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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 C\(_{60}\) 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 C\(_{60}\) fullerene noncovalent complexes with the commonly used anticancer drug doxorubicin. C\(_{60}\)-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 C\(_{60}\) fullerene considerable nanocarrier function.The results of this study indicated that C\(_{60}\) fullerene-based delivery nanocomplexes had a potential value for optimization of doxorubicin efficiency against leukemic cells.
Aspergillus is an important fungal genus containing economically important species, as well as pathogenic species of animals and plants. Using eighteen fungal species of the genus Aspergillus, we conducted a comprehensive investigation of conserved genes and their evolution. This also allows us to investigate the selection pressure driving the adaptive evolution in the pathogenic species A. fumigatus. Among single-copy orthologs (SCOs) for A. fumigatus and the closely related species A. fischeri, we identified 122 versus 50 positively selected genes (PSGs), respectively. Moreover, twenty conserved genes of unknown function were established to be positively selected and thus important for adaption. A. fumigatus PSGs interacting with human host proteins show over-representation of adaptive, symbiosis-related, immunomodulatory and virulence-related pathways, such as the TGF-β pathway, insulin receptor signaling, IL1 pathway and interfering with phagosomal GTPase signaling. Additionally, among the virulence factor coding genes, secretory and membrane protein-coding genes in multi-copy gene families, 212 genes underwent positive selection and also suggest increased adaptation, such as fungal immune evasion mechanisms (aspf2), siderophore biosynthesis (sidD), fumarylalanine production (sidE), stress tolerance (atfA) and thermotolerance (sodA). These genes presumably contribute to host adaptation strategies. Genes for the biosynthesis of gliotoxin are shared among all the close relatives of A. fumigatus as an ancient defense mechanism. Positive selection plays a crucial role in the adaptive evolution of A. fumigatus. The genome-wide profile of PSGs provides valuable targets for further research on the mechanisms of immune evasion, antimycotic targeting and understanding fundamental virulence processes.
Aspergillus fumigatus is a saprophytic, cosmopolitan fungus that attacks patients with a weak immune system. A rational solution against fungal infection aims to manipulate fungal metabolism or to block enzymes essential for Aspergillus survival. Here we discuss and compare different bioinformatics approaches to analyze possible targeting strategies on fungal-unique pathways. For instance, phylogenetic analysis reveals fungal targets, while domain analysis allows us to spot minor differences in protein composition between the host and fungi. Moreover, protein networks between host and fungi can be systematically compared by looking at orthologs and exploiting information from host–pathogen interaction databases. Further data—such as knowledge of a three-dimensional structure, gene expression data, or information from calculated metabolic fluxes—refine the search and rapidly put a focus on the best targets for antimycotics. We analyzed several of the best targets for application to structure-based drug design. Finally, we discuss general advantages and limitations in identification of unique fungal pathways and protein targets when applying bioinformatics tools.
Synergy of chemo- and photodynamic therapies with C\(_{60}\) Fullerene-Doxorubicin nanocomplex
(2019)
A nanosized drug complex was explored to improve the efficiency of cancer chemotherapy, complementing it with nanodelivery and photodynamic therapy. For this, nanomolar amounts of a non-covalent nanocomplex of Doxorubicin (Dox) with carbon nanoparticle C\(_{60}\) fullerene (C\(_{60}\)) were applied in 1:1 and 2:1 molar ratio, exploiting C\(_{60}\) both as a drug-carrier and as a photosensitizer. The fluorescence microscopy analysis of human leukemic CCRF-CEM cells, in vitro cancer model, treated with nanocomplexes showed Dox’s nuclear and C\(_{60}\)'s extranuclear localization. It gave an opportunity to realize a double hit strategy against cancer cells based on Dox's antiproliferative activity and C\(_{60}\)'s photoinduced pro-oxidant activity. When cells were treated with 2:1 C\(_{60}\)-Dox and irradiated at 405 nm the high cytotoxicity of photo-irradiated C\(_{60}\)-Dox enabled a nanomolar concentration of Dox and C\(_{60}\) to efficiently kill cancer cells in vitro. The high pro-oxidant and pro-apoptotic efficiency decreased IC\(_{50}\) 16, 9 and 7 × 10\(^3\)-fold, if compared with the action of Dox, non-irradiated nanocomplex, and C\(_{60}\)'s photodynamic effect, correspondingly. Hereafter, a strong synergy of therapy arising from the combination of C\(_{60}\)-mediated Dox delivery and C\(_{60}\) photoexcitation was revealed. Our data indicate that a combination of chemo- and photodynamic therapies with C\(_{60}\)-Dox nanoformulation provides a promising synergetic approach for cancer treatment.
