Effects of linker length on phase separation: lessons from the Rubisco-EPYC1 system of the algal pyrenoid.

Biomolecular condensates are membraneless organelles formed via phase separation of macromolecules, typically consisting of bond-forming "stickers" connected by flexible "linkers". Linkers have diverse roles, such as occupying space and facilitating interactions. To understand how linker length relative to other lengths affects condensation, we focus on the pyrenoid, which enhances photosynthesis in green algae. Specifically, we apply coarse-grained simulations and analytical theory to the pyrenoid proteins of Chlamydomonas reinhardtii: the rigid holoenzyme Rubisco and its flexible partner EPYC1. Remarkably, halving EPYC1 linker lengths decreases critical concentrations by ten-fold. We attribute this difference to the molecular "fit" between EPYC1 and Rubisco. Varying Rubisco sticker locations reveals that the native sites yield the poorest fit, thus optimizing phase separation. Surprisingly, shorter linkers mediate a transition to a gas of rods as Rubisco stickers approach the poles. These findings illustrate how intrinsically disordered proteins affect phase separation through the interplay of molecular length scales.

Single-image diffusion coefficient measurements of proteins in free solution.

Diffusion coefficient measurements are important for many biological and material investigations, such as studies of particle dynamics and kinetics, and size determinations. Among current measurement methods, single particle tracking (SPT) offers the unique ability to simultaneously obtain location and diffusion information about a molecule while using only femtomoles of sample. However, the temporal resolution of SPT is limited to seconds for single-color-labeled samples. By directly imaging three-dimensional diffusing fluorescent proteins and studying the widths of their intensity profiles, we were able to determine the proteins' diffusion coefficients using single protein images of submillisecond exposure times. This simple method improves the temporal resolution of diffusion coefficient measurements to submilliseconds, and can be readily applied to a range of particle sizes in SPT investigations and applications in which diffusion coefficient measurements are needed, such as reaction kinetics and particle size determinations.

Increase of hepatic fat accumulation by liver specific expression of Hepatitis B virus X protein in zebrafish.

The pathogenesis of fatty liver disease remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis in zebrafish by liver specific expression of Hepatitis B virus X protein (HBx). Transgenic zebrafish lines, GBXs, which selectively express the GBx transgene (GFP-fused HBx gene) in liver, were established. GBX Liver phenotypes were evaluated by histopathology and molecular analysis of fatty acid (FA) metabolism-related genes expression. Most GBXs (66-81%) displayed obvious emaciation starting at 4 months old. Over 99% of the emaciated GBXs developed hepatic steatosis or steatohepatitis, which in turn led to liver hypoplasia. The liver histology of GBXs displayed steatosis, lobular inflammation, and balloon degeneration, similar to non-alcoholic steatohepatitis (NASH). Oil red O stain detected the accumulation of fatty droplets in GBXs. RT-PCR and Q-rt-PCR analysis revealed that GBx induced hepatic steatosis had significant increases in the expression of lipogenic genes, C/EBP-alpha, SREBP1, ChREBP and PPAR-gamma, which then activate key enzymes of the de novo FA synthesis, ACC1, FAS, SCD1, AGAPT, PAP and DGAT2. In addition, the steatohepatitic GBX liver progressed to liver degeneration and exhibited significant differential gene expression in apoptosis and stress. The GBX models exhibited both the genetic and functional factors involved in lipid accumulation and steatosis-associated liver injury. In addition, GBXs with transmissible NASH-like phenotypes provide a promising model for studying liver disease.

Quantum chemical benchmark databases of gold-standard dimer interaction energies.

Advances in computational chemistry create an ongoing need for larger and higher-quality datasets that characterize noncovalent molecular interactions. We present three benchmark collections of quantum mechanical data, covering approximately 3,700 distinct types of interacting molecule pairs. The first collection, which we refer to as DES370K, contains interaction energies for more than 370,000 dimer geometries. These were computed using the coupled-cluster method with single, double, and perturbative triple excitations [CCSD(T)], which is widely regarded as the gold-standard method in electronic structure theory. Our second benchmark collection, a core representative subset of DES370K called DES15K, is intended for more computationally demanding applications of the data. Finally, DES5M, our third collection, comprises interaction energies for nearly 5,000,000 dimer geometries; these were calculated using SNS-MP2, a machine learning approach that provides results with accuracy comparable to that of our coupled-cluster training data. These datasets may prove useful in the development of density functionals, empirically corrected wavefunction-based approaches, semi-empirical methods, force fields, and models trained using machine learning methods.

Precision analysis for standard deviation measurements of immobile single fluorescent molecule images.

