Targeted mutagenesis of flavonoid biosynthesis pathway genes reveals functional divergence in seed coat colour, oil content and fatty acid composition in Brassica napus L.

Yellow-seed is widely accepted as a good-quality trait in Brassica crops. Previous studies have shown that the flavonoid biosynthesis pathway is essential for the development of seed colour, but its function in Brassica napus, an important oil crop, is poorly understood. To systematically explore the gene functions of the flavonoid biosynthesis pathway in rapeseed, several representative TRANSPARENT TESTA (TT) genes, including three structural genes (BnaTT7, BnaTT18, BnaTT10), two regulatory genes (BnaTT1, BnaTT2) and a transporter (BnaTT12), were selected for targeted mutation by CRISPR/Cas9 in the present study. Seed coat colour, lignin content, seed quality and yield-related traits were investigated in these Bnatt mutants together with Bnatt8 generated previously. These Bnatt mutants produced seeds with an elevated seed oil content and decreased pigment and lignin accumulation in the seed coat without any serious defects in the yield-related traits. In addition, the fatty acid (FA) composition was also altered to different degrees, i.e., decreased oleic acid and increased linoleic acid and α-linolenic acid, in all Bnatt mutants except Bnatt18. Furthermore, gene expression analysis revealed that most of BnaTT mutations resulted in the down-regulation of key genes related to flavonoid and lignin synthesis, and the up-regulation of key genes related to lipid synthesis and oil body formation, which may contribute to the phenotype. Collectively, our study generated valuable resources for breeding programs, and more importantly demonstrated the functional divergence and overlap of flavonoid biosynthesis pathway genes in seed coat colour, oil content and FA composition of rapeseed.

Drude Polarizable Lipid Force Field with Explicit Treatment of Long-Range Dispersion: Parametrization and Validation for Saturated and Monounsaturated Zwitterionic Lipids.

Accurate empirical force fields of lipid molecules are a critical component of molecular dynamics simulation studies aimed at investigating properties of monolayers, bilayers, micelles, vesicles, and liposomes, as well as heterogeneous systems, such as protein-membrane complexes, bacterial cell walls, and more. While the majority of lipid force field-based simulations have been performed using pairwise-additive nonpolarizable models, advances have been made in the development of the polarizable force field based on the classical Drude oscillator model. In the present study, we undertake further optimization of the Drude lipid force field, termed Drude2023, including improved treatment of the phosphate and glycerol linker region of PC and PE headgroups, additional optimization of the alkene group in monounsaturated lipids, and inclusion of long-range Lennard-Jones interactions using the particle-mesh Ewald method. Initial optimization targeted quantum mechanical (QM) data on small model compounds representative of the linker region. Subsequent optimization targeted QM data on larger model compounds, experimental data, and dihedral potentials of mean force from the CHARMM36 additive lipid force field using a parameter reweighting protocol. The use of both experimental and QM target data during the reweighting protocol is shown to produce physically reasonable parameters that reproduce a collection of experimental observables. Target data for optimization included surface area/lipid for DPPC, DSPC, DMPC, and DLPC bilayers and nuclear magnetic resonance (NMR) order parameters for DPPC bilayers. Validation data include prediction of membrane thickness, scattering form factors, electrostatic potential profiles, compressibility moduli, surface area per lipid, water permeability, NMR T1 relaxation times, diffusion constants, and monolayer surface tensions for a variety of saturated and unsaturated lipid mono- and bilayers. Overall, the agreement with experimental data is quite good, though the results are less satisfactory for the NMR T1 relaxation times for carbons near the ester groups. Notable improvements compared to the additive C36 force field were obtained for membrane dipole potentials, lipid diffusion coefficients, and water permeability with the exception of monounsaturated lipid bilayers. It is anticipated that the optimized polarizable Drude2023 force field will help generate more accurate molecular simulations of pure bilayers and heterogeneous systems containing membranes, advancing our understanding of the role of electronic polarization in these systems.

Molecular dynamics simulations of the human ocular lens with age and cataract.