The human-pathogenic bacterium Salmonella enterica adjusts and adapts to different environments while attempting colonization. In the course of infection nutrient availabilities change drastically. New techniques, "-omics" data and subsequent integration by systems biology improve our understanding of these changes. We review changes in metabolism focusing on amino acid and carbohydrate metabolism. Furthermore, the adaptation process is associated with the activation of genes of the Salmonella pathogenicity islands (SPIs). Anti-infective strategies have to take these insights into account and include metabolic and other strategies. Salmonella infections will remain a challenge for infection biology.
Epicardium-derived cells (EPDC) and atrial stromal cells (ASC) display cardio-regenerative potential, but the molecular details are still unexplored. Signals which induce activation, migration and differentiation of these cells are largely unknown. Here we have isolated rat ventricular EPDC and rat/human ASC and performed genetic and proteomic profiling. EPDC and ASC expressed epicardial/mesenchymal markers (WT-1, Tbx18, CD73,CD90, CD44, CD105), cardiac markers (Gata4, Tbx5, troponin T) and also contained phosphocreatine. We used cell surface biotinylation to isolate plasma membrane proteins of rEPDC and hASC, Nano-liquid chromatography with subsequent mass spectrometry and bioinformatics analysis identified 396 rat and 239 human plasma membrane proteins with 149 overlapping proteins. Functional GO-term analysis revealed several significantly enriched categories related to extracellular matrix (ECM), cell migration/differentiation, immunology or angiogenesis. We identified receptors for ephrin and growth factors (IGF, PDGF, EGF, anthrax toxin) known to be involved in cardiac repair and regeneration. Functional category enrichment identified clusters around integrins, PI3K/Akt-signaling and various cardiomyopathies. Our study indicates that EPDC and ASC have a similar molecular phenotype related to cardiac healing/regeneration. The cell surface proteome repository will help to further unravel the molecular details of their cardio-regenerative potential and their role in cardiac diseases.
Though several previous studies reported the in vitro and in vivo antioxidant effect of kinetin (Kn), details on its action in cisplatin-induced toxicity are still scarce. In this study we evaluated, for the first time, the effects of kinetin in cisplatin (cp)- induced liver and lymphocyte toxicity in rats. Wistar male albino rats were divided into nine groups: (i) the control (C), (ii) groups 2,3 and 4, which received 0.25, 0.5 and 1 mg/kg kinetin for 10 days; (iii) the cisplatin (cp) group, which received a single intraperitoneal injection of CP (7.0 mg/kg); and (iv) groups 6, 7, 8 and 9, which received, for 10 days, 0.25, 0.5 and 1 mg/kg kinetin or 200 mg/kg vitamin C, respectively, and Cp on the fourth day. CP-injected rats showed a significant impairment in biochemical, oxidative stress and inflammatory parameters in hepatic tissue and lymphocytes. PCR showed a profound increase in caspase-3, and a significant decline in AKT gene expression. Intriguingly, Kn treatment restored the biochemical, redox status and inflammatory parameters. Hepatic AKT and caspase-3 expression as well as CD95 levels in lymphocytes were also restored. In conclusion, Kn mitigated oxidative imbalance, inflammation and apoptosis in CP-induced liver and lymphocyte toxicity; therefore, it can be considered as a promising therapy.
