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Triflic acid treatment enables LC-MS/MS analysis of insoluble bacterial biomass

The lysis and extraction of soluble bacterial proteins from cells is a common practice for proteomics analyses, but insoluble bacterial biomasses are often left behind. Here, we show that with triflic acid treatment, the insoluble bacterial biomass of Gram- and Gram+ bacteria can be rendered soluble. We use LC-MS/MS shotgun proteomics to show that bacterial proteins in the soluble and insoluble post-lysis fractions differ significantly. Additionally, in the case of Gram- Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell envelope-associated proteins. Finally, we apply triflic acid to a human microbiome sample to show that this treatment is robust and enables the identification of a new, complementary subset of proteins from a complex microbial mixture.

systems biology

A mathematical model of the effect of natural selection on adaptation forms that implemented by disruptive coloration of Taurotragus oryx

In the study, results of mathematical modelling of the influence of natural selection on performance of various forms of animal adaptation to habitat conditions are presented. For a formalized description of the subject of study, we used a new class of mathematical models-- discrete models of dynamical systems. Sets of strategies of protective coloration of antelopes Taurotragus oryx are the subject of a formalized description. Various combinations of brightness of green and red components of gray-brown non-uniform protective coloration of different parts of the silhouette of these animals were considered as such strategies. The sets based on the material of digital pictures of the two groups of Taurotragus oryx were compared. The first group includes antelopes Taurotragus oryx from Serengeti National Park (Tanzania) exposed to natural selection. The second group includes Taurotragus oryx, actually domesticated in Askania-Nova reserve (Ukraine), for which natural selection is not active. The sets of above mention strategies-combinations, modelled for the two groups, were compared by the numbers of unique combinations of values of brightness of red and green colours, as well as combinations with closest values of these brightness. The adaptive role of combinations with different values of red and green colours was identified with the role of idioadaptations. The adaptive role of combinations with equal values of red and green colours was identified with a more wide performance of aromorphoses. In this connection, the notions \"quasi-idioadaptation\" and \"quasi-aromorphosis\" were introduced in the paper.\n\nIt is assumed that both quasi-aromorphoses and quasi-idiadaptations, in certain conditions, contribute to the destruction of an integral visual perception of the silhouette of an animal against a many-coloured background of vegetation. At that, assumed that an adaptation function of a quasi-aromorphosis can be implemented in a wider range of colorimetric parameters of a plant background. The results of modelling indicate that the coloration of Taurotragus oryx from Serengeti is characterized by a larger set of quasi-adaptations than coloration of Taurotragus oryx from Askania-Nova. In the coloration of the latter, there is no quasi-aromorphosis with maximum values of brightness of both red and green components. But there exists a quasi-aromorphosis in the coloration of Taurotragus oryx from Serengeti. Such results of mathematical modelling correspond to prevailing ideas about the influence of natural selection on the character of adaptive reactions of living beings.

systems biology

Mapping In Vivo O-Glycoproteome Using Site-specific Extraction of O-linked glycopeptides (EXoO)

Protein glycosylation is one of the most abundant post-translational modifications. However, detailed analysis of in vivo O-linked glycosylation, a major type of protein glycosylation, has been severely impeded by the scarcity of suitable methodologies. Here, we present a chemoenzymatic method for the site-specific extraction of O-linked glycopeptides (EXoO), which enabled the unambiguous mapping of over 3,000 O-linked glycosylation sites and definition of their glycans on over 1,000 proteins in human kidney tissues, T cells and serum. This large-scale localization of O-linked glycosylation sites nearly doubles the number of previously identified sites, demonstrating that EXoO is the most effective method to-date for defining the site-specific O-linked glycoproteome in different types of sample. Detailed structural analysis of the sites identified revealed conserved motifs and topological orientations facing extracellular space, the cell surface, the lumen of the ER and the Golgi. EXoO was also able to reveal significant differences in the in vivo O-linked glycoproteome of tumor and normal kidney tissues pointing to its broader use in clinical diagnostics and therapeutics.

systems biology

Fibroblast growth factor is predicted to dominate MAPK activation by pro-angiogenic factors

