PSN 2018 PROGRAMME

While insulin treatment was discovered over a century ago, it is still the only effective routine treatment for type 1 diabetes. In this talk we will describe our attempts to stop the progression of this disease with T regulatory cells (Tregs) and to restore insulin secretion with the transplantations of pancreatic islets. We will present long-term results of our trials discussing clinical, metabolic and immune background of the patients, which, in our opinion, influenced the efficacy of this treatment. These strategies pave a way towards better treatment for diabetic patients in the near future.

An increasingly digital environment we live in gives us many new ways of finding and accessing diverse information and views. It also enables an increase in the volume of various kinds of disinformation in circulation. In the 21st century the numbers of supporters of anti-vaccine, flat Earth, or creationism movements are increasing at astonishing rate. This panel will address questions why science and evidence-based medicine are losing the battle for the public trust and how scientists and physicians can counteract the flood of disinformation.

Thousands of genetic variants have been linked to common diseases that affect the immune system, such as type 1 diabetes, rheumatoid arthritis, celiac disease and inflammatory bowel disease. However, the molecular mechanisms by which genetic variants predispose an individual to the development of immune diseases are largely unknown. Many of these variants localise in non-coding parts of the genome, indicating that they may function through regulation of gene expression. As gene expression can be highly cell type specific it is important that functional follow-up studies are carried out in disease most relevant cell types. We have developed methods that integrate disease associated variants with histone marks to pinpoint critical disease cell types. One of the identified cell types is the CD4 regulatory T cell (Tregs). Using Tregs from 100 healthy blood donors we generated detailed map of gene expression regulation by mapping quantitative trait loci (QTL) for RNA-seq (gene transcripts), ATAC-seq (chromatin accessibility) and ChIP-seq (promoter and enhancer histone modifications). This approach allowed us to refine immune disease signals to functional variants and prioritise candidate causal genes.

In 2007, Ransohoff (Journal of Clinical Epidemiology) stated that “The search for molecular markers for cancer (…) many promising initial results have been found to be unreliable or not reproducible, and the larger process of discovery can seem slow and inefficient.” In 2017, Ioannidis and Bossuyt (Clinical Chemistry) wrote: “The current biomarker pipeline is too prone to failures.” These two quotations suggest that, in the decade separating the two publications, not much has changed regarding the quality of the process of discovering and validating markers obtained by using advanced ‘omics’ technologies. Yet, the concept of precision cancer medicine is very much linked to the use of such markers. In this presentation, we review some common errors related to the design and analysis of (bio)marker studies. Avoiding those (and similar) errors might speed the search for molecular markers up and make it more efficient.

 

INTRODUCTION:

Researchers have not agreed on a single data normalization strategy for microRNA molecules quantification in serum or plasma using RT-qPCR method. In contrast to mRNA quantification in specific types of tissues, a search for a stable, endogenous reference microRNA or a set of microRNAs did not bring a success – plentiful of candidates did not pass validation. In this work, we presented an ensemble approach to normalization, which aimed to find the most stable, endogenous housekeeping microRNA or set of microRNAs.

METHODS:

We conducted a literature search to gather available datasets of microRNA qPCR profiling from serum or plasma. We applied three different normalization algorithms (GeNorm, BestKeeper, NormFinder) to each dataset to calculate the most stable single microRNA. Then we repeated the analysis for all possible pairs in each dataset to find the most stable pair microRNA. We calculated a ranking showing the position of single microRNAs and pairs determined by stability value.

RESULTS:

We downloaded 11 datasets, which followed our inclusion criteria, found in Gene Expression Omnibus database. The median fractional ranking value was higher for single than combination of microRNAs (0.49vs0.26, p=0.0159). The 10 most stable pairs derived on average from 48.62% percent of single microRNAs (48.62 95%CI34.62-62.62%).

CONCLUSIONS:

We outlined a new normalization scheme and performed the analysis on 11 available datasets, which showed that pairs of microRNA are a better fit for a reference factor than a single microRNA. We also proved that the most stable pairs are derived from the most stable single microRNAs.

 

 

INTRODUCTION: Cataract is developed by the formation of opacifications of the crystalline lens, which becomes less transparent. Age-related cataracts are leading causes of blindness nowadays affecting more than half population of 75+ yo. The methods for detection and objective evaluation of cataract in vivo are crucial for a proper management of cataractous eyes. In this paper, we demonstrate comprehensive visualization of crystalline lens opacities in vivo in patients with different types of cataracts.

METHODS: We also present visualization strategies to enhance image contrast related to lens opacifications, and to identify features of lenticular macro- and micro-morphology in different types of cataracts.

RESULTS: We developed and optimized a OCT-based optical platform enabling imaging the entire anterior segment of the eye with micrometer resolution. We imaged 50 eyes of 30 participants (mean age: 60±18 yo; age range: 26-91 yo). The access to volumetric data allowed for generating virtually any cross-section as well as en-face and / or side projection maps contrasted with different parameters. We visualized and identified different features characteristic for cataract formation such as cortical spokes, water clefts, vacuoles and enhanced scattering in the nucleus. Finally, the OCT projection maps have been used for a quantitative analysis of opacification at different grades of cataracts.

CONCLUSIONS: We demonstrated that 3-D SS-OCT technology enables volumetric visualization of in vivo macro- and microstructural changes in the crystalline lens related to opacification. Access to volumetric data allows for contrast enhancement due to the increased scattering inside the lens. Quantification of opacities may help in diagnosing and grading cataract eyes.

 

Acute lymphoblastic leukemia (ALL) is the most common cancer type among children and adolescents. Overall survival in this disease in pediatric population reaching 90-95%, however, ALL relapse might occur in approximately 10-20% of patients with probability of long-term survival of 60%. Recent advances in understanding of molecular background of pediatric ALL revealed that this disease is very heterogeneous and some of the genetic lesion could be targetable by specific drugs. The most common high-risk subtype of B-cell precursor ALL (15-20%), which could be identified by genomic screening is BCR-ABL1-like leukemia. In this group of ALL, multicolor flow cytometry, RNA NGS-sequencing and dense SNP-arrays might show molecular defect for targeted therapy with tyrosine kinase inhibitors (TKI) in 60-70%. BCR-ABL1-like BCP-ALL is also heterozygous disease and many different genomic aberration might lead to activation of a few common targetable signaling pathways like JAK-STAT, MAP-ERK and ABL1/ABL2. This knowledge would be transferable into therapeutic strategies for pediatric ALL, which may further improve clinical outcome of the patients in near future.