Standard deviation measurements of intensity profiles of stationary single fluorescent molecules are useful for studying axial localization, molecular orientation, and a fluorescence imaging system's spatial resolution. Here we report on the analysis of the precision of standard deviation measurements of intensity profiles of single fluorescent molecules imaged using an EMCCD camera.We have developed an analytical expression for the standard deviation measurement error of a single image which is a function of the total number of detected photons, the background photon noise, and the camera pixel size. The theoretical results agree well with the experimental, simulation, and numerical integration results. Using this expression, we show that single-molecule standard deviation measurements offer nanometer precision for a large range of experimental parameters.

Structural Basis of AZD9291 Selectivity for EGFR T790M.

AZD9291 (Osimertinib) is highly effective in treating EGFR-mutated non-small-cell lung cancers (NSCLCs) with T790M-mediated drug resistance. Despite the remarkable success of AZD9291, its binding pose with EGFR T790M remains unclear. Here, we report unbiased, atomic-level molecular dynamics (MD) simulations in which spontaneous association of AZD9291 with EGFR kinases having WT and T790M mutant gatekeepers was observed. Simulation-generated structural models suggest that the binding pose of AZD9291 with T790M differs from its binding pose with the WT, and that AZD9291 interacts extensively with the gatekeeper residue (Met 790) in T790M but not with Thr 790 in the WT, which explains why AZD9291 binds T790M with higher affinity. The MD simulation-generated models were confirmed by experimentally determined EGFR/T790M complex crystal structures. This work may facilitate the rational design of drugs that can overcome resistance mutations to AZD9291, and more generally it suggests the potential of using unbiased MD simulation to elucidate small-molecule binding poses.

Interdisciplinary Approach to Treat Patients With Tooth Agenesis: A Case Report.

INTRODUCTION: Tooth agenesis is a type of dental anomaly in which individuals are missing teeth due to developmental failure as a result of genetic or environmental factors. With approximately one fourth of the population missing ≥1 third molar, tooth agenesis is considered a common dental anomaly. However, the severity of tooth agenesis can range from a missing single tooth to multiple teeth. When suffering from severe tooth agenesis, the patient's health and social relationships are often affected. CASE PRESENTATION: The patient in this report congenitally lost 11 teeth and suffered from compromised esthetics and impaired chewing function. In such a severe tooth agenesis case, interdisciplinary treatments involving orthodontics, periodontics, and prosthodontics were engaged to reconstruct the ideal biology, function, and esthetics for the patient. With an interdisciplinary approach, the periodontist played an important role in the rehabilitation of the edentulous regions with implants in combination with various hard and soft tissue augmentation procedures. In addition, the patient with severe tooth agenesis presented with additional dental anomalies. The periodontist, therefore, had to collaborate with other specialists to provide early detection and intervention to avoid future complications, such as the management of infraoccluded ankylosed deciduous molars and aberrant frenum. The patient at the end of treatment had a good occlusion with improved function and esthetics. CONCLUSION: This case report describes the interdisciplinary treatment approach used and points out the role of periodontists in the treatment of a patient with severe tooth agenesis.

Anisotropic adsorption of molecular assemblies on crystalline surfaces.

Orientational order of surfactant micelles and proteins on crystalline templates has been observed but, given that the template unit cell is significantly smaller than the characteristic size of the adsorbate, this order cannot be attributed to lattice epitaxy. We interpret the template-directed orientation of rodlike molecular assemblies as arising from anisotropic van der Waals interactions between the assembly and crystalline surfaces where the anisotropic van der Waals interaction is calculated using the Lifshitz methodology. Provided the assembly is sufficiently large, substrate anisotropy provides a torque that overcomes rotational Brownian motion near the surface. The probability of a particular orientation is computed by solving a Smoluchowski equation that describes the balance between van der Waals and Brownian torques. Torque aligns both micelles and protein fibrils; the interaction energy is minimized when the assembly lies perpendicular to a symmetry axis of a crystalline substrate. Theoretical predictions agree with experiments for both hemi-cylindrical micelles and protein fibrils adsorbed on graphite.

Functionalized single graphene sheets derived from splitting graphite oxide.

A process is described to produce single sheets of functionalized graphene through thermal exfoliation of graphite oxide. The process yields a wrinkled sheet structure resulting from reaction sites involved in oxidation and reduction processes. The topological features of single sheets, as measured by atomic force microscopy, closely match predictions of first-principles atomistic modeling. Although graphite oxide is an insulator, functionalized graphene produced by this method is electrically conducting.

[Successful preimplantation genetic diagnosis for beta-thalassemia using primer extension preamplification].