Angiogenesis is important in physiological and pathological conditions, as blood vessels provide nutrients and oxygen needed for tissue growth and survival. Therefore, targeting angiogenesis is a prominent strategy in both tissue engineering and cancer treatment. However, not all of the approaches to promote or inhibit angiogenesis lead to successful outcomes. Angiogenesis-based therapies primarily target pro-angiogenic factors such as vascular endothelial growth factor-A (VEGF) or fibroblast growth factor (FGF) in isolation, and there is a limited understanding of how these promoters combine together to stimulate angiogenesis. Thus, more quantitative insight is needed to understand their interactions. In this study, we have trained and validated a detailed mathematical model to quantitatively characterize the crosstalk of FGF and VEGF intracellular signaling. The model focuses on FGF- and VEGF-induced mitogen-activated protein kinase (MAPK) signaling and phosphorylation of extracellular regulated kinase (ERK), which promote cell proliferation. We apply the model to predict the dynamics of phosphorylated ERK (pERK) in response to the stimulation by FGF and VEGF individually and in combination. The model predicts that FGF plays a dominant role in promoting ERK phosphorylation, compared to VEGF. The modeling predictions show that VEGFR2 density and trafficking parameters significantly influence the level of VEGF-induced pERK. The model matches experimental data and is a framework to synthesize and quantitatively explain experimental studies. Ultimately, the model provides mechanistic insight into FGF and VEGF interactions needed to identify potential targets for pro-or anti-angiogenic therapies.

systems biology

Reconstruction of the Decidual Pathways in Human Endometrial Cells Using Single-Cell RNA-Seq

Breakdown of the feto-maternal interface in early pregnancy causes miscarriage. The cycling endometrium becomes poised to transition to a pregnant state during the midluteal implantation window, coinciding with differentiation of stromal cells into decidual cells (DC) and emergence of senescent decidual cells (snDC). Emerging evidence suggests that DC engage uterine natural killer cells to eliminate their senescent counterparts, thus enabling formation of a robust decidual matrix in pregnancy. To examine if failure to constrain snDC during the peri-implantation window increases the risk of miscarriage, we reconstructed the decidual pathway at single-cell level in vitro and demonstrated that, without immune surveillance, secondary senescence rapidly transforms DC into progesterone-resistant cells that abundantly express extracellular matrix remodelling factors. Additional single-cell analysis of midluteal endometrium identified DIO2 and SCARA5 as marker genes of a diverging decidual response in vivo. Finally, we report a conspicuous link between a pro-senescent decidual response in luteal phase endometrium and recurrent pregnancy loss, suggesting that pre-pregnancy screening and intervention may reduce the burden of miscarriage.

systems biology

Predicting the Outcomes of New Short-Course Regimens for Multi-Drug Resistant Tuberculosis Using Intrahost and Pharmacokinetic-Pharmacodynamic Modelling

Short-course regimens for multi-drug resistant tuberculosis (MDR-TB) are urgently needed. Limited data suggest that the new drug, bedaquiline (BDQ), may have the potential to shorten MDR-TB treatment to less than six months when used in conjunction with standard anti-TB drugs. However, the feasibility of BDQ in shortening MDR-TB treatment duration remains to be established. Mathematical modelling provides a platform to investigate different treatment regimens and predict their efficacy. We developed a mathematical model to capture the immune response to TB inside a human host environment. This model was then combined with a pharmacokinetic-pharmacodynamic model to simulate various short-course BDQ-containing regimens. Our modelling suggests that BDQ could reduce MDR-TB treatment duration to just 18 weeks (four months) while still maintaining a very high treatment success rate (100% for daily BDQ for two weeks, or 95% for daily BDQ for one week during the intensive phase). The estimated time to bacterial clearance of these regimens ranges from 27 to 33 days. Our findings provide the justification for empirical evaluation of short-course BDQ-containing regimens. If short-course BDQ-containing regimens are found to improve outcomes then we anticipate clear cost-savings and a subsequent improvement in the efficiency of national TB programs.

systems biology

Dynamic modelling suggests differential mechanisms for initiation of non-selective autophagy and mitophagy