Classical Hodgkin lymphoma (cHL) is a B-cell malignancy diagnosed in ~20,000 new patients in North America and Europe each year. Classical HLs include small numbers of malignant Reed–Sternberg (RS) cells within an extensive inflammatory infiltrate. R-S cells secrete a variety of chemokines and cytokines that attract immune system cells. However, despite the prominent inflammatory infiltrate in cHL, there is little evidence for effective immune response against tumor cells. We found that RS cells selectively overexpressed the immunoregulatory glycan-binding protein, galectin- 1 (LGALS1), thus suppressing anti-tumor Th1 responses and favoring expansion of Treg cells. More recently, we have demonstrated that R-S cells overexpress PD-L1 and PD-L2 ligands as key targets at the 9p24.1 amplification peak in cHL lines and in primary R-S cells. Since the NFkB and JAK/STATs pathways control the expression of multiple immunoregulatory proteins, therapeutic targeting of NFkB and STATs might decrease their expression, increasing immunogenicity of R-S cells. We found that PIM1/2/3 inhibition blocked JAK-STAT signaling and markedly attenuated NFkB-dependent gene expression. PIM inhibitor decreased the expression of multiple molecules engaged in developing the immunosuppressive microenvironment, including galectin-1 and PD-L1/2. These findings indicate that PIM kinases in cHL exhibit pleiotropic effects, orchestrating tumor immune escape and supporting RS cell survival. Inhibition of PIM kinases decreases RS cell viability and disrupts signaling circuits that link R-S cells. Taken together, R-S cell are genetically and functionally programmed to blunt host anti – tumor responses. Importantly, detailed understanding of these mechanisms can be effectively translated to game-changing therapies for cHL patients.

 

INTRODUCTION:Development of cancer metastases is related to dismal prognosis, still the mechanism behind is not well understood. Recently, reactivation of epithelial-mesenchymal transition (EMT) program was linked with increased metastatic abilities of cancer cells, also those which have already disseminated to blood  – circulating tumour cells (CTCs). The aim of our research was to isolate and molecularly characterize breast cancer tumor cells from different stages of metastatic cascade –primary tumours (PT), CTCs  and lymph nodes metastases (LNM).

METHODS: Analyses were performed on PT (N=107), matched LNM (N=52) and CTCs-enriched blood fractions (N=85) from 107 early breast cancer (BC) patients. We have developed a method for isolation of epithelial (before EMT), and mesenchymal CTCs (after EMT). Levels of markers related to EMT, cancer stem cell (CSCs) (ALDH1, CD44, CD133, OCT-4, NANOG) and invasion and metastasis (uPAR, CXCR4) were tested in the collected samples. Additionally, heterogeneity of CSCs markers expression was tested in PT and LNM.

RESULTS:Mesenchymal phenotype was consistently showing increased expression of CSCs markers in PT, LNM and CTCs. Mesenchymal CTCs displayed especially malignant phenotype, with increased CXCR4, uPAR, ALDH1, OCT-4, NANOG, CD44 in comparison to epithelial CTCs. Mesenchymal CTCs were also related to poor clinico-pathological characteristics (larger tumours, lymph node involvement) and 5.4-higher risk of death ALDH1, CD133, OCT-4 and CD44 heterogeneity was decreased in LNM in comparison to PT, indicating enrichment of malignant clones at the metastatic site.

CONCLUSIONS:Molecular profiling of cancer cells disseminated from PT can deliver additional clinically important information for BC patients.

 

This talk will introduce attendees to how epigenetic mechanisms (i.e. alterations in how the DNA sequence is being accessed) might play a role in bringing about persistent nervous system dysfunction. The audience will hear about mechanisms of chronic pain; how we can study epigenetics in chronic pain; the current state of the evidence; and finally a brief introduction to how some of this evidence can be of use to researchers everywhere.

The number of people with brain diseases (psychiatric and neurodegenerative disorders, traumatic injuries and stroke, and cancer) increases, and there is an urgent need for new or better treatments and drugs. To find them, cellular and animal models are being used in basic and preclinical studies. One of the emerging animal models for studying brain and its pathologies is zebrafish (Danio rerio). It has several features, which make it so attractive, such as high homology with human genome (up to 80%), transparent embryos and a small body, efficient breeding (one pair gives 300 embryos per week), no need for permission to work up to 5 days post fertilization, and possibility to perform automatic high throughput chemical and behavioral screenings. During the lecture a zebrafish model of Parkinson’s disease will be described to show the benefits of using this animal for understanding mechanisms of neurodegeneration. Also, the stim2 knockout zebrafish lines, which we generated by CRISPR/Cas9 technology, will be presented as a model to understand some features of sporadic Alzheimer’s disease. Although there is no perfect animal model that fully mimics human pathology, the studies using genetic or chemical zebrafish models allow us to understand the molecular and cellular mechanisms of diseases as well as to identify potential drug targets and to find chemicals that rescue particular phenotype.

 

The aim of the project was to develop pharmacological methods to stimulate tissue and skin regeneration. The essence of this innovative concept of regenerative medicine is the activation of regenerative response without transplanting tissues or cells from the patient’s body. Regenerative medicine is an opportunity for people who are waiting for an organ transplant, victims of serious accidents, such as spinal cord injury, patients suffering from civilization diseases such as diabetes, chronic wounds, cardiovascular diseases, pressure ulcers or effects of stroke.

In our project we designed, obtained and examined the pro-regenerative activity of peptides, peptidomimetics,  artificial proteins and low molecular weight compounds as well as to develop innovative delivery systems such as composite hydrogels, peptide fibrils and artificial proteins obtained by genetic engineering methods. As part of the project, experiments were conducted to develop a method for the stimulation of stem cells naturally occurring in the body (endogenous) as well as transplanted (exogenous) in skin wound healing.

The biological activity of substances with pro-regenerative activity has been studied in animals and on various cell types, both immortalized lines as well as primary cells collected from patients. In the investigations of skin wound healing, the in vitro tests of proliferation, differentiation and migration of human stem cells were carried out. In the animal studies, a model of dermal lesion and ear pinnae injury in the mouse were used to quantify the regenerative response.

