| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Ahlberg Gagnér, Viktor, 1989, et al.
(författare)
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Clustering of atomic displacement parameters in bovine trypsin reveals a distributed lattice of atoms with shared chemical properties
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322 .- 2045-2322. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Low-frequency vibrations are crucial for protein structure and function, but only a few experimental techniques can shine light on them. The main challenge when addressing protein dynamics in the terahertz domain is the ubiquitous water that exhibit strong absorption. In this paper, we observe the protein atoms directly using X-ray crystallography in bovine trypsin at 100 K while irradiating the crystals with 0.5 THz radiation alternating on and off states. We observed that the anisotropy of atomic displacements increased upon terahertz irradiation. Atomic displacement similarities developed between chemically related atoms and between atoms of the catalytic machinery. This pattern likely arises from delocalized polar vibrational modes rather than delocalized elastic deformations or rigid-body displacements. The displacement correlation between these atoms were detected by a hierarchical clustering method, which can assist the analysis of other ultra-high resolution crystal structures. These experimental and analytical tools provide a detailed description of protein dynamics to complement the structural information from static diffraction experiments. © 2019, The Author(s).
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| 3. |
- Ahlstrand, Emma, et al.
(författare)
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An interactive computer lab of the galvanic cell for students in biochemistry
- 2018
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Ingår i: Biochemistry and molecular biology education. - : Wiley-Blackwell. - 1470-8175 .- 1539-3429. ; 46:1, s. 58-65
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Tidskriftsartikel (refereegranskat)abstract
- We describe an interactive module that can be used to teach basic concepts in electrochemistry and thermodynamics to first year natural science students. The module is used together with an experimental laboratory and improves the students’ understanding of thermodynamic quantities such as ΔrG, ΔrH, and ΔrS that are calculated but not directly measured in the lab. We also discuss how new technologies can substitute some parts of experimental chemistry courses, and improve accessibility to course material. Cloud computing platforms such as CoCalc facilitate the distribution of computer codes and allow students to access and apply interactive course tools beyond the course's scope. Despite some limitations imposed by cloud computing, the students appreciated the approach and the enhanced opportunities to discuss study questions with their classmates and instructor as facilitated by the interactive tools.
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| 4. |
- Ahlstrand, Emma, et al.
(författare)
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Computer simulations of alkali-acetate solutions : Accuracy of the forcefields in difference concentrations
- 2017
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Ingår i: Journal of Chemical Physics. - : American Institute of Physics (AIP). - 0021-9606 .- 1089-7690. ; 147, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- When proteins are solvated in electrolyte solutions that contain alkali ions, the ions interact mostlywith carboxylates on the protein surface. Correctly accounting for alkali-carboxylate interactionsis thus important for realistic simulations of proteins. Acetates are the simplest carboxylates thatare amphipathic, and experimental data for alkali acetate solutions are available and can be comparedwith observables obtained from simulations. We carried out molecular dynamics simulations of alkali acetate solutions using polarizable and non-polarizable forcefields and examined the ionacetateinteractions. In particular, activity coefficients and association constants were studied in a range of concentrations (0.03, 0.1, and 1M). In addition, quantum-mechanics (QM) based energy decomposition analysis was performed in order to estimate the contribution of polarization, electrostatics, dispersion, and QM (non-classical) effects on the cation-acetate and cation-water interactions. Simulations of Li-acetate solutions in general overestimated the binding of Li+ and acetates. In lower concentrations, the activity coefficients of alkali-acetate solutions were too high, which is suggested to be due to the simulation protocol and not the forcefields. Energy decomposition analysis suggested that improvement of the forcefield parameters to enable accurate simulations of Li-acetate solution scan be achieved but may require the use of a polarizable forcefield. Importantly, simulations with some ion parameters could not reproduce the correct ion-oxygen distances, which calls for caution in thechoice of ion parameters when protein simulations are performed in electrolyte solutions.
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| 5. |
- Ahlstrand, Emma, et al.
