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Genomics technologies, especially transcriptional profiling, allows comparisons of gene expression within and across different organisms. By the use of the model group Tardigrada, also known as water bear, we try to understand the phenomenon of cryptobiosis. The dry organisms can survive for years without water. When re-exposed to water, the animals rehydrate and come back to life. The expression of genes in response to dehydratation and rehydratation is being examined in our laboratory through the generation of expressed sequence tags (ESTs), representational difference analysis (RDA) and subsequent microarray analysis. Molecular dissection of this complex phenomenom, including gene regulation, will allow the development of techniques for preservation and stabilisation of biological materials in a dried state.
Tardigrades have fascinated researchers for more than 300 years because of their extraordinary capability to undergo cryptobiosis and survive extreme environmental conditions. However, the survival mechanisms of tardigrades are still poorly understood mainly due to the absence of detailed knowledge about the proteome and genome of these organisms. Our study was intended to provide a basis for the functional characterization of expressed proteins in different states of tardigrades. High-throughput, high-accuracy proteomics in combination with a newly developed tardigrade specific protein database resulted in the identification of more than 3000 proteins in three different states: early embryonic state and adult animals in active and anhydrobiotic state. This comprehensive proteome resource includes protein families such as chaperones, antioxidants, ribosomal proteins, cytoskeletal proteins, transporters, protein channels, nutrient reservoirs, and developmental proteins. A comparative analysis of protein families in the different states was performed by calculating the exponentially modified protein abundance index which classifies proteins in major and minor components. This is the first step to analyzing the proteins involved in early embryonic development, and furthermore proteins which might play an important role in the transition into the anhydrobiotic state.
Background
Tardigrades represent an animal phylum with extraordinary resistance to environmental stress.
Results
To gain insights into their stress-specific adaptation potential, major clusters of related and similar proteins are identified, as well as specific functional clusters delineated comparing all tardigrades and individual species (Milnesium tardigradum, Hypsibius dujardini, Echiniscus testudo, Tulinus stephaniae, Richtersius coronifer) and functional elements in tardigrade mRNAs are analysed. We find that 39.3% of the total sequences clustered in 58 clusters of more than 20 proteins. Among these are ten tardigrade specific as well as a number of stress-specific protein clusters. Tardigrade-specific functional adaptations include strong protein, DNA- and redox protection, maintenance and protein recycling. Specific regulatory elements regulate tardigrade mRNA stability such as lox P DICE elements whereas 14 other RNA elements of higher eukaryotes are not found. Further features of tardigrade specific adaption are rapidly identified by sequence and/or pattern search on the web-tool tardigrade analyzer http://waterbear.bioapps.biozentrum.uni-wuerzburg.de. The work-bench offers nucleotide pattern analysis for promotor and regulatory element detection (tardigrade specific; nrdb) as well as rapid COG search for function assignments including species-specific repositories of all analysed data.
Conclusion
Different protein clusters and regulatory elements implicated in tardigrade stress adaptations are analysed including unpublished tardigrade sequences.
The phenomenon of desiccation tolerance, also called anhydrobiosis, involves the ability of an organism to survive the loss of almost all cellular water without sustaining irreversible damage. Although there are several physiological, morphological and ecological studies on tardigrades, only limited DNA sequence information is available. Therefore, we explored the transcriptome in the active and anhydrobiotic state of the tardigrade Milnesium tardigradum which has extraordinary tolerance to desiccation and freezing. In this study, we present the first overview of the transcriptome of M. tardigradum and its response to desiccation and discuss potential parallels to stress responses in other organisms.
Tardigrades are small, multicellular invertebrates which are able to survive times of unfavourable environmental conditions using their well-known capability to undergo cryptobiosis at any stage of their life cycle. Milnesium tardigradum has become a powerful model system for the analysis of cryptobiosis. While some genetic information is already available for Milnesium tardigradum the proteome is still to be discovered.