This work was supported by the grant no. STRATEGMED1/235077/9/NCBR/2014.

 

 

Multivariate data analysis is necessary in order to take advantage of the interdependence of the signal coming from multiple sensors and/or measurement techniques as well as the information hidden in the background and noise. We would like to present two examples of application of machine learning techniques for the analysis of neurobiological data.

In the first approach chemometric techniques are used for quantitative electrochemical analysis of neurotransmitters in mixtures containing also interferents such as uric and ascorbic acids. A novel electrochemical setup, namely the Rotating Droplet allows measurements in a very low volume of liquid (dozens of µL) which is applied between surfaces of the working electrode and a rotating rod. Hydrodynamic conditions of the measurement coupled with filtering of the signal from electrochemically irreversible interferents allow quantification of neurotransmitters in the nanomolar range.

The second application shows the potential of multivariate analysis to unambiguously discriminate between resilient, anhedonic and control specimens in a complex mice stress model. Simultaneous analysis of the data from different tests such as: light-dark box, sucrose preference, forced swim tests as well change of weight during the course of the experiment apart from discrimination allow also to choose variables of highest biological importance.

The aforementioned examples picture wide applicability of common machine learning techniques, which can be used to extract additional information from the background and separate overlapping signals, as in case of the neurotransmitter analysis, or confirm proper design of a complex animal model and choice of methods for its evaluation.

 

Cyclins and their catalytic partners, the cyclin-dependent kinases (CDKs), represent components of the core cell cycle machinery that drives cell proliferation.  Abnormal activation of these cell cycle proteins is seen very frequently in essentially all human tumor types.  In my talk, I will discuss our mouse genetic experiments that delineated the requirement for different cell cycle proteins in specific tumor types.  Our work has validated the cyclin D-CDK4/6 kinase as a therapeutic target for breast cancer treatment, and paved the way for clinical trials.  Currently, inhibitors of CDK4/6 are in clinical trials for breast cancer patients, with very promising results.  I will also discuss our studies, which revealed that CDK4/6 kinase plays important roles in controlling tumor cell metabolism as well as anti-tumor immune response. Lastly, I will discuss our ongoing work implicating other cell cycle proteins as potential therapeutic targets.

Both environmental sources and metabolic by-products in our body constantly damage cellular DNA.  To survive DNA damage, cells have to rapidly sense the DNA break and repair it in an error free manner. One of the primary causes of cancer is the incorrect repair of damaged DNA. Therefore an efficient and accurate DNA repair system needs to be in place to prevent the transformation of a normal cell to a tumor. However the converse is also true, the DNA repair machinery can be hijacked by cancer cells to negate the effects of therapy. Radiotherapy and most chemotherapeutic agents are directed to kill cancer cells by inducing irreparable DNA damage.  The cancer cells counter this treatment with changes in their DNA repair machinery, promptly repairing the damaged DNA. These cells become resistant to chemo and radio therapy. Therefore we need to have an in-depth understanding of DNA repair and the factors involved, both for gaining insight into the cause of cancer and for enhancing the efficacy of cancer therapy. Major focus of our research is to decipher the molecular mechanism of the cellular response to DNA damage, particularly DNA double strand breaks.

We developed Cancer Risk Assessment Method, which predicts the risk of getting cancer, based on the clinical data provided by the patient, and genomic data obtained by next-generation sequencing of all the genes associated with familial cancers. Our goal is to identify all people with high risk of cancer in whole Polish population of 38 million. We just started the population screening program of the scale unheard of mainly due to high costs of genomic sequencing. To make the screening program possible, we developed a novel method of genomic sequencing and analysis, which decreased 20-fold the price of the multigene test i.e. below a hundred Euros. The method has been designed and tested by the interdisciplinary research team at the University of Warsaw, and after validation immediately translated into clinical practice. It is estimated that there is at least a million women with high risk of getting breast cancer and the equal number of men with high risk of getting prostate cancer in Poland. We target at finding them all with the aim of introducing them into personalized prophylactic program. The patient’s prophylactic plan is personalized based on his/her medical history and genetic mutations found in any of 70 cancer-related genes screened. During the presentation we shall provide the preliminary results, including the prevalence and spectrum of gene mutations, based on first 15 000 patients.

In 2012 Department of Medical Genetics (Warsaw Medical University) has acquired Illumina HiSeq 1500 which allowed to establish whole exome sequencing (WES) as method for both research and diagnostic purposes. Since then we have performed > 1000 WES analyses, most of which aimed at finding diagnosis in patients suspected to suffer from rare disorders with a genetic basis. During the lecture selected findings will be presented illustrating how this approach enables discovery of novel diseases (i.e. those caused by mutations in genes not yet associated with known human disorder).

INTRODUCTION: A patient condition is often compromised by a lack of understanding of the disease and proposed therapy. It is a particular problem in Poland where the majority of medical personnel is convinced that it is not necessary to discuss with a patient about his or her conditions.

METHODS: Social media and educational platforms similar to Khan Academy offer unique opportunity to present a solid knowledge and valid interpretations of conditions and functioning of organs in a human body in an accessible and understandable manner.

RESULTS: I present results of localization of Health and Medicine chapter of Khan Academy in the Polish language together with its usage statistics.

CONCLUSIONS: Knowledgeable citizens and knowledgeable patients mean a better understanding of treatments and stricter following of rules.  Social media and educational platforms are natural tools for building a better understanding of therapies. A prerequisite is, however, that the healthcare community accepts the need and understands the advantages of dealing with better-informed patients.

 

 

Metagenomics is one of the widest developed area in Next Generation Sequencing. This method of nucleic acids analysis finds wide application in environmental, medical and industrial researches. The metagenomic profiling throught sequencing enables both complex environmental and host associated samples analysis. 

DNA for metagenomics analysis is isolated from different materials e.g. from salt, water, soil, saliva, stool or buccal, nasal and ear swabs. To identify and compare complex communities present within a given sample, 16S ribosomal RNA (rRNA) sequencing (Illumina) is used in our laboratory. For studying phylogeny and taxonomy of samples, amplicons of variable regions V3 and V4 of the 16S rRNA are amplified. Cause of its length, ~550bp, preferred sequencing platform is Illumina MiSeq in 2x300bp mode.

