Luminescence has found wide application in biology, biotechnology and medicine. Particularly, fluorescent and bioluminescent probes allow visualization of molecular targets at the cellular level and even macromolecules o...Luminescence has found wide application in biology, biotechnology and medicine. Particularly, fluorescent and bioluminescent probes allow visualization of molecular targets at the cellular level and even macromolecules or single small-molecule analytes. Among the most reliable tools for visualization of molecular targets are so-called responsive probes, which change the intensity and colour of the emitted signal after interaction with a molecular target. The majority of such probes allow detection of a single analyte. Meanwhile, most of the processes in the human body involve multiple elements. To better understand these mechanisms, it is possible to use several responsive probes simultaneously. However, this poses a risk of their different uptake by cells or different metabolism. In order to provide a more reliable response, so-called multi-analyte and multi-responsive probes are being developed. Examples of such probes will be discussed in the article, divided based on their response mechanism and detected changes within the cell.
Enzymes, as biocatalysts, are an important target of many therapies and are also of great industrial importance, which is why repeatable and accurate parameterization of enzymatic catalysis is very important. The most po...Enzymes, as biocatalysts, are an important target of many therapies and are also of great industrial importance, which is why repeatable and accurate parameterization of enzymatic catalysis is very important. The most popular spectrophotometric detection method in enzymology, despite its low cost and speed, often cannot be used directly due to the inappropriate spectral properties of substrates and products. It is then necessary to use auxiliary enzymes, chemical modification of substrates or post-reaction analysis, which may increase the cost of measurement, extend its time or affect the accuracy. Isothermal titration calorimetry is a method widely used mainly for the characterization of inter-molecular interactions, however, its use in enzyme kinetics is gaining more and more recognition due to the direct measurement of the reaction rate using the universal parameter of heat, high sensitivity and low reagent consumption. This work discusses two strategies for conducting a kinetic calorimetric experiment and their applications.
Human myeloid leukemia cells (HL-60/S4) exposed to hyperosmotic stress with sucrose undergo dehydration and cell shrinkage. Interphase chromatin and mitotic chromosomes congeal, exhibiting altered phase separation (demix...Human myeloid leukemia cells (HL-60/S4) exposed to hyperosmotic stress with sucrose undergo dehydration and cell shrinkage. Interphase chromatin and mitotic chromosomes congeal, exhibiting altered phase separation (demixing) of chromatin proteins. To investigate changes in the transcriptome, we exposed HL-60/S4 cells to hyperosmotic sucrose stress (~600 milliOsmolar) for 30 and 60 minutes. We employed RNA-Seq of polyA mRNA to identify genes with increased or decreased transcript levels relative to untreated control cells (i.e., differential gene expression). These genes were examined for over-representation of Gene Ontology (GO) terms. In stressed cells, multiple GO terms associated with transcription, translation, mitochondrial function and proteosome activity, as well as "replication-dependent histones", were over-represented among genes with increased transcript levels; whereas, genes with decreased transcript levels were over-represented with transcription repressors. The transcriptome profiles of hyperosmotically-stressed cells suggest acquisition of cellular rebuilding, a futile homeostatic response, as these cells are ultimately doomed to a dehydrated death.
Environmental carcinogens exert their carcinogenic effects by forming DNA adducts. This type of DNA damage can also be formed endogenously as a result of, e.g., oxidative damage. Unrepaired DNA adducts may induce mutati...Environmental carcinogens exert their carcinogenic effects by forming DNA adducts. This type of DNA damage can also be formed endogenously as a result of, e.g., oxidative damage. Unrepaired DNA adducts may induce mutations in critical genes, leading to the initiation of chemical carcinogenesis. Therefore, detection, identification, and quantification of DNA adducts is essential for cancer risk assessment. Over the last 50 years, the major DNA adducts formed by different classes of environmental carcinogens were characterized. With the development of techniques such as 32P-postlabeling, their measurement was implemented into molecular epidemiology. Advances in liquid chromatography-tandem mass spectrometry (LC-MS ) made the measurement of adducts more precise and allowed to gain knowledge about their identity and structures. Therefore, opened the way to DNA adductomics, the "omics" approach investigating DNA adducts comprehensively, similarly to proteomics. This review presents the historical perspective of DNA adducts research and the emerging field of adductomics.
