Engineered apoptotic nucleases for chromatin research

We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5

The role of NF-κB transcription factor in cellular response to ionizing radiation

The NF-κB transcription factor is involved in different aspects of the cellular response to stress, including atypical NF-κB pathway activated by damage induced by ionizing radiation. Moreover, NF-κB could be involved in the regulation of genes activated by other stress-responsive factors. Here we aimed to perform the integrative genomics screening to compare subsets of NF-κB-dependent genes induced by a pro-inflammatory stimulus and a high dose of ionizing radiation and also to identify new genes potentially co-regulated by NF-κB and p53 transcription factors in irradiated cells. Methods. The RelA-containing NF-κB dimers were activated by TNFα cytokine (classical proinflammatory pathway) and a single 4 or 10 Gy dose (atypical radiation-induced pathway) in human osteosarcoma cells. NF-κB-dependent and p53-dependent genes were identified using the gene expression profiling (by RNA-Seq) in cells with downregulated RELA or TP53 combined with the global profiling of RelA and p53 binding sites (by ChIP-Seq). Candidate genes were subsequently validated by quantitative PCR. Results: There were 37 NF-κB-dependent protein-coding genes identified: in all cases RelA bound in their regulatory regions upon activation while downregulation of RELA suppressed their stimulus-induced upregulation, which apparently indicated the positive regulation mode (this set of genes included a few “novel” NF-κB-dependent species). The kinetics of the NF-κB activation was slower in cells exposed to radiation than in cytokine-stimulated ones. However, subsets of NF-κB-dependent genes upregulated by both types of stimuli were essentially the same. Moreover, we identified a subset of radiation-modulated genes whose expression was affected by silencing of both TP53 and RELA, and a subset of radiation-upregulated genes where radiation stimulated binding of both p53 and RelA. For three genes an antagonistic effect of both transcription factors was observed: IL4I1 was activated by NF-κB and inhibited by p53, while CDKN1A and SERPINE1 were activated by p53 and inhibited by NF-κB. Moreover, RRAD was putatively co-activated by both factors. Conclusions: One could expect that similar cellular processes resulting from activation of the NF-κB pathway could be induced in cells responding to pro-inflammatory cytokines and in cells where so-called “sterile inflammation” response was initiated by radiation-induced damage. Moreover, certain stress-responsive genes induced by ionizing radiation could be co-regulated by NF-κB and p53.publishedVersio

Active heat shock transcription factor 1 supports migration of the melanoma cells via vinculin down-regulation

AbstractHeat shock transcription factor 1 (HSF1), the major regulator of stress response, is frequently activated in cancer and has an apparent role in malignant transformation. Here we analyzed the influence of the over-expression of a constitutively active transcriptionally-competent HSF1 mutant form on phenotypes of mouse and human melanoma cells. We observed that the expression of active HSF1 supported anchorage-independent growth in vitro, and metastatic spread in the animal model in vivo, although the proliferation rate of cancer cells was not affected. Furthermore, active HSF1 enhanced cell motility, reduced the adherence of cells to a fibronectin-coated surface, and affected the actin cytoskeleton. We found that although the expression of active HSF1 did not affect levels of epithelial-to-mesenchymal transition markers, it caused transcriptional down-regulation of vinculin, protein involved in cell motility, and adherence. Functional HSF1-binding sites were found in mouse and human Vcl/VCL genes, indicating a direct role of HSF1 in the regulation of this gene. An apparent association between HSF1-induced down-regulation of vinculin, increased motility, and a reduced adherence of cells suggests a possible mechanism of HSF1-mediated enhancement of the metastatic potential of cancer cells

Metabolomic Signature Discriminates Normal Human Cornea from Keratoconus - A Pilot GC/MS Study

The molecular etiology of keratoconus (KC), a pathological condition of the human cornea, remains unclear. The aim of this work was to perform profiling of metabolites and identification of features discriminating this pathology from the normal cornea. The combination of gas chromatography and mass spectrometry (GC/MS) techniques has been applied for profiling and identification of metabolites in corneal buttons from 6 healthy controls and 7 KC patients. An untargeted GC/MS-based approach allowed the detection of 377 compounds, including 46 identified unique metabolites, whose levels enabled the separation of compared groups of samples in unsupervised hierarchical cluster analysis. There were 13 identified metabolites whose levels di erentiated between groups of samples. Downregulation of several carboxylic acids, fatty acids, and steroids was observed in KC when compared to the normal cornea. Metabolic pathways associated with compounds that discriminated both groups were involved in energy production, lipid metabolism, and amino acid metabolism. An observed signature may reflect cellular processes involved in the development of KC pathology, including oxidative stress and inflammation

Changes of protein glycosylation in the course of radiotherapy

This is the first study of changes in protein glycosylation due to exposure of human subjects to ionizing radiation. Site specific glycosylation patterns of 7 major plasma proteins were analyzed; 171 glycoforms were identified; and the abundance of 99 of these was followed in the course of cancer radiotherapy in 10 individual patients. It was found that glycosylation of plasma proteins does change in response to partial body irradiation (~60 Gy), and the effects last during follow-up; the abundance of some glycoforms changed more than twofold. Both the degree of changes and their time-evolution showed large inter-individual variability

Harmonization of exosome isolation from culture supernatants for optimized proteomics analysis