During autophagy, the ULK complex nucleates autophagic precursors which give rise to autophagosomes. We analysed by live imaging and mathematical modelling translocation of ATG13 (part of ULK complex) to autophagic puncta in starvation-induced autophagy and ivermectin-induced mitophagy. In non-selective autophagy, the intensity and duration of ATG13 translocation approximated a normal distribution whereas wortmannin reduced this and shifted to a log-normal distribution. During mitophagy, multiple translocations of ATG13, with increasing time between peaks were observed. We hypothesised that these multiple translocations arise because engulfment of mitochondrial fragments requires successive nucleations of multiple phagophores on the same target, and a mathematical model based on this idea reproduced the oscillatory behaviour. Significantly, model and experimental data were also in agreement that the number of ATG13 translocations is directly proportional to the diameter of the targeted mitochondrial fragments. Our data provide novel insights into the early dynamics of selective and non-selective autophagy.

systems biology

Probabilistic graphical models and computational metabolic models applied to the analysis of metabolomics data in breast cancer

Metabolomics has a great potential in the development of new biomarkers in cancer. In this study, metabolomics and gene expression data from breast cancer tumor samples were analyzed, using (1) probabilistic graphical models to define associations using quantitative data without other a priori information; and (2) Flux Balance Analysis and flux activities to characterize differences in metabolic pathways. On the one hand, both analyses highlighted the importance of glutamine in breast cancer. Moreover, cell experiments showed that treating breast cancer cells with drugs targeting glutamine metabolism significantly affects cell viability. On the other hand, these computational methods suggested some hypotheses and have demonstrated their utility in the analysis of metabolomics data and in associating metabolomics with patients clinical outcome.

systems biology

Ultra-sensitive proteome profiling of FACS-isolated cell populations by data-independent acquisition-MS: Application to human hematopoietic stem and progenitor cells

Physiological processes in multicellular organisms depend on the function and interactions of a multitude of specialized cell types operating in context. Fluorescence-activated cell sorting (FACS) provides a powerful tool to determine the cell type composition of complex mixtures and to purify highly homogeneous cell populations using a small number of differentially expressed marker proteins. These populations can be further characterized, e.g. by phenotypic or molecular analyses.\n\nWe describe an ultra-sensitive mass spectrometric method for the robust quantitative and reproducible proteomic analysis of cohorts of FACS-isolated cell samples. It uses a minimum of post-sorting sample processing steps prior to data-independent acquisition MS on a current generation Orbitrap hybrid mass spectrometer. The method provides highly accurate and reproducible quantitative proteome profiles across the cohort with an average coefficient of variance <15% from as little as 150 ng of total peptide mass. We quantified the proteome of 25,000 sorted human hematopoietic stem/multipotent progenitor cell and three committed progenitor cell subpopulations (common myeloid progenitors, megakaryocyte-erythrocyte progenitors and granulocyte-macrophage progenitors) isolated from five healthy donors. On average, 5,851 protein groups were identified per sample. After stringent filtering, a subset of 4,131 protein groups ([&ge;]2 peptides) was used for differential comparison across the 20 samples, defining unique proteomic signatures for each cell type tested. A comparison of proteomic and transcriptomic profiles of the four cell types indicated hematopoietic stem/multipotent progenitor cell-specific divergent regulation of biochemical processes that are essential for maintaining stemness and were detected at the proteome rather than the transcriptome level.\n\nThe technology supports the generation of extensive and accurate quantitative proteomic profiles from low numbers of FACS-purified cells providing new information about the biochemical state of the analyzed cell types that is essential for basic and translational research.

systems biology

Drugs modulating stochastic gene expression affect the erythroid differentiation process

BackgroundTo understand how a metazoan cell makes the decision to differentiate, we assessed the role of stochastic gene expression (SGE) during the erythroid differentiation process. Our hypothesis is that stochastic gene expression has a role in single-cell decision-making. In agreement with this hypothesis, we and others recently showed that SGE significantly increased during differentiation. However, evidence for the causative role of SGE is still lacking. Such demonstration would require being able to experimentally manipulate SGE levels and analyze the resulting impact of these variations on cell differentiation.\n\nResultWe identified three drugs that modulate SGE in primary erythroid progenitor cells. Artemisinin and Indomethacin simultaneously decreased SGE and reduced the amount of differentiated cells. Inversely, -methylene-{gamma}-butyrolactone-3 (MB-3) simultaneously increased the level of SGE and the amount of differentiated cells. We then used a dynamical modelling approach which confirmed that differentiation rates were indeed affected by the drug treatment.\n\nConclusionUsing single-cell analysis and modeling tools, we provide experimental evidence that in a physiologically relevant cellular system, control of SGE can directly modify differentiation, supporting a causal link between the two.

