Use of top-down and bottom-up fourier transform ion cyclotron resonance mass spectrometry for mapping calmodulin sites modified by platinum anticancer drugs

Calmodulin (CaM) is a highly conserved, ubiquitous, calcium-binding protein; it binds to and regulates many different protein targets, thereby functioning as a calcium sensor and signal transducer. CaM contains 9 methionine (Met), 1 histidine (His), 17 aspartic acid (Asp), and 23 glutamine acid (Glu) residues, all of which can potentially react with platinum compounds; thus, one-third of the CaM sequence is a possible binding target of platinum anticancer drugs, which represents a major challenge for identification of specific platinum modification sites. Here, top-down electron capture dissociation (ECD) was used to elucidate the transition metal–platinum(II) modification sites. By using a combination of top-down and bottom-up mass spectrometric (MS) approaches, 10 specific binding sites for mononuclear complexes, cisplatin and [Pt(dien)Cl]Cl, and dinuclear complex [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] on CaM were identified. High resolution MS of cisplatin-modified CaM revealed that cisplatin mainly targets Met residues in solution at low molar ratios of cisplatin–CaM (2:1), by cross-linking Met residues. At a high molar ratio of cisplatin:CaM (8:1), up to 10 platinum(II) bind to Met, Asp, and Glu residues. [{cis-PtCl2(NH3)}2(μ-NH2(CH2)4NH2)] forms mononuclear adducts with CaM. The alkanediamine linker between the two platinum centers dissociates due to a trans-labilization effect. [Pt(dien)Cl]Cl forms {Pt(dien)}2+ adducts with CaM, and the preferential binding sites were identified as Met51, Met71, Met72, His107, Met109, Met124, Met144, Met145, Glu45 or Glu47, and Asp122 or Glu123. The binding of these complexes to CaM, particularly when binding involves loss of all four original ligands, is largely irreversible which could result in their failure to reach the target DNA or be responsible for unwanted side-effects during chemotherapy. Additionally, the cross-linking of cisplatin to CaM might lead to the loss of the biological function of CaM or CaM–Ca2+ due to limiting the flexibility of the CaM or CaM–Ca2+ complex to recognize target proteins or blocking the binding region of target proteins to CaM

Quantification of methane emissions from UK biogas plants

The rising number of operational biogas plants in the UK brings a new emissions category to consider for methane monitoring, quantification and reduction. Minimising methane losses from biogas plants to the atmosphere is critical not only because of their contribution of methane to global warming but also with respect to the sustainability of renewable energy production. Mobile greenhouse gas surveys were conducted to detect plumes of methane emissions from the biogas plants in southern England that varied in their size, waste feed input materials and biogas utilization. Gaussian plume modelling was used to estimate total emissions of methane from ten biogas plants based on repeat passes through the plumes. Methane emission rates ranged from 0.1 to 58.7 kg CH4 hr-1, and the percentage of losses relative to the calculated production rate varied between 0.02 and 8.1%. The average emission rate was 15.9 kg CH4 hr-1, and the average loss was 3.7%. In general, methane emission rates from smaller farm biogas plants were higher than from larger food waste biogas plants. We also suggest that biogas methane emissions may account for between 0.4 and 3.8%, with an average being 1.9% of the total methane emissions in the UK excluding the sewage sludge biogas plants

Fluctuation-Dissipation relations in Driven Granular Gases

We study the dynamics of a 2d driven inelastic gas, by means of Direct Simulation Monte Carlo (DSMC) techniques, i.e. under the assumption of Molecular Chaos. Under the effect of a uniform stochastic driving in the form of a white noise plus a friction term, the gas is kept in a non-equilibrium Steady State characterized by fractal density correlations and non-Gaussian distributions of velocities; the mean squared velocity, that is the so-called {\em granular temperature}, is lower than the bath temperature. We observe that a modified form of the Kubo relation, which relates the autocorrelation and the linear response for the dynamics of a system {\em at equilibrium}, still holds for the off-equilibrium, though stationary, dynamics of the systems under investigation. Interestingly, the only needed modification to the equilibrium Kubo relation is the replacement of the equilibrium temperature with an effective temperature, which results equal to the global granular temperature. We present two independent numerical experiment, i.e. two different observables are studied: (a) the staggered density current, whose response to an impulsive shear is proportional to its autocorrelation in the unperturbed system and (b) the response of a tracer to a small constant force, switched on at time $t_w$, which is proportional to the mean-square displacement in the unperturbed system. Both measures confirm the validity of Kubo's formula, provided that the granular temperature is used as the proportionality factor between response and autocorrelation, at least for not too large inelasticities.Comment: 11 pages, 7 figures, submitted for publicatio

Photosensing and Thermosensing by Phytochrome B Require Both Proximal and Distal Allosteric Features within the Dimeric Photoreceptor

Phytochromes (Phys) encompass a diverse collection of bilin-containing photoreceptors that help plants and microorganisms perceive light through photointerconversion between red light (Pr) and far-red light (Pfr)-absorbing states. In addition, Pfr reverts thermally back to Pr via a highly enthalpic process that enables temperature sensation in plants and possibly other organisms. Through domain analysis of the Arabidopsis PhyB isoform assembled recombinantly, coupled with measurements of solution size, photoconversion, and thermal reversion, we identified both proximal and distal features that influence all three metrics. Included are the downstream C-terminal histidine kinase-related domain known to promote dimerization and a conserved patch just upstream of an N-terminal Period/Arnt/Sim (PAS) domain, which upon removal dramatically accelerates thermal reversion. We also discovered that the nature of the bilin strongly influences Pfr stability. Whereas incorporation of the native bilin phytochromobilin into PhyB confers robust Pfr → Pr thermal reversion, that assembled with the cyanobacterial version phycocyanobilin, often used for optogenetics, has a dramatically stabilized Pfr state. Taken together, we conclude that Pfr acquisition and stability are impacted by a collection of opposing allosteric features that inhibit or promote photoconversion and reversion of Pfr back to Pr, thus allowing Phys to dynamically measure light, temperature, and possibly time