The human ocular lens consists primarily of elongated, static fibers characterized by high stability and low turnover, which differ dramatically in their composition and properties from other biological membranes. Cholesterol (Chol) and sphingolipids (SL) are present at high concentrations, including saturated SLs, such as dihyrosphingomyelin (DHSM). Past molecular dynamics simulations demonstrated that the presence of DHSM and high Chol concentration contributes to higher order in lipid membranes. This current study simulated more complex models of human lens membranes. Models were developed representing physiological compositions in cataractous lenses aged 74 ± 6 years and in healthy lenses aged 22 ± 4, 41 ± 6, and 69 ± 3 years. With older age, Chol and ceramide concentrations increase and glycerophospholipid concentration decreases. With cataract, ceramide concentration increases and Chol and glycerophospholipid concentrations decrease. Surface area per lipid, deuterium order parameters (SCD), sterol tilt angle, electron density profiles, bilayer thickness, chain interdigitation, two-dimensional radial distribution functions (2D-RDF), lipid clustering, and hydrogen bonding were calculated for all simulations. All systems exhibited low surface area per lipid and high bilayer thickness, indicative of strong vertical packing. SCD parameters suggest similarly, with saturated tails in the hydrophobic core of the membrane having elevated order. Vertical packing and acyl tail order increased with both age and cataract condition. Lateral diffusion decreased with age and cataracts, with the older and cataractous models demonstrating increased long-range structure by the 2D-RDF analysis. In future work examining the membrane proteins of the lens, these models can serve as a physiologically accurate representation of the lens lipidome.

Colchicine Is a Weapon for Managing the Heart Disease Among Interstitial Lung Disease With Viral Infection: Have We Found the Holy Grail?

Background: This study investigated the effect of colchicine use on the risks of heart disease (HD), pericarditis, endocarditis, myocarditis, cardiomyopathy, cardiac arrhythmia, and cardiac failure in patients having interstitial lung disease (ILD) with virus infection (ILD cohort). Methods: We retrospectively enrolled ILD cohort between 2000 and 2013 from the Longitudinal Health Insurance Database and divided them into colchicine users (n = 12,253) and colchicine non-users (n = 12,253) through propensity score matching. The event of interest was the diagnosis of HD. The incidence of HD was analyzed using multivariate Cox proportional hazards models between colchicine users and the comparison cohort after adjustment for age, sex, medication, comorbidities, and index date based on the time-dependent analysis. Results: Colchicine users had a significantly lower risk of HD (aHR = 0.87, 95% confidence interval (CI]) = 0.82-0.92) than did the colchicine non-user. For colchicine non-users as the reference, the aHR (95% CI) of the patients who received colchicine of 2-7, 8-30, 31-150, and > 150 days were 0.89 (0.81-0.98), 0.84 (0.76-0.94), 090 (0.80-0.99), and 0.83 (0.74-0.93), respectively; regardless of duration use, the lower risk of HD persisted in colchicine users. The cumulative incidence of HD in colchicine users was significantly lower than that in the colchicine non-users (log-rank p < 0.001). Conclusion: The addition of short-term or long-term colchicine to standard medical therapy may have benefits to prevent the HD among the ILD patients concurrent with a virus infection or comorbidities even in elderly patients.

Site-Directed Mutagenesis of the Carotenoid Isomerase Gene BnaCRTISO Alters the Color of Petals and Leaves in Brassica napus L.

The diversity of petal and leaf color can improve the ornamental value of rapeseed and promote the development of agriculture and tourism. The two copies of carotenoid isomerase gene (BnaCRTISO) in Brassica napus (BnaA09.CRTISO and BnaC08.CRTISO) was edited using the CRISPR/Cas9 system in the present study. The mutation phenotype of creamy white petals and yellowish leaves could be recovered only in targeted mutants of both BnaCRTISO functional copies, indicating that the redundant roles of BnaA09.CRTISO and BnaC08.CRTISO are vital for the regulation of petal and leaf color. The carotenoid content in the petals and leaves of the BnaCRTISO double mutant was significantly reduced. The chalcone content, a vital substance that makes up the yellow color, also decreased significantly in petals. Whereas, the contents of some carotenes (lycopene, α-carotene, γ-carotene) were increased significantly in petals. Further, transcriptome analysis showed that the targeted mutation of BnaCRTISO resulted in the significant down-regulation of important genes BnaPSY and BnaC4H in the carotenoid and flavonoid synthesis pathways, respectively; however, the expression of other genes related to carotenes and xanthophylls synthesis, such as BnaPDS3, BnaZEP, BnaBCH1 and BCH2, was up-regulated. This indicates that the molecular mechanism regulating petal color variation in B. napus is more complicated than those reported in Arabidopsis and other Brassica species. These results provide insight into the molecular mechanisms underlying flower color variation in rapeseed and provides valuable resources for rapeseed breeding.

