect against pathogens. The analysis determined signaling pathways controlling the induction of protective immunity, including the MAPK cascade, adipocytokine, cAMP, TNF, and Toll-like receptor signaling pathway. The AdV1+rRBD formulation induced IL-6, IL-8, and TNF. RNA-seq of the VERO E6 cell line showed differences in the apoptosis gene expression stimulated with the platforms vs. mock. In conclusion, AdV1+rRBD effectively generates T and B memory cell-mediated protection, presenting promising results in producing CD8+ platform-specific T cells and isotype-switched IgG memory B cells. The platform induces protective immunity by controlling the Th1, Th2, and Th17 cell differentiation gene expression patterns. Further studies are required to confirm its effectiveness.

Multi-scale phase separation by explosive percolation with single-chromatin loop resolution.

The 2 m-long human DNA is tightly intertwined into the cell nucleus of the size of 10 µm. The DNA packing is explained by folding of chromatin fiber. This folding leads to the formation of such hierarchical structures as: chromosomal territories, compartments; densely-packed genomic regions known as Topologically Associating Domains (TADs), or Chromatin Contact Domains (CCDs), and loops. We propose models of dynamical human genome folding into hierarchical components in human lymphoblastoid, stem cell, and fibroblast cell lines. Our models are based on explosive percolation theory. The chromosomes are modeled as graphs where CTCF chromatin loops are represented as edges. The folding trajectory is simulated by gradually introducing loops to the graph following various edge addition strategies that are based on topological network properties, chromatin loop frequencies, compartmentalization, or epigenomic features. Finally, we propose the genome folding model - a biophysical pseudo-time process guided by a single scalar order parameter. The parameter is calculated by Linear Discriminant Analysis of chromatin features. We also include dynamics of loop formation by using Loop Extrusion Model (LEM) while adding them to the system. The chromatin phase separation, where fiber folds in 3D space into topological domains and compartments, is observed when the critical number of contacts is reached. We also observe that at least 80% of the loops are needed for chromatin fiber to condense in 3D space, and this is constant through various cell lines. Overall, our in-silico model integrates the high-throughput 3D genome interaction experimental data with the novel theoretical concept of phase separation, which allows us to model event-based time dynamics of chromatin loop formation and folding trajectories.

Genomic Marks Associated with Chromatin Compartments in the CTCF, RNAPII Loop and Genomic Windows.

The nature of genome organization into two basic structural compartments is as yet undiscovered. However, it has been indicated to be a mechanism of gene expression regulation. Using the classification approach, we ranked genomic marks that hint at compartmentalization. We considered a broad range of marks, including GC content, histone modifications, DNA binding proteins, open chromatin, transcription and genome regulatory segmentation in GM12878 cells. Genomic marks were defined over CTCF or RNAPII loops, which are basic elements of genome 3D structure, and over 100 kb genomic windows. Experiments were carried out to empirically assess the whole set of features, as well as the individual features in classification of loops/windows, into compartment A or B. Using Monte Carlo Feature Selection and Analysis of Variance, we constructed a ranking of feature importance for classification. The best simple indicator of compartmentalization is DNase-seq open chromatin measurement for CTCF loops, H3K4me1 for RNAPII loops and H3K79me2 for genomic windows. Among DNA binding proteins, this is RUNX3 transcription factor for loops and RNAPII for genomic windows. Chromatin state prediction methods that indicate active elements like promoters, enhancers or heterochromatin enhance the prediction of loop segregation into compartments. However, H3K9me3, H4K20me1, H3K27me3 histone modifications and GC content poorly indicate compartments.

Intermingling of chromosome territories.

The importance of higher order nuclear structure and compartmentalization for the control of the cell life is now indisputable. The genome of higher eukaryotes is organized into definite chromosome territories, and the three-dimensional organization of these territories may be intently related to genomic function, global regulation of gene expression, and even formation of exchange aberrations. In this review, we discuss our current understanding of the chromosome territories phenomenon and briefly describe how genes relocation in three-dimensional arrangement of the genome may influence their functioning. We explain how the intermingling of the edges of chromosome territories allows the formation of rare long-range interchromosomal interactions. Moreover, we illustrate recent discoveries describing the mechanisms of physical proximity-based chromosome translocations and its clinical consequence for fusion genes formation and tumor development. Finally, we characterize the inner structure of the intermingled chromosomes briefly, and explain how chromosome intermingling affects gene expression regulation.

The structural variability of the influenza A hemagglutinin receptor-binding site.

Hemagglutinin (HA) is a transmembrane protein of the influenza A virus and a key component in its life cycle. The protein allows the virus to enter a host cell by recognizing specific glycans attached to transmembrane proteins of the host, which leads to viral endocytosis. In recent years, significant progress has been made in understanding the structural relationship between changes in the HA receptor-binding site (RBS) and the sialylated glycans that bind them. Several mutations were identified in the HA RBS that allows the virus to change host tropism. Their impact on binding the analogs of human and avian receptors was determined with X-ray crystallography. In this article, we provide a short overview of the HA protein structure and briefly discuss the adaptive mutations introduced to different HA subtypes.