            In data analysis, we use OpenSource tools like Qiime2 platform for bacterial and fungal metagenomics analysis and other tools like ViromeScan or FastViromeExplorer for virome analysis. The use of this tools allows us to analyse community richness, dissimilarity, taxonomic assignment and difference abundance.

            Our basic metagenomic analysis pipeline is based on Qiime2. These platform lets us perform all steps of data pre-processing and analysis in one environment. Based on our hypothesis we can chose the best metrics of α and β diversity. Taxonomic assignment is performed with reference sequence databases which are specially prepared for Qiime2.

            Summarizing metagenomics is a powerful tool which provide information about all prokaryotic organisms living in the environment of interest. Advanced bioinformatics tools give us chance to easy and effective analyse the metagenomic data.

 

Since the discovery of Philadelphia chromosome by Nowell and Hungerford in 1960, chronic myeloid leukemia (CML) became a model neoplastic disease, not only for the studies on leukemogenesis, but also in the search for an effective cancer therapy. This myeloproliferative disease is characterized by almost uniform genetic aberration, the presence of Philadelphia chromosome, a result of reciprocal translocation t(9;22)(q34;q11) and formation of fusion BCR-ABL1 gene, encoding chimeric protein – BCR-ABL1. BCR-ABL1 is a constitutively active oncogenic tyrosine kinase that phosphorylates a number of downstream target proteins and in effect, facilitates expansion of leukemic cells. Introduction of imatinib, tyrosine kinase inhibitor (TKI) , almost two decades ago, change dramatically the landscape of the CML therapy and outcome for the majority of patients. However, despite unquestionable success of TKIs in the treatment of CML, drug resistance remains a serious problem for a significant number of patients and progression to incurable acute phase (aka blastic phase or blast crisis) still occur. Current treatment also cannot eradicate the disease, because of the persistence of the leukemic stem cells (LSCs) and although patients are “operationally” cured, therapy must be continued indefinitely, except for a minority of patients, who can safely stop therapy. LSCs are intrinsically resistant to TKIs, since their survival and proliferative capabilities are not solely dependent on BCR-ABL1 oncogenic kinase. Recently, advances in genetics, especially next-generation sequencing (NGS), allowed us to look with high resolution at the clonal architecture of leukemia, including LSCs and find additional genetic and epigenetic aberrations responsible for drug resistance. We employed a custom-designed panel of more than a thousand genes involved in human cancer followed by targeted NGS and focus on patients with unfavorable course of CML. Additionally, we studied other Philadelphia-negative myeloproliferative neoplasms (MPNs), especially triple-negative MPNs, in respect to canonical mutations in JAK2, calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) and atypical myeloid leukemia. New discoveries in our understanding of genomic landscape of CML and MPNs help not only in routine diagnostics but may help to find a cure for these malignancies and will give again new inspiration in cancer research.

Introduction: One of the main problems in the current treatment of colon cancer is the resistance of tumor cells to chemotherapy. TRAIL is a natural protein that effectively eliminates many types of tumor cells and potentially may act synergistically with some chemotherapeutics. However, the biological half-life of TRAIL in mammalian organisms is very short, significantly affecting its therapeutic effectiveness. The aim of the study was to investigate, if genetically engineered Lactococcus lactis bacteria can be used as a safe carrier of the TRAIL protein, enabling both, the control of long-term TRAIL secrection and elimination of tumor cells.
Methods: Recombinant plasmid with codon optimized for hsTRAIL-cDNA was constructed and transformed via electroporation into L.lactis NZ9000 cells. Synthesis and secretion of hsTRAIL was determined in broth supernatants by PCR, ELISA and Western blot. Cytotoxic activity of hsTRAIL against tumor cells was determined in vitro after incubation of human colon cancer cell lines HCT116 and SW480 with broth supernatants from L. lactis culture or by co-culture of tumor cells with selected L. lactis clones alone or in combination with chemotherapeutics (5-Fluorouracil, irinotecan, puromycin). Apoptosis of cancer cells was confirmed by Annexin V binding and flow cytometry analysis.
Results: L.lactis(hsTRAIL+) bacteria effectively kill HCT116 and SW480 cells and acts synergistically with cytostatics, enhancing elimination of colon cancer cells.
Conclusion: L.lactis(hsTRAIL+) bacteria produce biologically active hsTRAIL with potential application in colon cancer immunotherapy.
Supported by the National Science Centre in Poland (UMO2014/15/B/NZ5/03484) and H2020-MSCA-RISE-2017 action no.777682 “CANCER”.

 

Vps4A and Vps4B, members of the AAA ATPase family, are the only enzymes of the ESCRT machinery, which mediate membrane remodeling events, such as endosomal cargo sorting, exosome secretion, autophagy, andcytokinesis. Recent studies have shown that expression of ESCRT proteins is changed in human pathologies, including cancer.

The VPS4B locus is localized to the chromosome 18q which undergoes frequent deletions in colorectal cancer (CRC). Our analysis of The Cancer Genome Atlas dataset revealed mono- and bi-allelic deletions of VPS4B at a frequency of 67% and 2% in CRC, respectively. Consequently, by testing clinical samples of CRC, we observed a significant downregulation of both mRNA and protein levels of Vps4B. Based on this data, we hypothesized that loss or decreased levels of Vps4B make CRC cells more dependent on the Vps4A activity. We confirmed that concomitant depletion of Vps4A and Vps4B generates a synthetic lethal phenotype in CRC cell lines grown in vitro and as xenografts in immunodeficient mice. Our transcriptomics analysis indicated a number of processes affected by lack of both Vps4 paralogs. Specifically, we confirmed that cell death induced by Vps4 depletion is accompanied by a strong induction of inflammatory response involving NF-κB activation.

Cumulatively, our data demonstrate a synthetic lethality between Vps4 paralogs. We believe that vulnerability of CRC cells to Vps4 inhibition may serve as a potential target for precision CRC therapy. This may further provide a rationale for the development of specific Vps4 inhibitors.