All-Atom Modeling of Complex Cellular Membranes.

Cell membranes are composed of a variety of lipids and proteins where they interact with each other to fulfill their roles. The first step in modeling these interactions in molecular simulations is to have reliable mimetics of the membrane's lipid environment. This Feature Article presents our recent efforts to model complex cellular membranes using all-atom force fields. A short review of the CHARMM36 (C36) lipid force field and its recent update to incorporate the long-range dispersion is presented. Key examples of model membranes mimicking various species and organelles are given. These include single-celled organisms such as bacteria (E. coli., chlamydia, and P. aeruginosa) and yeast (plasma membrane, endoplasmic reticulum, and trans-Golgi network) and more advanced ones such as plants (soybean and Arabidopsis thaliana) and mammals (ocular lens, stratum corneum, and peripheral nerve myelin). Leaflet asymmetry in composition has also been applied to some of these models. With the increased lipid diversity in the C36 lipid FF, these complex models can better reflect the structural, mechanical, and dynamic properties of realistic membranes and open an opportunity to study biological processes involving other molecules.

Symmetric and Asymmetric Models for the Arabidopsis thaliana Plasma Membrane: A Simulation Study.

Arabidopsis thaliana is an important model organism, which has attracted many biologists. While most research efforts have been on studying the genetics and proteins of this organism, a systematic study of its lipidomics is lacking. Here, we present a novel, asymmetric model of its cell membrane with its lipid composition consisting of five glycerophospholipids, two glycolipids, and sitosterol determined from multiple independent experiments. A typical lipid type in plant membranes is glycosyl inositol phosphoryl ceramide (GIPC), which accounts for about 10% of the total lipids in the outer leaflet in our model. Two symmetric models representing the inner and outer leaflets of the membrane were built and simulated until equilibrium was reached and then combined to form the asymmetric model. Our results indicate that the outer leaflet is more rigid and tightly packed compared to the inner leaflet. Pressure profiles for the two leaflets are overall similar though the outer leaflet exhibits larger oscillations. A special focus on lipid organization is discussed and the interplay between glycolipids and sitosterols is found to be important. The current model provides a baseline for future modeling of similar membranes and can be used to study partitioning of small molecules in the membrane or further developed to study the interaction between plant membrane proteins and lipids.

Simulations of Diabetic and Non-Diabetic Peripheral Nerve Myelin Lipid Bilayers.

The multilayered myelin sheath is a critical component of both central and peripheral nervous systems, forming a protective barrier against axonal damage and facilitating the movement of nervous impulses. It is primarily composed of cholesterol (CHL1), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin (SM), and galactosylceramide (GalCer) lipids. For rat sciatic nerve myelin (part of the peripheral nervous system, PNS), it has been found that cholesterol and unsaturated fatty acid contents are significantly lower in diabetic than in non-diabetic conditions. In this study, lipid compositions from experimental data are used to create four model rat sciatic nerve myelin lipid bilayers: PI-containing (non-diabetic and diabetic) and PS-containing (non-diabetic and diabetic), which were then simulated using the all-atom CHARMM36 force field. Simulation results of diabetic membranes indicate less rigid, more laterally expansive, and thinner bilayers as well as potentially reduced interactions between GalCer on opposing myelin leaflets, supporting a direct role of the cholesterol content decrease in instigating myelin deterioration and diabetic peripheral neuropathy. Compared to PI-lipids, PS-lipids were found to cause higher inter-lipid spacing and decreased order within membranes as a result of their smaller headgroup size and higher inter-lipid hydrogen bonding potential, which allow them to more frequently reside deeper in the membrane plane and produce pushing effects on other lipids. GalCer deuterium order parameters and non-diabetic headgroup-to-headgroup bilayer thicknesses were compared to experimental data, exhibiting close alignment, supporting the future usage of these models to study the PNS myelin sheath.