The olive tree is a venerable Mediterranean plant and often used in traditional medicine. The main aim of the present study was to evaluate the effect of Olea europaea L. cv. Arbosana leaf extract (OLE) and its encapsulation within a spanlastic dosage form on the improvement of its pro-oxidant and antiproliferative activity against HepG-2, MCF-7, and Caco-2 human cancer cell lines. The LC-HRESIMS-assisted metabolomic profile of OLE putatively annotated 20 major metabolites and showed considerable in vitro antiproliferative activity against HepG-2, MCF-7, and Caco-2 cell lines with IC\(_{50}\) values of 9.2 ± 0.8, 7.1 ± 0.9, and 6.5 ± 0.7 µg/mL, respectively. The encapsulation of OLE within a (spanlastic) nanocarrier system, using a spraying method and Span 40 and Tween 80 (4:1 molar ratio), was successfully carried out (size 41 ± 2.4 nm, zeta potential 13.6 ± 2.5, and EE 61.43 ± 2.03%). OLE showed enhanced thermal stability, and an improved in vitro antiproliferative effect against HepG-2, MCF-7, and Caco-2 (IC\(_{50}\) 3.6 ± 0.2, 2.3 ± 0.1, and 1.8 ± 0.1 µg/mL, respectively) in comparison to the unprocessed extract. Both preparations were found to exhibit pro-oxidant potential inside the cancer cells, through the potential inhibitory activity of OLE against glutathione reductase and superoxide dismutase (IC\(_{50}\) 1.18 ± 0.12 and 2.33 ± 0.19 µg/mL, respectively). These inhibitory activities were proposed via a comprehensive in silico study to be linked to the presence of certain compounds in OLE. Consequently, we assume that formulating such a herbal extract within a suitable nanocarrier would be a promising improvement of its therapeutic potential.
Background:
Commensal bacteria like Neisseria meningitidis sometimes cause serious disease. However, genomic comparison of hyperinvasive and apathogenic lineages did not reveal unambiguous hints towards indispensable virulence factors. Here, in a systems biological approach we compared gene expression of the invasive strain MC58 and the carriage strain α522 under different ex vivo conditions mimicking commensal and virulence compartments to assess the strain-specific impact of gene regulation on meningococcal virulence.
Results:
Despite indistinguishable ex vivo phenotypes, both strains differed in the expression of over 500 genes under infection mimicking conditions. These differences comprised in particular metabolic and information processing genes as well as genes known to be involved in host-damage such as the nitrite reductase and numerous LOS biosynthesis genes. A model based analysis of the transcriptomic differences in human blood suggested ensuing metabolic flux differences in energy, glutamine and cysteine metabolic pathways along with differences in the activation of the stringent response in both strains. In support of the computational findings, experimental analyses revealed differences in cysteine and glutamine auxotrophy in both strains as well as a strain and condition dependent essentiality of the (p)ppGpp synthetase gene relA and of a short non-coding AT-rich repeat element in its promoter region.
Conclusions:
Our data suggest that meningococcal virulence is linked to transcriptional buffering of cryptic genetic variation in metabolic genes including global stress responses. They further highlight the role of regulatory elements for bacterial virulence and the limitations of model strain approaches when studying such genetically diverse species as N. meningitidis.