OBJECTIVE: To achieve preimplantation genetic diagnosis (PGD) of the couples at risk of having children with beta-thalassemia, as an alternative to prenatal diagnosis. METHODS: Two couples carrying different thalassemia mutations of codon 41/42 and codon intervening sequence 2 position 654 received standard in vitro fertilization treatment and intracytoplasmic sperm injection, embryo biopsy and the whole genome was amplified by primer extension preamplification (PEP). Nested polymerase chain reaction was then used to amplify two mutation sites separately. Both were detected by reverse dot-blot. RESULTS: A total of 35 oocytes were retrieved from the two patients. Among them, 87% showed two pronuclei, and embryo biopsy was performed on 16 of these embryos and 25 blastomeres were obtained. The amplification efficacy was 84%. The genotype study of non-transferred and surplus embryos showed 15% of allele drop-out rate. Five embryos were transferred to the uterus of both patients. One pregnancy achieved, resulted in live healthy twin births, which confirmed the results of PGD. CONCLUSIONS: This unaffected pregnancy resulting from PGD by PEP for beta-thalassemia demonstrates that this technique can be a effective diagnostic tool for carrier couples who desire a healthy child.

Protein sliding and hopping kinetics on DNA.

Using Monte Carlo simulations, we deconvolved the sliding and hopping kinetics of GFP-LacI proteins on elongated DNA from their experimentally observed seconds-long diffusion trajectories. Our simulations suggest the following results: (i) in each diffusion trajectory, a protein makes on average hundreds of alternating slides and hops with a mean sliding time of several tens of milliseconds; (ii) sliding dominates the root-mean-square displacement of fast diffusion trajectories, whereas hopping dominates slow ones; (iii) flow and variations in salt concentration have limited effects on hopping kinetics, while in vivo DNA configuration is not expected to influence sliding kinetics; and (iv) the rate of occurrence for hops longer than 200 nm agrees with experimental data for EcoRV proteins.

Bending properties of single functionalized graphene sheets probed by atomic force microscopy.

We probe the bending characteristics of functionalized graphene sheets with the tip of an atomic force microscope. Individual sheets are transformed from a flat into a folded configuration. Sheets can be reversibly folded and unfolded multiple times, and the folding always occurs at the same location. This observation suggests that the folding and bending behavior of the sheets is dominated by pre-existing kink (or even fault) lines consisting of defects and/or functional groups.

Liver-specific expression of p53-negative regulator mdm2 leads to growth retardation and fragile liver in zebrafish.

Tumorigenesis requires inactivation of the p53 tumor suppressor pathway, likely involving the negative regulator Mdm2 protein. To analyze the possible roles of Mdm2 in oncogenesis and other functions during zebrafish hepatogenesis, we generated transgenic zebrafish by liver-specific Mdm2 over-expression utilizing a fusion between genes encoding GFP and mdm2, GFP::Mdm2. Over-expression of GFP::Mdm2 in the zebrafish liver did not interrupt normal liver development in the larval stages but approximately 30% of the adult fish raised from the same larvae displayed obvious growth retardation at 16 weeks of age. Most growth-retarded adults displayed liver atrophy, contraction, or hypoplasia, which proved lethal within 4 to 8 months. Histologically, over-expression of GFP::Mdm2 in Gmdm2-liver leading to liver degeneration may in some way have been due to an increased cell apoptosis accompanied by a slightly interrupted cell cycle or hepatocyte proliferation. Liver degeneration or other transgenic phenotypes were not associated with liver cancer; however, liver-degenerated phenotypes could be passed to wild-type zebrafish. In this study, we generated transgenic zebrafish lines with a "fragile liver." The "fragile liver" zebrafish can provide a model for molecular pathology of liver diseases and for screening small molecules that affect mdm2-related pathways.

Hydrophobic interaction and hydrogen-bond network for a methane pair in liquid water.

We employ fully quantum-mechanical molecular dynamics simulations to evaluate the force between two methanes dissolved in water, as a model for hydrophobic association. A stable configuration is found near the methane-methane contact separation, while a shallow second potential minimum occurs for the solvent-separated configuration. The strength and shape of the potential of mean force are in conflict with earlier classical force-field simulations but agree well with a simple hydrophobic burial model which is based on solubility experiments. Examination of solvent dynamics reveals stable water cages at several specific methane-methane separations.

Oxygen-driven unzipping of graphitic materials.

Optical microscope images of graphite oxide (GO) reveal the occurrence of fault lines resulting from the oxidative processes. The fault lines and cracks of GO are also responsible for their much smaller size compared with the starting graphite materials. We propose an unzipping mechanism to explain the formation of cracks on GO and cutting of carbon nanotubes in an oxidizing acid. GO unzipping is initiated by the strain generated by the cooperative alignment of epoxy groups on a carbon lattice. We employ two small GO platelets to show that through the binding of a new epoxy group or the hopping of a nearby existing epoxy group, the unzipping process can be continued during the oxidative process of graphite. The same epoxy group binding pattern is also likely to be present in an oxidized carbon nanotube and cause its breakup.