Next-Generation Sequencing of Circulating Tumor DNA Reveals Frequent Alterations in Advanced Hepatocellular Carcinoma.

BACKGROUND: Because imaging has a high sensitivity to diagnose hepatocellular carcinoma (HCC) and tissue biopsies carry risks such as bleeding, the latter are often not performed in HCC. Blood-derived circulating tumor DNA (ctDNA) analysis can identify somatic alterations, but its utility has not been characterized in HCC. MATERIALS AND METHODS: We evaluated 14 patients with advanced HCC (digital ctDNA sequencing [68 genes]). Mutant relative to wild-type allele fraction was calculated. RESULTS: All patients (100%) had somatic alterations (median = 3 alterations/patient [range, 1-8]); median mutant allele fraction, 0.29% (range, 0.1%-37.77%). Mutations were identified in several genes: TP53 (57% of patients), CTNNB1 (29%), PTEN (7%), CDKN2A (7%), ARID1A (7%), and MET (7%); amplifications, in CDK6 (14%), EGFR (14%), MYC (14%), BRAF (7%), RAF1 (7%), FGFR1 (7%), CCNE1 (7%), PIK3CA (7%), and ERBB2/HER2 (7%). Eleven patients (79%) had ≥1 theoretically actionable alteration. No two patients had identical genomic portfolios, suggesting the need for customized treatment. A patient with a CDKN2A-inactivating and a CTNNB1-activating mutation received matched treatment: palbociclib (CDK4/6 inhibitor) and celecoxib (COX-2/Wnt inhibitor); des-gamma-carboxy prothrombin level decreased by 84% at 2 months (1,410 to 242 ng/mL [normal: ≤7.4 ng/mL]; alpha fetoprotein [AFP] low at baseline). A patient with a PTEN-inactivating and a MET-activating mutation (an effect suggested by in silico molecular dynamic simulations) received sirolimus (mechanistic target of rapamycin inhibitor) and cabozantinib (MET inhibitor); AFP declined by 63% (8,320 to 3,045 ng/mL [normal: 0-15 ng/mL]). CONCLUSION: ctDNA derived from noninvasive blood tests can provide exploitable genomic profiles in patients with HCC. IMPLICATIONS FOR PRACTICE: This study reports that blood-derived circulating tumor DNA can provide therapeutically exploitable genomic profiles in hepatocellular cancer, a malignancy that is known to be difficult to biopsy.

Water exit pathways and proton pumping mechanism in B-type cytochrome c oxidase from molecular dynamics simulations.

Cytochrome c oxidase (CcO) is a vital enzyme that catalyzes the reduction of molecular oxygen to water and pumps protons across mitochondrial and bacterial membranes. While proton uptake channels as well as water exit channels have been identified for A-type CcOs, the means by which water and protons exit B-type CcOs remain unclear. In this work, we investigate potential mechanisms for proton transport above the dinuclear center (DNC) in ba3-type CcO of Thermus thermophilus. Using long-time scale, all-atom molecular dynamics (MD) simulations for several relevant protonation states, we identify a potential mechanism for proton transport that involves propionate A of the active site heme a3 and residues Asp372, His376 and Glu126(II), with residue His376 acting as the proton-loading site. The proposed proton transport process involves a rotation of residue His376 and is in line with experimental findings. We also demonstrate how the strength of the salt bridge between residues Arg225 and Asp287 depends on the protonation state and that this salt bridge is unlikely to act as a simple electrostatic gate that prevents proton backflow. We identify two water exit pathways that connect the water pool above the DNC to the outer P-side of the membrane, which can potentially also act as proton exit transport pathways. Importantly, these water exit pathways can be blocked by narrowing the entrance channel between residues Gln151(II) and Arg449/Arg450 or by obstructing the entrance through a conformational change of residue Tyr136, respectively, both of which seem to be affected by protonation of residue His376.

A de novo compound targeting α-synuclein improves deficits in models of Parkinson's disease.