systems biology

Transcriptional crosstalk varies between regulatory strategies

Genes differ in the frequency at which they are expressed and in the form of regulation used to control their activity. In particular, positive or negative regulation can lead to activation of a gene in response to an external signal. Previous works proposed that the form of regulation of a gene correlates with its frequency of usage: positive regulation when the gene is frequently expressed and negative regulation when infrequently expressed. Such network design means that, in the absence of their regulators, the genes are found in their least required activity state, hence regulatory intervention is often necessary. Due to the multitude of genes and regulators, spurious binding and unbinding events, called \"crosstalk\", could occur. To determine how the form of regulation affects the global crosstalk in the network, we used a mathematical model that includes multiple regulators and multiple target genes. We found that crosstalk depends non-monotonically on the availability of regulators. Our analysis showed that excess use of regulation entailed by the formerly suggested network design caused high crosstalk levels in a large part of the parameter space. We therefore considered the opposite idle design, where the default unregulated state of genes is their frequently required activity state. We found, that idle design minimized the use of regulation and thus minimized crosstalk. In addition, we estimated global crosstalk of S. cerevisiae using transcription factors binding data. We demonstrated that even partial network data could suffice to estimate its global crosstalk, suggesting its applicability to additional organisms. We found that S. cerevisiae estimated crosstalk is lower than that of a random network, suggesting that natural selection reduces crosstalk. In summary, our study highlights a new type of protein production cost which is typically overlooked: that of regulatory interference caused by the presence of excess regulators in the cell. It demonstrates the importance of whole-network descriptions, which could show effects missed by single-gene models.\n\nAuthor SummaryGenes differ in the frequency at which they are expressed and in the form of regulation used to control their activity. The basic level of regulation is mediated by different types of DNA-binding proteins, where each type regulates particular gene(s). We distinguish between two basic forms of regulation: positive - if a gene is activated by the binding of its regulatory protein, and negative - if it is active unless bound by its regulatory protein. Due to the multitude of genes and regulators, spurious binding and unbinding events, called \"crosstalk\", could occur. How does the form of regulation, positive or negative, affect the extent of regulatory crosstalk? To address this question, we used a mathematical model integrating many genes and many regulators. As intuition suggests, we found that in most of the parameter space, crosstalk increased with the availability of regulators. We propose, that crosstalk is usually reduced when networks are designed such that minimal regulation is needed, which we call the idle design. In other words: a frequently needed gene will use negative regulation and conversely, a scarcely needed gene will employ positive regulation. In both cases, the requirement for the regulators is minimized. In addition, we demonstrate how crosstalk can be calculated from available datasets and discuss the technical challenges in such calculation, specifically data incompleteness.

systems biology

Long-range memory of growth and cycle progression correlates cell cycles in lineage trees

Mammalian cell proliferation is controlled by mitogens. However, how proliferation is coordinated with cell growth is poorly understood. Here we show that statistical properties of cell lineage trees - the cell-cycle length correlations within and across generations - reveal how cell growth controls proliferation. Analyzing extended lineage trees with latent-variable models, we find that two antagonistic heritable variables account for the observed cycle-length correlations. Using molecular perturbations of mTOR and MYC we identify these variables as cell size and regulatory license to divide, which are coupled through a minimum-size checkpoint. The checkpoint is relevant only for fast cell cycles, explaining why growth control of mammalian cell proliferation has remained elusive. Thus, correlated fluctuations of the cell cycle encode its regulation.

systems biology

Genome-scale metabolic construction of the stress-tolerant hybrid yeast Zygosaccharomyces parabailii