<div><p>Exosomes, the smallest subset of extracellular vesicles (EVs), have recently attracted much attention in the scientific community. Their involvement in intercellular communication and molecular reprogramming of different cell types created a demand for a stringent characterization of the proteome which exosomes carry and deliver to recipient cells. Mass spectrometry (MS) has been extensively used for exosome protein profiling. Unfortunately, no standards have been established for exosome isolation and their preparation for MS, leading to accumulation of artefactual data. These include the presence of high-abundance exosome-contaminating serum proteins in culture media which mask low-abundance exosome-specific components, isolation methods that fail to yield “pure” vesicles or variability in protein solubilization protocols. There is an unmet need for the development of standards for exosome generation, harvesting, and isolation from cellular supernatants and for optimization of protein extraction methods before proteomics analysis by MS. In this communication, we illustrate the existing problems in this field and provide a set of recommendations that are expected to harmonize exosome processing for MS and provide the faithful picture of the proteomes carried by exosomes. The recommended workflow for effective and specific identification of proteins in exosomes released by the low number of cells involves culturing cells in medium with a reduced concentration of exosome-depleted serum, purification of exosomes by size-exclusion chromatography, a combination of different protein extraction method and removal of serum-derived proteins from the final dataset using an appropriate sample of cell-unexposed medium as a control. Application of this method allowed detection of >250 vesicle-specific proteins in exosomes from 10 mL of culture medium.</p></div

Mass spectrometry-based analysis of therapy-related changes in serum proteome patterns of patients with early-stage breast cancer

<p>Abstract</p> <p>Background</p> <p>The proteomics approach termed proteome pattern analysis has been shown previously to have potential in the detection and classification of breast cancer. Here we aimed to identify changes in serum proteome patterns related to therapy of breast cancer patients.</p> <p>Methods</p> <p>Blood samples were collected before the start of therapy, after the surgical resection of tumors and one year after the end of therapy in a group of 70 patients diagnosed at early stages of the disease. Patients were treated with surgery either independently (26) or in combination with neoadjuvant chemotherapy (5) or adjuvant radio/chemotherapy (39). The low-molecular-weight fraction of serum proteome was examined using MALDI-ToF mass spectrometry, and then changes in intensities of peptide ions registered in a mass range between 2,000 and 14,000 Da were identified and correlated with clinical data.</p> <p>Results</p> <p>We found that surgical resection of tumors did not have an immediate effect on the mass profiles of the serum proteome. On the other hand, significant long-term effects were observed in serum proteome patterns one year after the end of basic treatment (we found that about 20 peptides exhibited significant changes in their abundances). Moreover, the significant differences were found primarily in the subgroup of patients treated with adjuvant therapy, but not in the subgroup subjected only to surgery. This suggests that the observed changes reflect overall responses of the patients to the toxic effects of adjuvant radio/chemotherapy. In line with this hypothesis we detected two serum peptides (registered m/z values 2,184 and 5,403 Da) whose changes correlated significantly with the type of treatment employed (their abundances decreased after adjuvant therapy, but increased in patients treated only with surgery). On the other hand, no significant correlation was found between changes in the abundance of any spectral component or clinical features of patients, including staging and grading of tumors.</p> <p>Conclusions</p> <p>The study establishes a high potential of MALDI-ToF-based analyses for the detection of dynamic changes in the serum proteome related to therapy of breast cancer patients, which revealed the potential applicability of serum proteome patterns analyses in monitoring the toxicity of therapy.</p

Combining low-dose CT-based radiomics and metabolomics for early lung cancer screening support

Due to its predominantly asymptomatic or mildly symptomatic progression, lung cancer is often diagnosed in advanced stages, resulting in poorer survival rates for patients. As with other cancers, early detection significantly improves the chances of successful treatment. Early diagnosis can be facilitated through screening programs designed to detect lung tissue tumors when they are still small, typically around 3mm in size. However, the analysis of extensive screening program data is hampered by limited access to medical experts. In this study, we developed a procedure for identifying potential malignant neoplastic lesions within lung parenchyma. The system leverages machine learning (ML) techniques applied to two types of measurements: low-dose Computed Tomography-based radiomics and metabolomics. Using data from two Polish screening programs, two ML algorithms were tested, along with various integration methods, to create a final model that combines both modalities to support lung cancer screening

Establishing propositional truth-value in counterfactual and real-world contexts during sentence comprehension: Differential sensitivity of the left and right inferior frontal gyri

What makes a proposition true or false has traditionally played an essential role in philosophical and linguistic theories of meaning. A comprehensive neurobiological theory of language must ultimately be able to explain the combined contributions of real-world truth-value and discourse context to sentence meaning. This fMRI study investigated the neural circuits that are sensitive to the propositional truth-value of sentences about counterfactual worlds, aiming to reveal differential hemispheric sensitivity of the inferior prefrontal gyri to counterfactual truth-value and real-world truth-value. Participants read true or false counterfactual conditional sentences (“If N.A.S.A. had not developed its Apollo Project, the first country to land on the moon would be Russia/America”) and real-world sentences (“Because N.A.S.A. developed its Apollo Project, the first country to land on the moon has been America/Russia”) that were matched on contextual constraint and truth-value. ROI analyses showed that whereas the left BA 47 showed similar activity increases to counterfactual false sentences and to real-world false sentences (compared to true sentences), the right BA 47 showed a larger increase for counterfactual false sentences. Moreover, whole-brain analyses revealed a distributed neural circuit for dealing with propositional truth-value. These results constitute the first evidence for hemispheric differences in processing counterfactual truth-value and real-world truth-value, and point toward additional right hemisphere involvement in counterfactual comprehension