The review analyzes the role of physicochemical processes in the formation of the function-dependent architecture of the cell nucleus, built on the platform of a folded genome. The main attention is paid to various forms...The review analyzes the role of physicochemical processes in the formation of the function-dependent architecture of the cell nucleus, built on the platform of a folded genome. The main attention is paid to various forms of the phase separation process, primarily the processes of liquid-liquid phase separation and polymer-polymer phase separation. The role of these processes in the formation of chromatin compartments and maintenance of three-dimensional genome architecture is discussed in detail. The relationship between genome activity and the creation of functional compartments in the cell nucleus is also analyzed.
MicroRNAs are small RNAs that enable parts of the genome to regulate the other parts of the genome by RNA::RNA complementarity. Genes that encode microRNAs function as trans-acting regulators of hundreds of other genes,...MicroRNAs are small RNAs that enable parts of the genome to regulate the other parts of the genome by RNA::RNA complementarity. Genes that encode microRNAs function as trans-acting regulators of hundreds of other genes, primarily by inhibiting the production of protein from mRNAs to which the microRNAs can bind by base pairing. MicroRNAs and their Argonaute partner proteins constitute a regulatory complex (the miRISC) that exhibits astonishing regulatory versatility. microRNAs have been shown to perform diverse roles in genetic regulatory networks (GRNs) - to control developmental switches, to dampen gene expression noise, to coordinate multigene functional modules, and more broadly, to confer robustness and resilience to developmental and homeostatic processes. Genetic analysis reveals that the function of particular microRNAs can be conditional, such that the microRNA is required under particular environmental or physiological conditions, but relatively dispensable under other conditions. The diversity and versatility of microRNA function in animal systems reflects the many ways that miRISC can be regulated by cellular signaling pathways, and the structure-function interplay among microRNA, target, and Argonaute.
The article has been written for the occasion of 25 Anniversary of Gliwice Scientific Meetings (GSN). For this reason, I am going to present scientific contacts of the Institute of Oncology at Gliwice with the Institute...The article has been written for the occasion of 25 Anniversary of Gliwice Scientific Meetings (GSN). For this reason, I am going to present scientific contacts of the Institute of Oncology at Gliwice with the Institute of Human Genetics of the Polish Academy of Sciences at Poznań not only at conference occasions but also in regular research manner.
The p53 tumor suppressor protein is best known as an activator of cell cycle arrest and apoptosis. Only a fraction of p53-activated genes encode proteins affecting cellular replication and various forms of cell death (ap...The p53 tumor suppressor protein is best known as an activator of cell cycle arrest and apoptosis. Only a fraction of p53-activated genes encode proteins affecting cellular replication and various forms of cell death (apoptosis, ferroptosis, autophagy). The p53-regulated genes can be divided into so-called the core transcriptional program, which comprises genes activated in most cell types by most activators, and into the group of genes activated in in cell- or stress-specific manner. Activation of p53 occurs via the extensive set of posttranslational modifications, which adjust its stability, interaction with other transcription regulators, and its ability to form a tetramer. Surprisingly, in mouse models, the activation of the best-studied p53 target genes encoding the inhibitor of the cell cycle (CDKN1A) or the inducers of apoptosis (e.g. NOXA, PUMA) is dispensable for protection against cancers. Thus, the non-classical functions of p53 must be studied to better understand its tumor suppressive mechanisms.