Genome-scale metabolic models are powerful tools to understand and engineer cellular systems facilitating their use as cell factories. This is especially true for microorganisms with known genome sequences from which nearly complete sets of enzymes and metabolic pathways are determined, or can be inferred. Yeasts are highly diverse eukaryotes whose metabolic traits have long been exploited in industry, and although many of their genome sequences are available, few genome-scale metabolic models have so far been produced. For the first time, we reconstructed the genome-scale metabolic model of the hybrid yeast Zygosaccharomyces parabailii, which is a member of the Z. bailii sensu lato clade notorious for stress-tolerance and therefore relevant to industry. The model comprises 3096 reactions, 2091 metabolites, and 2413 genes. Our own laboratory data were then used to establish a biomass synthesis reaction, and constrain the extracellular environment. Through constraint-based modeling, our model reproduces the co-consumption and catabolism of acetate and glucose posing it as a promising platform for understanding and exploiting the metabolic potential of Z. parabailii.

systems biology

Genome-scale metabolic network reconstruction of the chloroform-respiring Dehalobacter restrictus strain CF

BackgroundOrganohalide-respiring bacteria (OHRB) play an important role in the global halogen cycle and bioremediation of industrial sites contaminated with chlorinated organics. One notable OHRB is Dehalobacter restrictus strain CF, which is capable of respiring chloroform to dichloromethane. Improved bioremediation strategies could be employed with a greater understanding of D. restrictus metabolism in isolate and community cultures. To this end, we reconstructed the genome-scale metabolic network of D. restrictus to study its metabolism in future studies using flux balance analysis.\n\nMethodThe RAST annotation server and Model SEED framework were used to obtain a draft metabolic network reconstruction. Additional curation was required for its acetyl-CoA sources, the Wood-Ljungdahl pathway, TCA cycle, electron transport chain, hydrogenase complexes, and formate dehydrogenase complexes.\n\nResultsiHH623 is the first curated genome-scale metabolic model in the Peptococcaceae family. It spans 1087 reactions and 983 metabolites, covering 623 genes (21% of all ORFs). Its potential sources of acetyl-CoA are pyruvate ferredoxin oxidoreductase, pyruvate formate lyase, acetyl-CoA synthetase, phosphate acetyltransferase, and CO-methylating acetyl-CoA synthase. NADPH may be regenerated by isocitrate dehydrogenase, malic enzyme, NADP-reducing hydrogenase, cytosolic formate dehydrogenase, ferredoxin-dependent bifurcating transhydrogenase, 5-methyltetrahydrofolate dehydrogenase, and 5-10-methylenetetrahydrofolate. Additional reactions that were added or removed to the D. restrictus reconstruction are discussed.\n\nConclusionsWe reconstructed the genome-scale metabolic network of D. restricus by obtaining an initial draft with the RAST server and Model SEED framework. Curation was required for D. restricus acetyl-CoA sources, TCA cycle, electron transport chain, hydrogenase complexes, and formate dehydrogenase complexes. This metabolic model can be used to decipher D. restrictus metabolism in isolate and community cultures in future studies, or as a template to reconstruct the metabolic network of other Peptococcaceae species. The extensive curation of the draft metabolic network reconstruction highlights the need to be cautious of automated metabolic network reconstruction.

systems biology

Stochastic temporal timing for intracellular events to cross dynamically fluctuating thresholds

Fractional killing, which is a significant impediment to successful chemotherapy, is observed even in a population of genetically identical cancer cells exposed to apoptosis-inducing agents. This phenomenon arises not from genetic mutation but from cell-to-cell variation in the activation timing and level of the proteins that regulate apoptosis. To understand the mechanism behind the phenomenon, we formulate complex fractional killing processes as a first-passage time (FPT) problem with a stochastically fluctuating boundary. Analytical calculations are performed for the FPT distribution in a toy model of stochastic p53 gene expression, where the cancer cell is killed only when the p53 expression level crosses an activity apoptotic threshold. Counterintuitively, we find that threshold fluctuations can effectively enhance cellular killing by significantly decreasing the mean time that the p53 protein reaches the threshold level for the first time. Moreover, faster fluctuations lead to the killing of more cells. These qualitative results imply that dynamic variability in threshold is an unneglectable stochastic source, and can be taken as a strategy for combating fractional killing of cancer cells.