Photodynamic therapy (PDT) employs light activation of tissue-localized photosensitizer in an oxygen-dependent process which initiates oxidative stress, inflammation, and cell death. Laser systems, which are mostly used in PDT as light sources can be costly and oversized. light-emitting diodes (LEDs) equipment has a high potential to simplify technical part of phototriggered therapies and to reduce its costs. We develop the LED-based system that includes the control and irradiation units. The system provides the same power density at any irradiation point. Among the advantages of the device is a possibility to change the irradiation area and tune the irradiation dose. PDT experiments with cancer cells in vitro treated with two different photosensitizers demonstrated a possibility to use the developed LED-based system as a low-cost light source in PDT.
The isoquinoline quaternary alkaloid Berberine possesses a variety of pharmacological properties that suggests its promising application for an anticancer delivery system design utilizing its ability to intercalate DNA. In the current work, we have investigated the effects of Berberine on the human T cell leukemia cell line in vitro. Fluorescent microscopy of leukemic cells revealed Berberine nuclear localization. The results showed that Berberine inhibited leukemic cell growth in a time- and dose-dependent manner, that was associated with reactive oxygen species production intensification and caspase 3/7 activity increase with followed apoptosis induction. Berberine was used as a toxic and phototoxic agent for triple system synthesis along with DNA as a carrier and nanosilver as a plasmonic accelerator of Berberine electronic transitions and high energy emission absorbent centers. The proposed method allows to obtain the complex of DNA with Berberine molecules and silver nanoparticles. The optical properties of free components as well as their various combinations, including the final triple system DNA-Nanosilver-Berberine, were investigated. Obtained results support the possibility to use the triple system DNA-Nanosilver-Berberine as an alternative therapeutic agent for cancer treatment.
Complexation with C60 Fullerene Increases Doxorubicin Efficiency against Leukemic Cells In Vitro
(2019)
Conventional anticancer chemotherapy is limited because of severe side effects as well as a quickly evolving multidrug resistance of the tumor cells. To address this problem, we have explored a C60 fullerene-based nanosized system as a carrier for anticancer drugs for an optimized drug delivery to leukemic cells.
Here, we studied the physicochemical properties and anticancer activity of C60 fullerene noncovalent complexes with the commonly used anticancer drug doxorubicin. C60-Doxorubicin complexes in a ratio 1:1 and 2:1 were characterized with UV/Vis spectrometry, dynamic light scattering, and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The obtained analytical data indicated that the 140-nm complexes were stable and could be used for biological applications. In leukemic cell lines (CCRF-CEM, Jurkat, THP1 and Molt-16), the nanocomplexes revealed ≤ 3.5 higher cytotoxic potential in comparison with the free drug in a range of nanomolar concentrations. Also, the intracellular drug’s level evidenced C60 fullerene considerable nanocarrier function.
The results of this study indicated that C60 fullerene-based delivery nanocomplexes had a potential value for optimization of doxorubicin efficiency against leukemic cells.
Tardigrades have unique stress-adaptations that allow them to survive extremes of cold, heat, radiation and vacuum.
To study this, encoded protein clusters and pathways from an ongoing transcriptome study on the tardigrade Milnesium tardigradum were analyzed using bioinformatics tools and compared to expressed sequence tags (ESTs) from Hypsibius dujardini, revealing major pathways involved in resistance against extreme environmental conditions.
ESTs are available on the Tardigrade Workbench along with software and databank updates. Our analysis reveals that RNA stability motifs for M. tardigradum are different from typical motifs known from higher animals. M. tardigradum and H. dujardini protein clusters and conserved domains imply metabolic storage pathways for glycogen, glycolipids and specific secondary metabolism as well as stress response pathways (including heat shock proteins, bmh2, and specific repair pathways). Redox-, DNA-, stress- and protein protection pathways complement specific repair capabilities to achieve the strong robustness of M. tardigradum.
These pathways are partly conserved in other animals and their manipulation could boost stress adaptation even in human cells. However, the unique combination of resistance and repair pathways make tardigrades and M. tardigradum in particular so highly stress resistant.