Background: Chagas disease (CD) is a major burden in Latin America, expanding also to non-endemic countries. A gold standard to detect the CD causing pathogen Trypanosoma cruzi is currently not available. Existing real time polymerase chain reactions (RT-PCRs) lack sensitivity and/or specificity. We present a new, highly specific RT-PCR for the diagnosis and monitoring of CD. Material and Methods: We analyzed 352 serum samples from Indigenous people living in high endemic CD areas of Colombia using three leading RT-PCRs (k-DNA-, TCZ-, 18S rRNA-PCR), the newly developed one (NDO-PCR), a Rapid Test/enzyme-linked immuno sorbent assay (ELISA), and immunofluorescence. Eighty-seven PCR-products were verified by sequence analysis after plasmid vector preparation. Results: The NDO-PCR showed the highest sensitivity (92.3%), specificity (100%), and accuracy (94.3%) for T. cruzi detection in the 87 sequenced samples. Sensitivities and specificities of the kDNA-PCR were 89.2%/22.7%, 20.5%/100% for TCZ-PCR, and 1.5%/100% for the 18S rRNA-PCR. The kDNA-PCR revealed a 77.3% false positive rate, mostly due to cross-reactions with T. rangeli (NDO-PCR 0%). TCZ- and 18S rRNA-PCR showed a false negative rate of 79.5% and 98.5% (NDO-PCR 7.7%), respectively. Conclusions: The NDO-PCR demonstrated the highest specificity, sensitivity, and accuracy compared to leading PCRs. Together with serologic tests, it can be considered as a reliable tool for CD detection and can improve CD management significantly.
Cushing’s disease (CD) is a rare condition caused by adrenocorticotropic hormone (ACTH)-producing adenomas of the pituitary, which lead to hypercortisolism that is associated with high morbidity and mortality. Treatment options in case of persistent or recurrent disease are limited, but new insights into the pathogenesis of CD are raising hope for new therapeutic avenues. Here, we have performed a meta-analysis of the available sequencing data in CD to create a comprehensive picture of CD’s genetics. Our analyses clearly indicate that somatic mutations in the deubiquitinases are the key drivers in CD, namely USP8 (36.5%) and USP48 (13.3%). While in USP48 only Met415 is affected by mutations, in USP8 there are 26 different mutations described. However, these different mutations are clustering in the same hotspot region (affecting in 94.5% of cases Ser718 and Pro720). In contrast, pathogenic variants classically associated with tumorigenesis in genes like TP53 and BRAF are also present in CD but with low incidence (12.5% and 7%). Importantly, several of these mutations might have therapeutic potential as there are drugs already investigated in preclinical and clinical setting for other diseases. Furthermore, network and pathway analyses of all somatic mutations in CD suggest a rather unified picture hinting towards converging oncogenic pathways.
To enable a sustainable supply of chemicals, novel biotechnological solutions are required that replace the reliance on fossil resources. One potential solution is to utilize tailored biosynthetic modules for the metabolic conversion of CO2 or organic waste to chemicals and fuel by microorganisms. Currently, it is challenging to commercialize biotechnological processes for renewable chemical biomanufacturing because of a lack of highly active and specific biocatalysts. As experimental methods to engineer biocatalysts are time- and cost-intensive, it is important to establish efficient and reliable computational tools that can speed up the identification or optimization of selective, highly active, and stable enzyme variants for utilization in the biotechnological industry. Here, we review and suggest combinations of effective state-of-the-art software and online tools available for computational enzyme engineering pipelines to optimize metabolic pathways for the biosynthesis of renewable chemicals. Using examples relevant for biotechnology, we explain the underlying principles of enzyme engineering and design and illuminate future directions for automated optimization of biocatalysts for the assembly of synthetic metabolic pathways.
Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a promising approach for cancer therapy. Recently, we showed that the oncolytic vaccinia virus GLV-1h68 has a therapeutic potential in treating human prostate and hepatocellular carcinomas in xenografted mice. In this study, we describe the use of dynamic boolean modeling for tumor growth prediction of vaccinia virus-injected human tumors. Antigen profiling data of vaccinia virus GLV-1h68-injected human xenografted mice were obtained, analyzed and used to calculate differences in the tumor growth signaling network by tumor type and gender. Our model combines networks for apoptosis, MAPK, p53, WNT, Hedgehog, the T-killer cell mediated cell death, Interferon and Interleukin signaling networks. The in silico findings conform very well with in vivo findings of tumor growth. Similar to a previously published analysis of vaccinia virus-injected canine tumors, we were able to confirm the suitability of our boolean modeling for prediction of human tumor growth after virus infection in the current study as well. In summary, these findings indicate that our boolean models could be a useful tool for testing of the efficacy of VACV-mediated cancer therapy already before its use in human patients.