(författare)
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Interaction energies between metal ions (Zn2+ and Cd2+) and biologically relevant ligands
- 2013
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Ingår i: International Journal of Quantum Chemistry. - Hoboken, NJ : Wiley. - 0020-7608 .- 1097-461X. ; 113:23, s. 2554-2562
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Tidskriftsartikel (refereegranskat)abstract
- Interactions between the group XII metals Zn2+ and Cd2+ and amino acid residues play an important role in biology due to the prevalence of the first and the toxicity of the second. Estimates of the interaction energies between the ions and relevant residues in proteins are however difficult to obtain. This study reports on calculated interaction energy curves for small complexes of Zn2+ or Cd2+ and amino acid mimics (acetate, methanethiolate, and imidazole) or water. Given that many applications and models (e.g., force fields, solvation models, etc.) begin with and rely on an accurate description of gas-phase interaction energies, this is where our focus lies in this study. Four density functional theory (DFT)-functionals and MP2 were used to calculate the interaction energies not only at the respective equilibrium distances but also at a relevant range of ion–ligand separation distances. The calculated values were compared with those obtained by CCSD(T). All DFT-methods are found to overestimate the magnitude of the interaction energy compared to the CCSD(T) reference values. The deviation was analyzed in terms of energy components from localized molecular orbital energy decomposition analysis scheme and is mostly attributed to overestimation of the polarization energy. MP2 shows good agreement with CCSD(T) [root mean square error (RMSE) = 1.2 kcal/mol] for the eight studied complexes at equilibrium distance. Dispersion energy differences at longer separation give rise to increased deviations between MP2 and CCSD(T) (RMSE = 6.4 kcal/mol at 3.0 Å). Overall, the results call for caution in applying DFT methods to metalloprotein model complexes even with closed-shell metal ions such as Zn2+ and Cd2+, in particular at ion–ligand separations that are longer than the equilibrium distances.
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| 6. |
- Ahlstrand, Emma, et al.
(författare)
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Interaction Energies in Complexes of Zn and Amino Acids : A Comparison of Ab Initio and Force Field Based Calculations
- 2017
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Ingår i: Journal of Physical Chemistry A. - : American Chemical Society (ACS). - 1089-5639 .- 1520-5215. ; 121:13, s. 2643-2654
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Tidskriftsartikel (refereegranskat)abstract
- Zinc plays important roles in structural stabilization of proteins, eniyine catalysis, and signal transduction. Many Zn binding sites are located at the interface between the protein and the cellular fluid. In aqueous solutions, Zn ions adopt an octahedral coordination, while in proteins zinc can have different coordinations, with a tetrahedral conformation found most frequently. The dynainics of Zn binding to proteins and the formation of complexes that involve Zn are dictated by interactions between Zn and its binding partners. We calculated the interaction energies between Zn and its ligands in complexes that mimic protein binding sites and in Zn complexes of water and one or two amino acid moieties, using quantum mechanics (QM) and molecular mechanics (MM). It was found that MM calculations that neglect or only approximate polarizability did not reproduce even the relative order of the QM interaction energies in these complexes. Interaction energies calculated with the CHARMM-Diode polarizable force field agreed better with the ab initio results,:although the deviations between QM and MM were still rather large (40-96 kcallmol). In order to gain further insight into Zn ligand interactions, the free energies of interaction were estimated by QM calculations with continuum solvent representation, and we performed energy decomposition analysis calculations to examine the characteristics of the different complexes. The ligand-types were found to have high impact on the relative strength of polarization and electrostatic interactions. Interestingly, ligand ligand interactions did not play a significant role in the binding of Zn. Finally) analysis of ligand exchange energies suggests that carboxylates could be exchanged with water molecules, which explains the flexibility in Zn:binding dynamics. An exchange between earboxylate (Asp/Glii) and imidazole (His) is less likely.
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| 7. |
- Becconi, Olga, et al.
(författare)
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Protein-ion Interactions : Simulations of Bovine Serum Albumin in Physiological Solutions of NaCl, KCl and LiCl
- 2017
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Ingår i: Israel Journal of Chemistry. - : Wiley-Blackwell. - 0021-2148. ; 57:5, s. 403-412
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Tidskriftsartikel (refereegranskat)abstract
- Specific interactions that depend on the nature of electrolytes are observed when proteins and other molecules are studied by potentiometric, spectroscopic and theoretical methods at high salt concentrations. More recently, it became clear that such interactions may also be observed in solutions that can be described by the Debye-Hückel theory, i.e., at physiological (0.1 mol dm−3) and lower concentrations. We carried out molecular dynamics simulations of bovine serum albumin in physiological solutions at T=300 and 350 K. Analysis of the simulations revealed some differences between LiCl solutions and those of NaCl and KCl. The binding of Li+ ions to the protein was associated with a negative free energy of interaction whereas much fewer Na+ and K+ ions were associated with the protein surface. Interestingly, unlike other proteins BSA does not show a preference to Na+ over K+. Quantum chemical calculations identified a significant contribution from polarisation to the hydration of Li+ and (to a lesser degree) Na+, which may indicate that polarisable force-fields will provide more accurate results for such systems.
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| 8. |
- Buetti-Dinh, Antoine, 1984-, et al.