 

 

Particularity of Mycobacterium tuberculosis (MTB) as a pathogen lies in a small percentage of infected people developing active disease, whereas in most of them the bacteria remain in an asymptomatic ‘latent’ state even for decades. It is hypothesized that in latent infection bacteria re-programme their metabolism and enter a ‘persister’ state, thus avoiding immune response and antibiotics. 

Molecular mechanisms of persistence, and metabolic switching between replicating and non-replicating state, are so far poorly understood. They constitute though a potential target for new improved treatments, especially where antibiotic therapy is unsuccessful.

Recent identification of leaderless transcripts (lacking SD sequence) in MTB in significantly higher proportion than in other bacteria, and discovery of ‘specialised’ ribosomes preferentially translating leaderless mRNAs in Escherichia coli in response to antibiotic stress, suggest that metabolic re-programming could occur at the translational level.  

Leaderless transcripts in MTB encode proteins with secondary adaptive functions, like toxin-antitoxin systems, known to be activated in ‘persistence’ models. We observed efficient translation of MTB leaderless mRNAs in stress conditions, i.e. in starvation model of growth arrest. Leaderless translation was preferentially enhanced in these conditions comparing to SD transcripts. This indicates that phenotypic adaptation of MTB relies on translational regulation and raises the questions of ribosome specialization.  

Here we present recent advances in the work of our team on identification of trigger factors and mechanisms of canonical versus leaderless translation in MTB by means of translational fusions with reporter genes, of nascent chain purification and of ribosome profiling.

 

Laboratory is not the only environment for a scientist, clinic in not the only environment for a practitioner. The society expects from us active role in innovative economy, in opinion media or in general education, each of the activities are equally important. Invited panelists will describe how their research interests sparked action in other areas, how they translate science to general public or to specific business models. Moreover, those beyond-science activities may frequently inspire future research ideas.

Effective motivation of the research team and the ability to inspire junior team members are what defines a good Leader. In this panel, two established team leaders will share their thoughts on mentoring junior team members, directing them towards new research areas and motivating them to take upon themselves high-risk ventures and succeed. Once the group grows and the scientific interests of team members diverge, the question arises how can one coordinate projects that bridge several disciplines? Such interdisciplinary projects require a broad scientific perspective and the ability of the Leader to effectively manage specialists from different fields. This panel’s aim will be to present how such interdisciplinary projects can be effectively coordinated, what are the potential gains of such efforts and what difficulties arise when basic research meets application-driven science. Whether the results to undertake such endeavors are worth it or not will be discussed with the audience during this panel.

Diversity is essential in science. It recruits new ideas, different perspectives, and fresh approaches to problem-solving — elements necessary for creativity and innovation. In recent years women are increasingly working in male-biased fields like physics or engineering however equal numbers of men and women working in senior roles are still quite a unfulfilled dream.  How to approach towards gender equality? Female panellist, all leading experts in the filed of oncology, biology, chemistry, physics as well as computer sciences will address the issue of how to counteract the gender gap.

Proteolysis is one of the most important and ancient reactions in biology. Enzymes that catalyze this reaction are called proteases. Proteases as “good guys” perform many significant biological processes like cellular quality control, apoptosis, blood coagulation or signal transduction. However they can be also “bad guys” contributing to pathological events like cancer, diabetes, coagulopathies, inflammation, infectious or degenerative diseases. It is estimated that 5–10% of all pharmaceutical targets being pursued for drug development are proteases.
Despite significant progress in recent years, one of the biggest problems in the investigation of proteases is their similar activity and location. Due to the overlapping substrate specificity (preference in the recognition of natural amino acids) it is very hard to distinguish many major proteolytic enzymes families using chemical tools developed using classic screening technologies. This also very often limits discovery of selective drug or marker for specific activity monitoring.
Our group has recently developed technology to obtain new types of ultrasensitive chemical tools (substrates, inhibitors, activity-based probes) for major families of medically important proteases. Using this novel, unique and very efficient technology called Hybrid Combinatorial Substrate Library (HyCoSuL) we have demonstrated that protease substrate specificity can be significantly enlarged by the use of unnatural amino acids in peptide sequence. An overview of major strategies to develop very active and selective chemical tools, which can be used for reliable investigation of activity and location of proteases in health and disease will be presented.

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GPCRs (G-protein coupled receptors) are a key part of the cell interface between its external and internal environments. As GPCRs are involved in etiology of many diseases, despite that they are targets of many existing drugs, a lot of efforts are still focused on development of new medicines acting selectively on a given receptor subtype, having a specific receptors profile or operating through other than orthosteric mechanism of action (like e.g. allosteric modulation).
Since the first application of our multistep virtual screening (VS) protocol to search for new serotonergic 5-HT7 receptor ligands, similar VS procedures were used in search of agents acting on many different targets (e.g., 5-HT6, 5-HT2B, GABA-B, metabotropic glutamate receptors). All the VS filters are constantly upgraded using the most recent data of ligands of a given target (for the ligand-based stage) as well as available crystal structures for homology model development and docking (for the structure-based stage). The evolution of the VS protocol, the development of different tools for docking results analysis and several applications of the in silico platform will be presented.

Many rapid diagnostic tests (RDT) used on suspected malaria cases are based on the detection of the protein encoded by Plasmodium falciparum histidine-rich protein-2 (pfhrp2) gene. Parasite samples lacking pfhrp2 and pfhrp3 genes have recently emerged, but a comprehensive genetic analysis of these variants is lacking. With this purpose, genomic data from experimental P. falciparum genetic crosses between different laboratory lines  were first analysed. The data provided insights into the segregation pattern of these deletions and the fitness of the parental genotypes in each mating experiment. The frequency of pfhrp2 deletions was consistent with a Mendelian prediction in HB3 x DD2. The pfhrp2 and pfhrp3 deletions also appear to segregate independently of each other. Analysis of 3D7 x HB3 and 7G8 x GB4 estimated the probability of spontaneously generating a pfhrp2 deletion during sexual recombination to be less than 6.2%. Next, whole genome sequence data from 1,970 P. falciparum isolates collected globally were analysed. Nine samples displayed evidence of pfhrp2deletions whereas twenty-eight isolates had evidence of pfhrp3 deletions, which are widespread in Southeast Asia. Finally, a meta-analysis of six studies from South America and four studies from Africa revealed a positive mean association between the frequencies of pfhrp2 and pfhrp3 deletions in each continent. This may reflect a selective pressure jointly acting upon both loci. In conclusion, evidence of genetic selection on both pfhrp2 and pfhrp3 gene deletions is presented, but experimental crosses do not provide evidence of a fitness cost of these variants.