The early stress response by AP-1 (FOS/JUN), supported by upregulation of c-Myc and involved in cell-fate changes and adaptation to hostile environments, is increased in cancer. The review shows the biphasic character of...The early stress response by AP-1 (FOS/JUN), supported by upregulation of c-Myc and involved in cell-fate changes and adaptation to hostile environments, is increased in cancer. The review shows the biphasic character of this response with negative feed-back typically lasting a few hours as a feature of the genome regulation by self-organising criticality. It involves rapid splitting of the pericentromeric heterochromatin clusters, opening of the active chromatin, and a massive transcription acceleration wave. Phylostratigraphic analysis revealed that AP-1 genes evolved in the Cambrian explosion ~500 Mya integrating the protein interaction networks of reproduction including proto-placenta intertwined with cytokine and immunity pathways, sex determination, oocyte maturation, and embryonal stemness. The peak of this response as part of accelerated cell senescence led by AP-1 and IL-1β was found in breast cancer cell-line resistant to doxorubicin. Adaptability of aggressive cancer to treatments can be explained by emergent stress response evolutionarily protecting reproduction.
MicroRNAs (miRNAs) are generated from stem-loop-structured double-stranded RNA precursors by the consecutive action of the two RNase III-type endoribonuclease Drosha and Dicer. However, such structures are very common on...MicroRNAs (miRNAs) are generated from stem-loop-structured double-stranded RNA precursors by the consecutive action of the two RNase III-type endoribonuclease Drosha and Dicer. However, such structures are very common on cellular transcripts and specific features have evolved that guide and regulate processing of stem-loop-structured hairpins into mature and functional miRNAs. These features include sequence motifs and local RNA structures but also trans-acting factors such as RNA binding proteins. The menu of features required for miRNA biogenesis is summarized in this review.
Information coded in DNA is replicated, modified and transmitted from the origins of protein-based life. Analogies of these processes to information processing, transmission and storage in computer systems is straightfor...Information coded in DNA is replicated, modified and transmitted from the origins of protein-based life. Analogies of these processes to information processing, transmission and storage in computer systems is straightforward and can be utilized both in analysis of biological data and in development of biologically based technical systems. Transcription and translation processes are regulated by extremely complex regulatory networks, providing control of cell growth, cell cycle and cellular responses to stress. As such, they constitute engineering control systems exerting their actions at many levels of time scale and spatial organization. This work presents an engineering perspective on DNA-related information processing and biochemical process control in living cells, followed by a review of two-way crosstalk between engineering and biology.
Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major proble...Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.
This essay is in memoriam of Professor Mieczysław Chorąży (1925 - 2021). Eminent Man, outstanding scientist, soldier of the Warsaw Uprising, moral authority for generations of fellow researchers and an exceptionally warm...This essay is in memoriam of Professor Mieczysław Chorąży (1925 - 2021). Eminent Man, outstanding scientist, soldier of the Warsaw Uprising, moral authority for generations of fellow researchers and an exceptionally warm person. His character and life works are recalled here against the background of the times he lived in.
70 Years - from DNA Double Helix via Approaching Systems Genomics to a Generalized Unified Evolution Theory.70 Years - from DNA Double Helix via Approaching Systems Genomics to a Generalized Unified Evolution Theory.
Vacuolar processing enzymes (VPEs) are plant proteases belonging to the C13 protease family. The specific activity of VPEs was characterized by comparing them to animal caspases. VPEs perform many important functions at...Vacuolar processing enzymes (VPEs) are plant proteases belonging to the C13 protease family. The specific activity of VPEs was characterized by comparing them to animal caspases. VPEs perform many important functions at various stages of plant ontogenesis, playing a role not only in the proper development of the plant organism but also in plant reactions to biotic and abiotic stress factors. A particularly important role of VPEs is noted in the processing of vacuolar proteins, enabling the production of their mature and active forms. VPEs are involved in programmed cell death, but despite residual evidence, we also suggest that VPEs are involved in autophagy. Based on literature data on autophagy in yeast, we formulate a hypothesis that VPEs during autophagy in plant cells are involved in the degradation of autophagic bodies - one of the final stages of autophagy.