systems biology

Overdosage of balanced protein complexes reduces proliferation rate in aneuploid cells

Cells with complex aneuploidies, such as tumor cells, display a wide range of phenotypic abnormalities. However, molecular basis for this has been mainly studied in trisomic (2n+1) and disomic (n+1) cells. To determine how karyotype affects proliferation rate in cells with complex aneuploidies we generated forty 2n+x yeast strains in which each diploid cell has an extra 5 to 12 chromosomes and found that these strains exhibited abnormal cell-cycle progression. Proliferation rate was negatively correlated with the number of protein complexes in which all subunits were at the 3-copy level, but not with the number of imbalanced complexes made up of a mixture of 2-copy and 3-copy genes. Proteomics revealed that most 3-copy members of imbalanced complexes were expressed at only 2n protein levels whereas members of complexes in which all subunits are stoichiometrically balanced at 3 copies per cell had 3n protein levels. We identified individual protein complexes for which overdosage reduces proliferation rate, and found that deleting one copy of each member partially restored proliferation rate in cells with complex aneuploidies. Lastly, we validated this finding using orthogonal datasets from both yeast and from human cancers. Taken together, our study provides a novel explanation how aneuploidy affects phenotype.

systems biology

Gene expression patterns in synchronized islet populations

In vivo levels of insulin are oscillatory with a period of ~5-10 minutes, implying that the numerous islets of Langerhans within the pancreas are synchronized. While the synchronizing factors are still under investigation, one result of this behavior is expected to be coordinated intracellular [Ca2+] ([Ca2+]i) oscillations throughout the islet population. The role that coordinated [Ca2+]i oscillations have on controlling gene expression within pancreatic islets was examined by comparing gene expression levels in islets that were synchronized using a low amplitude glucose wave and an unsynchronized population. The [Ca2+]i oscillations in the synchronized population were homogeneous and had a significantly lower drift in their oscillation period as compared to unsynchronized islets. This reduced drift in the synchronized population was verified by comparing the drift of in vivo and in vitro profiles from published reports. Microarray profiling indicated a number of Ca2+-dependent genes were differentially regulated between the two islet populations. Gene set enrichment analysis revealed that the synchronized population had reduced expression of gene sets related to protein translation, protein turnover, energy expenditure, and insulin synthesis, while those that were related to maintenance of cell morphology were increased. It is speculated that these gene expression patterns in the synchronized islets results in a more efficient utilization of intra-cellular resources and response to environmental changes.

systems biology

Benchmarking Time-Series Data Discretization on Inference Methods

The rapid development in quantitatively measuring DNA, RNA, and protein has generated a great interest in the development of reverse-engineering methods, that is, data-driven approaches to infer the network structure or dynamical model of the system. Many reverse-engineering methods require discrete quantitative data as input, while many experimental data are continuous. Some studies have started to reveal the impact that the choice of data discretization has on the performance of reverse-engineering methods. However, more comprehensive studies are still greatly needed to systematically and quantitatively understand the impact that discretization methods have on inference methods. Furthermore, there is an urgent need for systematic comparative methods that can help select between discretization methods. In this work, we consider 4 published intracellular networks inferred with their respective time-series datasets. We discretized the data using different discretization methods. Across all datasets, changing the data discretization to a more appropriate one improved the reverse-engineering methods performance. We observed no universal best discretization method across different time-series datasets. Thus, we propose DiscreeTest, a two-step evaluation metric for ranking discretization methods for time-series data. The underlying assumption of DiscreeTest is that an optimal discretization method should preserve the dynamic patterns observed in the original data across all variables. We used the same datasets and networks to show that DiscreeTest is able to identify an appropriate discretization among several candidate methods. To our knowledge, this is the first time that a method for benchmarking and selecting an appropriate discretization method for time-series data has been proposed.\n\nAvailabilityAll the datasets, reverse-engineering methods and source code used in this paper are available in Vera-Liconas lab Github repository: https://github.com/VeraLiconaResearchGroup/Benchmarking_TSDiscretizations

systems biology