Background
Processing and analysis of DNA sequences obtained from next-generation sequencing (NGS) face some difficulties in terms of the correct prediction of DNA sequencing outcomes without the implementation of bioinformatics approaches. However, algorithms based on NGS perform inefficiently due to the generation of long DNA fragments, the difficulty of assembling them and the complexity of the used genomes. On the other hand, the Sanger DNA sequencing method is still considered to be the most reliable; it is a reliable choice for virtual modeling to build all possible consensus sequences from smaller DNA fragments.
Results
In silico and in vitro experiments were conducted: (1) to implement and test our novel sequencing algorithm, using the standard cloning vectors of different length and (2) to validate experimentally virtual shotgun sequencing using the PCR technique with the number of cycles from 1 to 9 for each reaction.
Conclusions
We applied a novel algorithm based on Sanger methodology to correctly predict and emphasize the performance of DNA sequencing techniques as well as in de novo DNA sequencing and its further application in synthetic biology. We demonstrate the statistical significance of our results.
Synthetically designed alternative photorespiratory pathways increase the biomass of tobacco and rice plants. Likewise, some in planta–tested synthetic carbon-concentrating cycles (CCCs) hold promise to increase plant biomass while diminishing atmospheric carbon dioxide burden. Taking these individual contributions into account, we hypothesize that the integration of bypasses and CCCs will further increase plant productivity. To test this in silico, we reconstructed a metabolic model by integrating photorespiration and photosynthesis with the synthetically designed alternative pathway 3 (AP3) enzymes and transporters. We calculated fluxes of the native plant system and those of AP3 combined with the inhibition of the glycolate/glycerate transporter by using the YANAsquare package. The activity values corresponding to each enzyme in photosynthesis, photorespiration, and for synthetically designed alternative pathways were estimated. Next, we modeled the effect of the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA cycle (CETCH), which is a set of natural and synthetically designed enzymes that fix CO₂ manifold more than the native Calvin–Benson–Bassham (CBB) cycle. We compared estimated fluxes across various pathways in the native model and under an introduced CETCH cycle. Moreover, we combined CETCH and AP3-w/plgg1RNAi, and calculated the fluxes. We anticipate higher carbon dioxide–harvesting potential in plants with an AP3 bypass and CETCH–AP3 combination. We discuss the in vivo implementation of these strategies for the improvement of C3 plants and in natural high carbon harvesters.
Candida auris is a globally emerging fungal pathogen responsible for causing nosocomial outbreaks in healthcare associated settings. It is known to cause infection in all age groups and exhibits multi-drug resistance with high potential for horizontal transmission. Because of this reason combined with limited therapeutic choices available, C. auris infection has been acknowledged as a potential risk for causing a future pandemic, and thus seeking a promising strategy for its treatment is imperative. Here, we combined evolutionary information with reverse vaccinology approach to identify novel epitopes for vaccine design that could elicit CD4+ T-cell responses against C. auris. To this end, we extensively scanned the family of proteins encoded by C. auris genome. In addition, a pathogen may acquire substitutions in epitopes over a period of time which could cause its escape from the immune response thus rendering the vaccine ineffective. To lower this possibility in our design, we eliminated all rapidly evolving genes of C. auris with positive selection. We further employed highly conserved regions of multiple C. auris strains and identified two immunogenic and antigenic T-cell epitopes that could generate the most effective immune response against C. auris. The antigenicity scores of our predicted vaccine candidates were calculated as 0.85 and 1.88 where 0.5 is the threshold for prediction of fungal antigenic sequences. Based on our results, we conclude that our vaccine candidates have the potential to be successfully employed for the treatment of C. auris infection. However, in vivo experiments are imperative to further demonstrate the efficacy of our design.