 

Presenting author: Dr. Filip Stefaniak, Ph.D. (fstefaniak@genesilico.pl)

Computational methods play a pivotal role in the early stages of small molecule drug discovery, and are widely applied in virtual screening, structure optimization, and compound activity profiling. Over the last decades in medicinal chemistry, almost all the attention has been directed to protein–ligand binding and computational tools were created with such targets in mind. However, with growing discoveries of functional RNAs and their possible applications, RNA macromolecules have gained considerable attention as possible drug targets. This flow of discovery was followed by adapting existing computational tools for RNA applications, as well as active development of new RNA-tailored methods. However, due to the different nature of RNA, especially its tendency to use morphological plasticity (conformational change in ligand binding), the modeling of RNA still remains a challenging task ([1], Figure 1).

Figure 1. RNA flexibility in response to ligand-binding: NMR structures of decoding region A-Site.

The evolution of ‘protein-based’ drug discovery, and related computational methods, offers some clues on possible future directions and developments in modeling RNA interactions with small molecule ligands. We will present a new computational tool for predicting RNA-ligand interactions, which uses a coarse grained representation of both interacting partners. We will also present the plans for the future development of a predicting methods which takes into account the full flexibility of the RNA and ligand.

 

Depletion of essential (L-tryptophan) or semi-essential (L-arginine) amino acids has been shown to suppress antitumor immune responses. Arginase-1 (Arg-1) is a cytosolic enzyme catalyzing degradation of L-arginine to L-ornithine and urea, depleting tumor microenvironment of this compound. T cells need arginine to support their proliferation in the lymph nodes and to promote their ability to kill tumor cells. Arginine deprivation is associated with decreased proliferation potential of activated T cells as well as with down-regulation of CD3 zeta, a major signal transducer from the T cell receptor (TCR). Thus, arginine deprivation due to increased Arg-1 activity is a very efficient strategy of the tumor to avoid T cell-mediated effector mechanisms and, at the same time, one of the potential targets of anti-tumor therapy. Arg-1 is overexpressed not only by cancer-associated fibroblasts (CAFs), myeloid-derived suppressory cells (MDSCs) but also numerous cancer cells such as renal cell carcinoma, breast carcinoma, prostate cancer and colorectal cancer. We have recently discovered that exosomes, a double-layered small vesicles produced by ovarian cancer cells, contain enzymatically active Arg-1. Exosome-derived Arg-1 suppresses proliferation of CD4 and CD8-positive T cells activated with anti-CD3/anti-CD28 antibodies as well as T cells activated in the antigen-specific manner. All these in vitro effects are reversed by addition of an arginase inhibitor. Arg-1 containing tumor-derived exosomes are efficiently being engulfed by the dendritic cells and transported to the draining lymph nodes to create immunosuppressive environment at the site of the development of the immune response. Moreover, tumor-derived Arg-1 is detectable in the ascites of ovarian cancer-bearing animals. Arg-1-expressing ovarian cancer grows faster in vivo and its growth is slowed down by the treatment of animals with the arginase inhibitor. In vivo, in Arg-1-expressing ovarian cancer cells arginase inhibition results in maturation of the peritoneal dendritic cells and their enhanced ability to engulf and present tumor-derived proteins. Altogether, our findings provide the first evidence for the role of Arg-1 in the formation of an immunosuppressive microenvironment in ovarian cancer. We identify a novel mechanism of exosomal Arg-1 distribution from the tumor cells to antigen presenting cells. Moreover, inhibition of Arg-1 activity may be an attractive novel anti-cancer strategy. The latter idea will be further discussed in this presentation.

Funding: National Science Center – OPUS 6 Program 2013/11/B/NZ6/02790, National Science Center – OPUS 12 Program 2016/23/B/NZ6/03463, National Center for Research and Development – STRATEGMED2/265503/3/NCBIR/15, and European Commission Horizon 2020 Programme 692180-STREAMH2020-TWINN-2015.

The aim of the MOBIT project is to create a novel integrated system of Personalized Tumor Diagnostics based on high quality biobanking and integrated analysis of ‘omics’ analysis of the patients with the most common tumors, mostly non-small cell lung cancer. The integrated analysis of genomic, transcriptomic, proteomic and metabolomic biomarkers (including analysis of tumor heterogeneity) and PET/MRI imaging as a tool for the invidualized therapy has been performed on model group of patients with non-small cell lung cancer. MOBIT team is using the most advanced research equipment for high-throughput studies and it has an experience in analysis of large-scale data, access to the know-how in the field of biobanking and the hybrid PET/MRI system. The result of the development phase of the project will be the reference model of personalized tumor diagnosis (the creation of commercial services ONCOSup) and the creation of a unique software platform for the collection, integration and analysis of omics and clinical data (SmartBioBase) for use in the implementation of individualized therapy.

 

INTRODUCTION: In muscular dystrophies endogenous pool of myogenic precursors, i.e. satellite cells, declines and is insufficient to effectively repair damaged tissue. Transplantation of stem cells, like embryonic stem cells (ESCs), that could replenish satellite cells population and support muscle regeneration could be considered as possible therapy of such diseases. Understanding the molecular mechanisms that drive the differentiation of ESCs is crucial to make foundation and strengthen the basis for the therapeutic use of these cells in the future. Pax7 is the key factor that drives the specification of skeletal muscle precursor cells, controls embryonic and fetal muscle differentiation in the developing embryo, and is responsible for maintaining satellite cells in adult muscle.

METHODS: The aim of our research was to determine the role of Pax7 at the early and advanced stages of in vivo myogenic differentiation of ESCs. Two types of ESCs: control (Pax7+/+) and lacking functional Pax7 (Pax7-/-) were transplanted under the skin of mice to generate teratomas. Such model allowed us to analyze terminal myogenic differentiation, including the formation of myoblasts, myotubes, and innervated mature muscle fibers.