3DFlu: database of sequence and structural variability of the influenza hemagglutinin at population scale.

The influenza virus type A (IVA) is an important pathogen which is able to cause annual epidemics and even pandemics. This fact is the consequence of the antigenic shifts and drifts capabilities of IVA, caused by the high mutation rate and the reassortment capabilities of the virus. The hemagglutinin (HA) protein constitutes the main IVA antigen and has a crucial role in the infection mechanism, being responsible for the recognition of host-specific sialic acid derivatives. Despite the relative abundance of HA sequence and serological studies, comparative structure-based analysis of HA are less investigated. The 3DFlu database contains well annotated HA representatives: 1192 models and 263 crystallographic structures. The relations between these proteins are defined using different metrics and are visualized as a network in the provided web interface. Moreover structural and sequence comparison of the proteins can be explored. Metadata information (e.g. protein identifier, IVA strain, year and location of infection) can enhance the exploration of the presented data. With our database researchers gain a useful tool for the exploration of high quality HA models, viewing and comparing changes in the HA viral subtypes at several information levels (sequence, structure, ESP). The complete and integrated view of those relations might be useful to determine the efficiency of transmission, pathogenicity and for the investigation of evolutionary tendencies of the influenza virus.Database URL: http://nucleus3d.cent.uw.edu.pl/influenza.

DIS3 shapes the RNA polymerase II transcriptome in humans by degrading a variety of unwanted transcripts.

Human DIS3, the nuclear catalytic subunit of the exosome complex, contains exonucleolytic and endonucleolytic active domains. To identify DIS3 targets genome-wide, we combined comprehensive transcriptomic analyses of engineered HEK293 cells that expressed mutant DIS3, with Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) experiments. In cells expressing DIS3 with both catalytic sites mutated, RNAs originating from unannotated genomic regions increased ∼2.5-fold, covering ∼70% of the genome and allowing for thousands of novel transcripts to be discovered. Previously described pervasive transcription products, such as Promoter Upstream Transcripts (PROMPTs), accumulated robustly upon DIS3 dysfunction, representing a significant fraction of PAR-CLIP reads. We have also detected relatively long putative premature RNA polymerase II termination products of protein-coding genes whose levels in DIS3 mutant cells can exceed the mature mRNAs, indicating that production of such truncated RNA is a common phenomenon. In addition, we found DIS3 to be involved in controlling the formation of paraspeckles, nuclear bodies that are organized around NEAT1 lncRNA, whose short form was overexpressed in cells with mutated DIS3. Moreover, the DIS3 mutations resulted in misregulation of expression of ∼50% of transcribed protein-coding genes, probably as a secondary effect of accumulation of various noncoding RNA species. Finally, cells expressing mutant DIS3 accumulated snoRNA precursors, which correlated with a strong PAR-CLIP signal, indicating that DIS3 is the main snoRNA-processing enzyme. EXOSC10 (RRP6) instead controls the levels of the mature snoRNAs. Overall, we show that DIS3 has a major nucleoplasmic function in shaping the human RNA polymerase II transcriptome.

Probabilistic approach to predicting substrate specificity of methyltransferases.

We present a general probabilistic framework for predicting the substrate specificity of enzymes. We designed this approach to be easily applicable to different organisms and enzymes. Therefore, our predictive models do not rely on species-specific properties and use mostly sequence-derived data. Maximum Likelihood optimization is used to fine-tune model parameters and the Akaike Information Criterion is employed to overcome the issue of correlated variables. As a proof-of-principle, we apply our approach to predicting general substrate specificity of yeast methyltransferases (MTases). As input, we use several physico-chemical and biological properties of MTases: structural fold, isoelectric point, expression pattern and cellular localization. Our method accurately predicts whether a yeast MTase methylates a protein, RNA or another molecule. Among our experimentally tested predictions, 89% were confirmed, including the surprising prediction that YOR021C is the first known MTase with a SPOUT fold that methylates a substrate other than RNA (protein). Our approach not only allows for highly accurate prediction of functional specificity of MTases, but also provides insight into general rules governing MTase substrate specificity.

Genomic positions of co-expressed genes: echoes of chromosome organisation in gene expression data.

BACKGROUND: The relationships between gene expression and nuclear structure, chromosome territories in particular, are currently being elucidated experimentally. Each chromosome occupies an individual, spatially-limited space with a preferential position relative to the nuclear centre that may be specific to the cell and tissue type. We sought to discover whether patterns in gene expression databases might exist that would mirror prevailing or recurring nuclear structure patterns, chromosome territory interactions in particular. RESULTS: We used human gene expression datasets, both from a tissue expression atlas and from a large set including diverse types of perturbations. We identified groups of positional gene clusters over-represented in gene expression clusters. We show that some pairs of chromosomes and pairs of 10 Mbp long chromosome regions are significantly enriched in the expression clusters. The functions of genes involved in inter-chromosome co-expression relationships are non-random and predominantly related to cell-cell communication and reaction to external stimuli. CONCLUSIONS: We suggest that inter-chromosomal gene co-expression can be interpreted in the context of nuclear structure, and that even expression datasets that include very diverse conditions and cell types show consistent relationships.