Estimating localization of various statins within a POPC bilayer.

As a class of drugs prescribed to heart disease patients, statins are among the most popular prescription drugs in the world. Over the years, statins have been shown to have beneficial effects on patients via pathways independent of their effect on cholesterol. These pleiotropic effects vary across the different statins, and a growing hypothesis is that they are related to the localization of the statins in and their effect on the membrane. In this study, we use molecular dynamics (MD) simulations with the CHARMM36 all-atom force field to investigate the localization of statins (atorvastatin, cerivastatin, lovastatin, and pravastatin) in a POPC bilayer and how they affect the acyl chain order parameters (SCD), surface area per lipid (APL), and thicknesses of the bilayer. The data obtained from 500 ns simulations suggests that lovastatin is localized deepest in the membrane, mostly interacting with the hydrophobic core, cerivastatin is slightly closer to the bilayer/solvent interface than lovastatin and interacts with the headgroups via its dihydroxy acid group, and pravastatin is found closest to the bilayer/solvent interface, its hydrophobic rings interacting mostly with the region around the acyl's carbonyl and its dihydroxy acid interacting with the solvent and the headgroups. Consistent binding of atorvastatin to the bilayer is not observed during our simulation due to self-aggregation. The statins differentially alter the SCD and APL and most of the bilayer thicknesses, but these effects are modest. Overall, as expected, the localization of statins seems to follow their hydrophilicity, and given previous data showing the relationship between statins' hydrophobicity and pleiotropic effects, one would expect statins that localize and interact with different regions of the membrane to have different effects. This research provides some important insight into statin localization in a simplified model of a cellular membrane.

CHARMM36 Lipid Force Field with Explicit Treatment of Long-Range Dispersion: Parametrization and Validation for Phosphatidylethanolamine, Phosphatidylglycerol, and Ether Lipids.

Long-range Lennard-Jones (LJ) interactions have been incorporated into the CHARMM36 (C36) lipid force field (FF) using the LJ particle-mesh Ewald (LJ-PME) method in order to remove the inconsistency of bilayer and monolayer properties arising from the exclusion of long-range dispersion [Yu, Y.; Semi-automated Optimization of the CHARMM36 Lipid Force Field to Include Explicit Treatment of Long-Range Dispersion. J. Chem. Theory Comput. 2021, 10.1021/acs.jctc.0c01326. (preceding article in this issue)]. The new FF is denoted C36/LJ-PME. While the first optimization was based on three phosphatidylcholines (PCs), this work extends the validation and parametrization to more lipids including PC, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and ether lipids. The agreement with experimental structure data is excellent for PC, PE, and ether lipids. C36/LJ-PME also compares favorably with scattering data of PG bilayers but less so with NMR deuterium order parameters of 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG) at 303.15 K, indicating a need for future optimization regarding PG-specific parameters. Frequency dependence of NMR T1 spin-lattice relaxation times is well-described by C36/LJ-PME, and the overall agreement with experiment is comparable to C36. Lipid diffusion is slower than C36 due to the added long-range dispersion causing a higher viscosity, although it is still too fast compared to experiment after correction for periodic boundary conditions. When using a 10 Å real-space cutoff, the simulation speed of C36/LJ-PME is roughly equal to C36. While more lipids will be incorporated into the FF in the future, C36/LJ-PME can be readily used for common lipids and extends the capability of the CHARMM FF by supporting monolayers and eliminating the cutoff dependence.

Semi-automated Optimization of the CHARMM36 Lipid Force Field to Include Explicit Treatment of Long-Range Dispersion.