RESULTS:Skeletal muscle tissue was formed in all teratomas. However, teratomas arising from Pax7-/- ESCs differed from the control ones in the expression of certain mesodermal and myogenic markers. Interestingly, in Pax7-/- teratomas smaller area was occupied by cells/fibers expressing skeletal myosin.

CONCLUSIONS: In the absence of functional Pax7 initiation of myogenic differentiation of ESCs is modulated. Pax7 role in the advanced myogenesis stages was also revealed by using tratoma model.

 

 

The Retinoid X Receptor alpha (RXRa) is a nuclear receptor (NR), with an ability to control gene expression, therefore maintain diverse biological processes. That capability is regulated by post-translational modifications, SUMOylation, which involves Small Ubiquitin-Like Modifiers (SUMO) binding to the sequences within the NR. The SUMO-acceptor site is created by lysine (K) residue, surrounded by consensus motif: Ψ-K-x-E/D, where Ψ is a hydrophobic amino acid, and x any amino acid.

The methodology included protein sequences files for RXR (a,b,g) homo sapiens and applying online tools to perform multiple protein sequence alignment to assess conservation patterns in RXR family. The SUMOylation sites predictors indicated candidates for lysine modification in isoforms. Potent sites in RXRs were evaluated, in terms of polymorphisms reported in in Exome Variant Server.

The identified mutant, RXRa E247K, was assessed by site-directed mutagenesis Polymer Chain Reaction (PCR). Then, the sequence was confirmed by Sanger Sequencing. Furthermore, the cell-based SUMOylation assays in vitro will explore SNP effect on RXR ability to undergo SUMOylation.

The findings indicated high degree of similarity and identity between RXRs. The verified SUMOylation sites present in RXRa, K245 and K108, are conserved in isoform RXRb and RXRg. The use of SUMO-predictors discovered new sites in RXRb, K435; and in RXRg, K205.

Outcome from EVS searching indicated the missense mutation was present in African population for K245 in RXRa, which caused protein change: p.(E247K), and might have damaging effect on its function.

 

Animal behavior is an expression of the central nervous system function, and its importance in providing insight into physiology of neuronal circuits and neurochemistry is still growing. While there are many reliable assays available, some aspects of behavior have been difficult to study. One common limitation of automated behavior quantification is image analysis, since for complex behaviors standard 2D video-tracking methods are not sufficient. Most of current tools either focus on precise limb position and gait analysis within limited movement range or allow for free movement but are limited by low spatiotemporal resolution. We have demonstrated how high-resolution 3D kinematic tracking allows analysis of variety of behaviors without losing spatiotemporal precision. Such tools for assessment of motor function could be extremely useful in research on neurological and pain-related disorders. On the other hand, interoceptive aspects of animal experience can be approached by behavioral transcriptomics, which allows for direct linking recent experiences of an animal with simple transcriptional signatures uniquely characterizing each experience. This tool allows for decoding past experiences and infer about interoceptive state of an animal but also provides a simple method to identify new genes and brain regions involved in particular behaviors and allowing to manipulate them. These tools provide whole spectrum of new possibilities in studying both motor and interoceptive aspects of behavior that could be applied both in basic and translational research and will likely contribute to exciting discoveries in biology and medicine.

 

INTRODUCTION:

Large number of disorders which previously where thought to be multifactorial are now supposed to be rare monogenic diseases with unknown genetic background. Next Generation Sequencing (NGS) technology with advanced data analysis software may bring us closer to answer the question of the genetic cause of rare neurological diseases. We used NGS in attempt to find variants causing neurological disorders in patients from the Clinic of Neurology Jagiellonian University Medical College.

METHODS:

First group of patients was diagnosed as suffering from hereditary spastic paraplegia (HSP) – inherited rare brain disorder characterized by lower extremity spasticity and weakness, often accompanied by other systemic or neurologic abnormalities such as ataxia, seizures, cognitive impairment, dementia, amyotrophy, extrapyramidal disturbance, or peripheral neuropathy. Second group manifested more blurred neurological phenotype, making the precise clinical diagnosis challening.

RESULTS: We have identified potentially pathogenic variants in 9 of 12 patients with HSP. Some of them were previously described as pathogenic with proven pathogenicity and shared between affected family members. Moreover we have made several interesting findings in group of patients without initial precise diagnosis.

CONCLUSIONS:

Here we report on our in-house data analysis pipeline and filtering schemes built to boost discovery power and make drawing clinical conclusions easier and variants which are selected as potentially causal in patients with neurological disorders.

 

 

INTRODUCTION: Omega-3 polyunsaturated fatty acids (ω-3PUFAs) DHA and EPA play an important role in the function of neurons and glial cells. They have immunomodulatory effects and as a precursors of proresolving mediators also exert neuroprotective effects. Our previous studies revealed that the increase in content of DHA and EPA in astrocyte membranes significantly improves their antioxidant defense and energy metabolism of mitochondria. The aim of the current study was to determine the changes in the protein levels of COX-2 and iNOS and in the profile and amount of cytokines released by astrocytes preincubated with PUFAs after stimulation with IL-1β, LPS and compounds released by activated microglia. 

RESULTS: The results showed reduced expression of COX-2 in LPS-treated astrocytes enriched with DHA, but not with EPA and decreased expression of iNOS only in cells incubated with EPA. In IL-1β-stimulated astrocytes preincubated with both PUFAs expression of COX-2 and iNOS was significantly reduced. Moreover incubation of cells with DHA and EPA before stimulation with IL-1β significantly inhibited the release of several proinflammatory mediators, and increased the release of anti-inflammatory cytokines. Similar results were obtained when PUFAs-enriched astrocytes grown in a medium conditioned with microglia previously activated with LPS.

CONCLUSIONS: This results can be a basis for the development of therapy in which -3PUFA-modified astrocytes could be used as a support for the brain tissue affected by inflammation.