For SARS-CoV-2, R0 calculations in the range of 2–3 dominate the literature, but much higher estimates have also been published. Because capacity for RT-PCR testing increased greatly in the early phase of the Covid-19 pandemic, R0 determinations based on these incidence values are subject to strong bias. We propose to use Covid-19-induced excess mortality to determine R0 regardless of RT-PCR testing capacity. We used data from the Robert Koch Institute (RKI) on the incidence of Covid cases, Covid-related deaths, number of RT-PCR tests performed, and excess mortality calculated from data from the Federal Statistical Office in Germany. We determined R0 using exponential growth estimates with a serial interval of 4.7 days. We used only datasets that were not yet under the influence of policy measures (e.g., lockdowns or school closures). The uncorrected R0 value for the spread of SARS-CoV-2 based on RT-PCR incidence data was 2.56 (95% CI 2.52–2.60) for Covid-19 cases and 2.03 (95% CI 1.96–2.10) for Covid-19-related deaths. However, because the number of RT-PCR tests increased by a growth factor of 1.381 during the same period, these R0 values must be corrected accordingly (R0corrected = R0uncorrected/1.381), yielding 1.86 for Covid-19 cases and 1.47 for Covid-19 deaths. The R0 value based on excess deaths was calculated to be 1.34 (95% CI 1.32–1.37). A sine-function-based adjustment for seasonal effects of 40% corresponds to a maximum value of R0January = 1.68 and a minimum value of R0July = 1.01. Our calculations show an R0 that is much lower than previously thought. This relatively low range of R0 fits very well with the observed seasonal pattern of infection across Europe in 2020 and 2021, including the emergence of more contagious escape variants such as delta or omicron. In general, our study shows that excess mortality can be used as a reliable surrogate to determine the R0 in pandemic situations.
Apart from some model organisms, the interactome of most organisms is largely unidentified. High-throughput experimental techniques to determine protein-protein interactions (PPIs) are resource intensive and highly susceptible to noise. Computational methods of PPI determination can accelerate biological discovery by identifying the most promising interacting pairs of proteins and by assessing the reliability of identified PPIs. Here we present a first in-depth study describing a global view of the ant Camponotus floridanus interactome. Although several ant genomes have been sequenced in the last eight years, studies exploring and investigating PPIs in ants are lacking. Our study attempts to fill this gap and the presented interactome will also serve as a template for determining PPIs in other ants in future. Our C. floridanus interactome covers 51,866 non-redundant PPIs among 6,274 proteins, including 20,544 interactions supported by domain-domain interactions (DDIs), 13,640 interactions supported by DDIs and subcellular localization, and 10,834 high confidence interactions mediated by 3,289 proteins. These interactions involve and cover 30.6% of the entire C. floridanus proteome.
Recent progress in nanobiotechnology has attracted interest to a biomedical application of the carbon nanostructure C60 fullerene since it possesses a unique structure and versatile biological activity. C60 fullerene potential application in the frame of cancer photodynamic therapy (PDT) relies on rapid development of new light sources as well as on better understanding of the fullerene interaction with cells.
The aim of this study was to analyze C60 fullerene effects on human leukemic cells (CCRF-CEM) in combination with high power single chip light-emitting diodes (LEDs) light irradiation of different wavelengths: ultraviolet (UV, 365 nm), violet (405 nm), green (515 nm) and red (632 nm). The time-dependent accumulation of fullerene C60 in CCRF-CEM cells up to 250 ng/106 cells at 24 h with predominant localization within mitochondria was demonstrated with immunocytochemical staining and liquid chromatography mass spectrometry. In a cell viability assay we studied photoexcitation of the accumulated C60 nanostructures with ultraviolet or violet LEDs and could prove that significant phototoxic effects did arise. A less pronounced C60 fullerene phototoxic effect was observed after irradiation with green, and no effect was detected with red light. A C60 fullerene photoactivation with violet light induced substantial ROS generation and apoptotic cell death, confirmed by caspase3/7 activation and plasma membrane phosphatidylserine externalization. Our work proved C60 fullerene ability to induce apoptosis of leukemic cells after photoexcitation with high power single chip 405 nm LED as a light source. This underlined the potential for application of C60 nanostructure as a photosensitizer for anticancer therapy.