The development of the CHARMM lipid force field (FF) can be traced back to the early 1990s with its current version denoted CHARMM36 (C36). The parametrization of C36 utilized high-level quantum mechanical data and free energy calculations of model compounds before parameters were manually adjusted to yield agreement with experimental properties of lipid bilayers. While such manual fine-tuning of FF parameters is based on intuition and trial-and-error, automated methods can identify beneficial modifications of the parameters via their sensitivities and thereby guide the optimization process. This work introduces a semi-automated approach to reparametrize the CHARMM lipid FF with consistent inclusion of long-range dispersion through the Lennard-Jones particle-mesh Ewald (LJ-PME) approach. The optimization method is based on thermodynamic reweighting with regularization with respect to the C36 set. Two independent optimizations with different topology restrictions are presented. Targets of the optimizations are primarily liquid crystalline phase properties of lipid bilayers and the compression isotherm of monolayers. Pair correlation functions between water and lipid functional groups in aqueous solution are also included to address headgroup hydration. While the physics of the reweighting strategy itself is well-understood, applying it to heterogeneous, complex anisotropic systems poses additional challenges. These were overcome through careful selection of target properties and reweighting settings allowing for the successful incorporation of the explicit treatment of long-range dispersion, and we denote the newly optimized lipid force field as C36/LJ-PME. The current implementation of the optimization protocol will facilitate the future development of the CHARMM and related lipid force fields.

Update of the CHARMM36 United Atom Chain Model for Hydrocarbons and Phospholipids.

Accurate lipid force field (FF) parameters used in molecular dynamics (MD) simulations are crucial for understanding the properties of lipid-containing systems and biological processes related to lipids. The last update of the CHARMM36 united atom chain model (C36UA) was in 2013 [Lee, S. J. Phys. Chem. B 2014, 118, 547 556]; it utilized CHARMM36 (C36) lipid FF parameters for headgroups and OPLS-UA Lennard-Jones (LJ) parameters for tails. Simulations with the FF were able to reproduce many experimental observables of lipid bilayers accurately, but to be more applicable for a wide range of lipids, additional FF parameter optimization was needed. In this work, we present an update of the model, named C36UAr. The parameterization included the LJ parameters for hydrocarbons and related dihedrals. Bulk liquid properties (density, heat of vaporization, isothermal compressibility, and diffusion constant) of model compounds were used as targets for the LJ parameter fitting, and dihedrals were fit to either quantum mechanical (QM) or potential of mean force (PMF) calculations using C36. Thermodynamic reweighting was used to further improve the parameters. Bilayer simulations of various lipid headgroups (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol) and tails (saturated, monounsaturated, and polyunsaturated) were performed to validate the model, and significant improvements were seen in bilayer properties, including surface area, membrane thicknesses, NMR deuterium order parameters, and density profiles. C36UAr was also compared to the hydrogen mass repartitioning (HMR) method. The high accuracy and competitive efficiency shown in this study make C36UAr one of the best choices for studies of membrane structure and membrane-associated proteins.

Modeling Pseudomonas aeruginosa inner plasma membrane in planktonic and biofilm modes.

Pseudomonas aeruginosa changes its growth modes under different conditions. The bacteria in biofilm is more resistant to environmental stress compared to the planktonic mode of growth. The compositions of the inner plasma membrane for the two modes are noticeably different. Major lipid types are chosen from experiment to model the membrane in both modes of growth, and molecular dynamics simulation is used to study the properties of the membrane. The CHARMM36 lipid force field is used and tested against several experimental results. Our models include lipids containing cyclopropane in the middle of the sn-2 tail, namely, 1-palmitoyl-2-cis-11,12-methylene-stearic-acid-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-cis-11,12-methylene-stearic-acid-sn-glycero-3-phosphoglycerol. The PE:PG ratio for the two model membranes is close, but the fraction of lipids composed of long-chain and cyclopropane-containing fatty acids changes significantly, causing differences between the two models. Compared to previous model membranes built for Escherichia coli, the inner membrane of P. aeruginosa has a longer averaged lipid tail length and a higher percentage of PG lipids, which are responsible for the changes in membrane properties like membrane thickness and stiffness. Most importantly, the comparison to experiments shows good agreements and encourages the model's use to study the behavior of proteins from P. aeruginosa associated with the membrane.