Alzheimer’s disease (AD) is the fifth leading cause of death among patients aged 65 and older. This fatal, neurodegenerative disease has complex nature reflected in many factors involved in its pathogenesis. Among several well-known hallmarks of AD, amyloid-β plaques and neurofibrillary tangles are of great interest to researchers as biological targets for new anti-Alzheimer’s agents.¹ In the search for novel therapies for AD, multi-target-directed ligand (MTDL) strategy seems to be very attractive approach as it provides compounds acting on several targets simultaneously.²

Our research team deals with design, synthesis and biological evaluation of novel derivatives with multi-target-directed activity. Recently, we reported a series of 1-benzylamino-2-hydroxyalkyl derivatives combining a unique inhibitory activity against butyrylcholinesterase, β-secretase, β-amyloid and tau protein aggregation. The most promising compound from this series was characterized by balanced activity against both disease-modifying as well as symptomatic targets.³ Successful results of 5-HT6 receptor (5-HT6R) antagonists in Phase II clinical trials promted us to design molecules aiming at inhibiting cholinesterases and blocking 5-HT6R. One of the recently published compounds was blood-brain barrier permeable antagonist of 5-HT6R with cholinesterases and amyloid β aggregation inhibitory activities.⁴

Several groups of novel MTDLs aiming at symptoms and causes of Alzheimer’s disease will be presented.

Acknowledgments: Part of the research was done in co-operation with University of Ljubljana and University of Barcelona. Financial support: NCN Grants 2016/23/D/NZ7/01328 and 2016/21/B/NZ7/01744.

¹ Citron, M. Nat. Rev. Drug Discov., 2010, 9, 387-398. ² Cavalli, A. et al. J. Med. Chem., 2008, 51(3), 347-72. ³ Panek, D. et al. ACS Chem. Neurosci., 2018, In Press. ⁴ Więckowska, A. et al. ACS Chem. Neurosci., 2018, In Press.

INTRODUCTION:  Neurodegenerative ocular pathologies are not limited to the retina but extend to other parts of the brain. It could be crucial for development of new therapeutic strategies to reliably monitor extra-retinal brain neurodegeneration and possible neuroprotective effects. We have developed a MR-based setting for preclinical evaluation of novel therapies in mouse model of glaucoma.

METHODS: DBA/2J mice that develop spontaneous age-dependent glaucoma-like pathology and age-matched controls were scanned with 7T MR scanner. The protocol included high resolution T2-weighted imaging, localized single voxel spectroscopy (MRS) and evaluation of anterograde axonal transport with MEMRI technique.

RESULTS: We observed age-dependent changes in eyeball morphology in DBA/2J mice – deepening of anterior eye chamber. The visual cortex volume was decreasing in ‘glaucoma’ mice in an age-dependent manner. MRS revealed biochemical changes in the visual cortex of the ‘glaucoma’ mice: decrease in taurine and glutamine levels and increase in glutamate. Anterograde axonal transport impairment was detected at an early stage of pathology and was dramatic in mice with advanced pathology.

CONCLUSIONS:DBA/2J mouse model of glaucoma develop pathologies that are associated with early changes in the brain that could be revealed by magnetic resonance imaging and spectroscopy. Our approach may serve for evaluation of new treatment strategies in ocular disorders that involve neurodegeneration.

Acknowledgments: The project was supported by Polish National Science Centre, grant No. DEC-2012/07/D/NZ4/04199. Project carried out with the use of the CePT infrastructure financed by the European Union – the European Regional Development Fund within the Operational Programme “Innovative economy” for 2007-2013.

Matrix metalloproteinase 9, MMP-9 is an extracellularly operating enzyme that has been demonstrated as important regulatory molecule in control of synaptic plasticity, learning and memory. We have shown that either genetic or pharmacological inhibition of MMP-9 impairs late phase of long-term potentiation at various pathways, as well as appetitive and spatial memory formation, although aversive learning remains apparently intact in MMP-9 KO mice. MMP-9 is locally translated and released from the excitatory synapses in response to neuronal activity. Extrasynaptic MMP-9 is required for growth and maturation of the dendritic spines to accumulate and immobilize AMPA receptors, making the excitatory synapses more efficacious. Our studies on animal models have implicated MMP-9 in such neuropsychiatric conditions, as e.g., epileptogenesis, autism spectrum disorders, development of addiction, and depression. We have also reported that in humans MMP-9 appears to contribute to epilepsy, alcohol addiction, Fragile X Syndrome, schizophrenia and bipolar disorder. In aggregate, all those conditions may be considered as relying on alterations of dendritic spines/excitatory synapses and thus understanding the role played by MMP-9 in the synaptic plasticity may allow to elucidate the underpinnings of major neuropsychiatric disorders.

Mitochondria must import the large majority of their proteome. We aim to understand cellular consequences of defects in the mitochondrial protein import. Two main arms of the cellular response to protein import dysfunction include the inhibition of cytosolic translation and activation of the major protein degradation machinery, the proteasome. The stimulation of the proteasome is driven by its more efficient assembly as a direct response to the amount of mistargeted proteins. The mechanism is beneficial for cells. Interestingly, activation of the proteasome could be uncoupled from translational inhibition by mistargeting of mitochondrial proteins and in the presence of healthy mitochondria. Under these conditions only proteasomal activity, and not the cellular protein synthesis, was modulated. The synthesis of cellular proteins is regulated by the signals, which come directly from the dysfunctional mitochondria. To understand translational inhibition, a site-specific redox proteomic analysis to delineate the yeast redoxome was performed. Increased levels of intracellular reactive oxygen species (ROSO caused by the mitochondria serve as a signal to attenuate global protein synthesis. Mapping of redox-active thiols in proteins revealed ROS-sensitive sites in several components of the translation apparatus. Thus, the increased levels of intracellular ROS caused by dysfunctional mitochondria serve as a signal to attenuate global protein synthesis.

Retinal photoreceptor cells can respond to light throughout our lives because they continuously regenerate a light-sensitive chromophore and certain essential structures. This series of reactions takes place in photoreceptor and the retinal pigment epithelium (RPE) cells. Defects in many proteins involved in these processes cause photoreceptor degeneration. For example, mutations in the rhodopsin gene may cause human diseases like retinitis pigmentosa (RP) that usually result in late-onset blindness. Our long-term goal is to elucidate the molecular mechanisms of phototransduction and retinal degeneration to discover therapeutics for inherited human blinding diseases caused by mutations in phototransduction genes. This is a necessary prerequisite for developing evidence-based therapeutic approaches for treatment of these pathological conditions. Combining disciplines such as state-of-the art imaging, bioinformatics, genomics, and structural biology with classical histopathological, physiological, and biochemical methods can dramatically increase understanding of causes of inherited human retinopathies.