Tracer diffusion in granular shear flows

Tracer diffusion in a granular gas in simple shear flow is analyzed. The analysis is made from a perturbation solution of the Boltzmann kinetic equation through first order in the gradient of the mole fraction of tracer particles. The reference state (zeroth-order approximation) corresponds to a Sonine solution of the Boltzmann equation, which holds for arbitrary values of the restitution coefficients. Due to the anisotropy induced in the system by the shear flow, the mass flux defines a diffusion tensor $D_{ij}$ instead of a scalar diffusion coefficient. The elements of this tensor are given in terms of the restitution coefficients and mass and size ratios. The dependence of the diffusion tensor on the parameters of the problem is illustrated in the three-dimensional case. The results show that the influence of dissipation on the elements $D_{ij}$ is in general quite important, even for moderate values of the restitution coefficients. In the case of self-diffusion (mechanically equivalent particles), the trends observed in recent molecular dynamics simulations are similar to those obtained here from the Boltzmann kinetic theory.Comment: 5 figure

Sensitivity of the air-sea CO2 exchange in the Baltic Sea and Danish inner waters to atmospheric short-term variability

Minimising the uncertainties in estimates of air–sea CO₂ exchange is an important step toward increasing the confidence in assessments of the CO₂ cycle. Using an atmospheric transport model makes it possible to investigate the direct impact of atmospheric parameters on the air–sea CO₂ flux along with its sensitivity to, for example, short-term temporal variability in wind speed, atmospheric mixing height and atmospheric CO₂ concentration. With this study, the importance of high spatiotemporal resolution of atmospheric parameters for the air–sea CO₂ flux is assessed for six sub-basins within the Baltic Sea and Danish inner waters. A new climatology of surface water partial pressure of CO₂ (<i>p</i>CO₂^w) has been developed for this coastal area based on available data from monitoring stations and on-board <i>p</i>CO₂^w measuring systems. Parameterisations depending on wind speed were applied for the transfer velocity to calculate the air–sea CO₂ flux. Two model simulations were conducted – one including short-term variability in atmospheric CO₂ (VAT), and one where it was not included (CAT). <br><br> A seasonal cycle in the air–sea CO₂ flux was found for both simulations for all sub-basins with uptake of CO₂ in summer and release of CO₂ to the atmosphere in winter. During the simulated period 2005–2010, the average annual net uptake of atmospheric CO₂ for the Baltic Sea, Danish straits and Kattegat was 287 and 471 Gg C yr⁻¹ for the VAT and CAT simulations, respectively. The obtained difference of 184 Gg C yr⁻¹ was found to be significant, and thus ignoring short-term variability in atmospheric CO₂ does have a sizeable effect on the air–sea CO₂ exchange. The combination of the atmospheric model and the new <i>p</i>CO₂^w fields has also made it possible to make an estimate of the marine part of the Danish CO₂ budget for the first time. A net annual uptake of 2613 Gg C yr⁻¹ was found for the Danish waters. <br><br> A large uncertainty is connected to the air–sea CO₂ flux in particular caused by the transfer velocity parameterisation and the applied <i>p</i>CO₂^w climatology. However, as a significant difference of 184 Gg C yr⁻¹ is obtained between the VAT and CAT simulations, the present study underlines the importance of including short-term variability in atmospheric CO₂ concentration in future model studies of the air–sea exchange in order to minimise the uncertainty

Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA

In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action

Drug-induced liver injury associated with lopinavir-ritonavir in patients with COVID-19: a disproportionality analysis of U.S. food and drug administration adverse event reporting system (FAERS) data

Background Liver injury has been documented independently in novel coronavirus disease 2019 (COVID-19) patients and patients treated with lopinavir-ritonavir. Objective to investigate the drug-induced liver injury associated with lopinavir-ritonavir among the patients with COVID-19. Methods We conducted a disproportionality analysis of US Food and Drug Administration Adverse Event Reporting System (FAERS) between 2020Q1 and 2021Q1 to evaluate the association between lopinavir-ritonavir and risk of drug-induced liver injury (or severe drug-induced liver injury) and calculated their reporting odds ratios (RORs) with 95% confidence intervals (CIs). Results A total of 3,425 cases of drug-induced liver injury were reported in 19,782 patients with COVID-19. The ROR for drug-induced liver injury was 2.99 (2.59–3.46), 3.16 (2.68–3.73), and 5.39 (4.63–6.26) when comparing lopinavir-ritonavir with all other drugs, hydroxychloroquine/chloroquine only, and remdesivir, respectively. For severe drug-induced liver injury, RORs for lopinavir-ritonavir provided evidence of an association compared with all other drugs (3.98; 3.15–5.05), compared with hydroxychloroquine/chloroquine only (5.33; 4.09–6.94), and compared with remdesivir (3.85; 3.03–4.89). Conclusions In the FAERS, we observed a disproportional signal for drug-induced liver injury associated with lopinavir-ritonavir in patients with COVID-19