(författare)
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A computational study of hedgehog signalling involved in basal cell carcinoma reveals the potential and limitation of combination therapy
- 2018
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Ingår i: BMC Cancer. - : BioMed Central. - 1471-2407 .- 1471-2407. ; 18:1, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- Background: The smoothened (SMO) receptor is an essential component of the Sonic hedgehog (SHH) signalling, which is associated with the development of skin basal cell carcinoma (BCC). SMO inhibitors are indicated for BCC patients when surgical treatment or radiation therapy are not possible. Unfortunately, SMO inhibitors are not always well tolerated due to severe side effects, and their therapeutical success is limited by resistance mutations. Methods: We investigated how common are resistance-causing mutations in two genomic databases which are not linked to BCC or other cancers, namely 1000 Genomes and ExAC. To examine the potential for combination therapy or other treatments, we further performed knowledge-based simulations of SHH signalling, in the presence or absence of SMO and PI3K/Akt inhibitors. Results: The database analysis revealed that of 18 known mutations associated with Vismodegib-resistance, three were identified in the databases. Treatment of individuals carrying such mutations is thus liable to fail a priori. Analysis of the simulations suggested that a combined inhibition of SMO and the PI3K/Akt signalling pathway may provide an effective reduction in tumour proliferation. However, the inhibition dosage of SMO and PI3K/Akt depended on the activity of phosphodiesterases (PDEs). Under high PDEs activities, SMO became the most important control node of the network. By applying PDEs inhibition, the control potential of SMO decreased and P13K appeared as a significant factor in controlling tumour proliferation. Conclusions: Our systems biology approach employs knowledge-based computer simulations to help interpret the large amount of data available in public databases, and provides application-oriented solutions for improved cancer resistance treatments.
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| 9. |
- Buetti-Dinh, Antoine, 1984-, et al.
(författare)
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Computer simulations of the signalling network in FLT3+-acute myeloid leukaemia : indications for an optimal dosage of inhibitors against FLT3 and CDK6
- 2018
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Ingår i: BMC Bioinformatics. - London : BioMed Central. - 1471-2105. ; 19, s. 1-13
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundMutations in the FMS-like tyrosine kinase 3 (FLT3) are associated with uncontrolled cellular functions that contribute to the development of acute myeloid leukaemia (AML). We performed computer simulations of the FLT3-dependent signalling network in order to study the pathways that are involved in AML development and resistance to targeted therapies.ResultsAnalysis of the simulations revealed the presence of alternative pathways through phosphoinositide 3 kinase (PI3K) and SH2-containing sequence proteins (SHC), that could overcome inhibition of FLT3. Inhibition of cyclin dependent kinase 6 (CDK6), a related molecular target, was also tested in the simulation but was not found to yield sufficient benefits alone.ConclusionsThe PI3K pathway provided a basis for resistance to treatments. Alternative signalling pathways could not, however, restore cancer growth signals (proliferation and loss of apoptosis) to the same levels as prior to treatment, which may explain why FLT3 resistance mutations are the most common resistance mechanism. Finally, sensitivity analysis suggested the existence of optimal doses of FLT3 and CDK6 inhibitors in terms of efficacy and toxicity.
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| 10. |
- Buetti-Dinh, Antoine, 1984-, et al.
(författare)
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Reverse engineering directed gene regulatory networks from transcriptomics and proteomics data of biomining bacterial communities with approximate Bayesian computation and steady-state signalling simulations
- 2020
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Ingår i: BMC Bioinformatics. - : BioMed Central (BMC). - 1471-2105. ; 21:1, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Background: Network inference is an important aim of systems biology. It enables the transformation of OMICs datasets into biological knowledge. It consists of reverse engineering gene regulatory networks from OMICs data, such as RNAseq or mass spectrometry-based proteomics data, through computational methods. This approach allows to identify signalling pathways involved in specific biological functions. The ability to infer causality in gene regulatory networks, in addition to correlation, is crucial for several modelling approaches and allows targeted control in biotechnology applications. Methods: We performed simulations according to the approximate Bayesian computation method, where the core model consisted of a steady-state simulation algorithm used to study gene regulatory networks in systems for which a limited level of details is available. The simulations outcome was compared to experimentally measured transcriptomics and proteomics data through approximate Bayesian computation. Results: The structure of small gene regulatory networks responsible for the regulation of biological functions involved in biomining were inferred from multi OMICs data of mixed bacterial cultures. Several causal inter- and intraspecies interactions were inferred between genes coding for proteins involved in the biomining process, such as heavy metal transport, DNA damage, replication and repair, and membrane biogenesis. The method also provided indications for the role of several uncharacterized proteins by the inferred connection in their network context. Conclusions: The combination of fast algorithms with high-performance computing allowed the simulation of a multitude of gene regulatory networks and their comparison to experimentally measured OMICs data through approximate Bayesian computation, enabling the probabilistic inference of causality in gene regulatory networks of a multispecies bacterial system involved in biomining without need of single-cell or multiple perturbation experiments. This information can be used to influence biological functions and control specific processes in biotechnology applications.
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