Novel higher-order epigenetic regulation of the Bdnf gene upon seizures.

Studies in cultured cells have demonstrated the existence of higher-order epigenetic mechanisms, determining the relationship between expression of the gene and its position within the cell nucleus. It is unknown, whether such mechanisms operate in postmitotic, highly differentiated cell types, such as neurons in vivo. Accordingly, we examined whether the intranuclear positions of Bdnf and Trkb genes, encoding the major neurotrophin and its receptor respectively, change as a result of neuronal activity, and what functional consequences such movements may have. In a rat model of massive neuronal activation upon kainate-induced seizures we found that elevated neuronal expression of Bdnf is associated with its detachment from the nuclear lamina, and translocation toward the nucleus center. In contrast, the position of stably expressed Trkb remains unchanged after seizures. Our study demonstrates that activation-dependent architectural remodeling of the neuronal cell nucleus in vivo contributes to activity-dependent changes in gene expression in the brain.

The spirit of competition: to win or not to win.

A competition is a contest between individuals or groups. The gain is often an award or recognition, which serves as a catalyst to motivate individuals to put forth their very best. Such events for recognition and success are part of many International Society for Computational Biology (ISCB) Student Council Regional Student Groups (RSGs) activities. These include a popular science article contest, a Wikipedia article competition, travel grants, poster and oral presentation awards during conferences, and quizzes at social events. Organizing competitions is no different than any other event; they require a lot of hard work to be successful. Each event gives remarkable organizational and social experience for students running it, while at the same time the participants of the competitions are rewarded by prizes and recognition. It gives everybody involved an opportunity to demonstrate their extraordinary talents and skills. Competitions are unique because they bring out both the best and worst in people.

A novel protein kinase-like domain in a selenoprotein, widespread in the tree of life.

Selenoproteins serve important functions in many organisms, usually providing essential oxidoreductase enzymatic activity, often for defense against toxic xenobiotic substances. Most eukaryotic genomes possess a small number of these proteins, usually not more than 20. Selenoproteins belong to various structural classes, often related to oxidoreductase function, yet a few of them are completely uncharacterised.Here, the structural and functional prediction for the uncharacterised selenoprotein O (SELO) is presented. Using bioinformatics tools, we predict that SELO protein adopts a three-dimensional fold similar to protein kinases. Furthermore, we argue that despite the lack of conservation of the "classic" catalytic aspartate residue of the archetypical His-Arg-Asp motif, SELO kinases might have retained catalytic phosphotransferase activity, albeit with an atypical active site. Lastly, the role of the selenocysteine residue is considered and the possibility of an oxidoreductase-regulated kinase function for SELO is discussed.The novel kinase prediction is discussed in the context of functional data on SELO orthologues in model organisms, FMP40 a.k.a.YPL222W (yeast), and ydiU (bacteria). Expression data from bacteria and yeast suggest a role in oxidative stress response. Analysis of genomic neighbourhoods of SELO homologues in the three domains of life points toward a role in regulation of ABC transport, in oxidative stress response, or in basic metabolism regulation. Among bacteria possessing SELO homologues, there is a significant over-representation of aquatic organisms, also of aerobic ones. The selenocysteine residue in SELO proteins occurs only in few members of this protein family, including proteins from Metazoa, and few small eukaryotes (Ostreococcus, stramenopiles). It is also demonstrated that enterobacterial mchC proteins involved in maturation of bactericidal antibiotics, microcins, form a distant subfamily of the SELO proteins.The new protein structural domain, with a putative kinase function assigned, expands the known kinome and deserves experimental determination of its biological role within the cell-signaling network.

A widespread peroxiredoxin-like domain present in tumor suppression- and progression-implicated proteins.

BACKGROUND: Peroxide turnover and signalling are involved in many biological phenomena relevant to human diseases. Yet, all the players and mechanisms involved in peroxide perception are not known. Elucidating very remote evolutionary relationships between proteins is an approach that allows the discovery of novel protein functions. Here, we start with three human proteins, SRPX, SRPX2 and CCDC80, involved in tumor suppression and progression, which possess a conserved region of similarity. Structure and function prediction allowed the definition of P-DUDES, a phylogenetically widespread, possibly ancient protein structural domain, common to vertebrates and many bacterial species. RESULTS: We show, using bioinformatics approaches, that the P-DUDES domain, surprisingly, adopts the thioredoxin-like (Thx-like) fold. A tentative, more detailed prediction of function is made, namely, that of a 2-Cys peroxiredoxin. Incidentally, consistent overexpression of all three human P-DUDES genes in two public glioblastoma microarray gene expression datasets was discovered. This finding is discussed in the context of the tumor suppressor role that has been ascribed to P-DUDES proteins in several studies. Majority of non-redundant P-DUDES proteins are found in marine metagenome, and among the bacterial species possessing this domain a trend for a higher proportion of aquatic species is observed. CONCLUSIONS: The new protein structural domain, now with a broad enzymatic function predicted, may become a drug target once its detailed molecular mechanism of action is understood in detail.