Abnormal accumulation and propagation of the neuronal protein α-synuclein has been hypothesized to underlie the pathogenesis of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Here we report a de novo-developed compound (NPT100-18A) that reduces α-synuclein toxicity through a novel mechanism that involves displacing α-synuclein from the membrane. This compound interacts with a domain in the C-terminus of α-synuclein. The E83R mutation reduces the compound interaction with the 80-90 amino acid region of α-synuclein and prevents the effects of NPT100-18A. In vitro studies showed that NPT100-18A reduced the formation of wild-type α-synuclein oligomers in membranes, reduced the neuronal accumulation of α-synuclein, and decreased markers of cell toxicity. In vivo studies were conducted in three different α-synuclein transgenic rodent models. Treatment with NPT100-18A ameliorated motor deficits in mThy1 wild-type α-synuclein transgenic mice in a dose-dependent manner at two independent institutions. Neuropathological examination showed that NPT100-18A decreased the accumulation of proteinase K-resistant α-synuclein aggregates in the CNS and was accompanied by the normalization of neuronal and inflammatory markers. These results were confirmed in a mutant line of α-synuclein transgenic mice that is prone to generate oligomers. In vivo imaging studies of α-synuclein-GFP transgenic mice using two-photon microscopy showed that NPT100-18A reduced the cortical synaptic accumulation of α-synuclein within 1 h post-administration. Taken together, these studies support the notion that altering the interaction of α-synuclein with the membrane might be a feasible therapeutic approach for developing new disease-modifying treatments of Parkinson's disease and other synucleinopathies.

Simulation of lipid bilayer self-assembly using all-atom lipid force fields.

In this manuscript we expand significantly on our earlier communication by investigating the bilayer self-assembly of eight different types of phospholipids in unbiased molecular dynamics (MD) simulations using three widely used all-atom lipid force fields. Irrespective of the underlying force field, the lipids are shown to spontaneously form stable lamellar bilayer structures within 1 microsecond, the majority of which display properties in satisfactory agreement with the experimental data. The lipids self-assemble via the same general mechanism, though at formation rates that differ both between lipid types, force fields and even repeats on the same lipid/force field combination. In addition to zwitterionic phosphatidylcholine (PC) and phosphatidylethanolamine (PE) lipids, anionic phosphatidylserine (PS) and phosphatidylglycerol (PG) lipids are represented. To our knowledge this is the first time bilayer self-assembly of phospholipids with negatively charged head groups is demonstrated in all-atom MD simulations.

BRAF mutation as a novel driver of eosinophilic cystitis.

Eosinophilic cystitis is a rare manifestation of hypereosinophilia and a cause of morbidity, including dysuria and hematuria. Although some cases can be attributed to infection or allergy, most cases are assessed to be idiopathic and treated with corticosteroids. However, hypereosinophilia can also be due to actionable clonal molecular alterations in the haematopoietic cells, similar to other myeloproliferative neoplasms. Common mutations associated with eosonophilic syndromes are of platelet-derived growth factor receptor α or ß or c-kit, though other pathogenic mutations have been found by next generation sequencing. Determination of a specific mutation may therefore identify clonality and refine treatment of some cases. Here we review the molecular features of eosinophilic disorders. We also describe the use of a liquid biopsy of circulating cell-free DNA in the workup of a case of eosinophilic cystitis in which next generation sequencing of cell-free DNA showed a BRAF I463T mutation. In silico modeling supports the functional impact and potential clinical relevance of BRAF I463T.

Data for molecular dynamics simulations of B-type cytochrome c oxidase with the Amber force field.

Cytochrome c oxidase (CcO) is a vital enzyme that catalyzes the reduction of molecular oxygen to water and pumps protons across mitochondrial and bacterial membranes. This article presents parameters for the cofactors of ba3-type CcO that are compatible with the all-atom Amber ff12SB and ff14SB force fields. Specifically, parameters were developed for the CuA pair, heme b, and the dinuclear center that consists of heme a3 and CuB bridged by a hydroperoxo group. The data includes geometries in XYZ coordinate format for cluster models that were employed to compute proton transfer energies and derive bond parameters and point charges for the force field using density functional theory. Also included are the final parameter files that can be employed with the Amber leap program to generate input files for molecular dynamics simulations with the Amber software package. Based on the high resolution (1.8 Å) X-ray crystal structure of the ba3-type CcO from Thermus thermophilus (Protein Data Bank ID number PDB: 3S8F), we built a model that is embedded in a POPC lipid bilayer membrane and solvated with TIP3P water molecules and counterions. We provide PDB data files of the initial model and the equilibrated model that can be used for further studies.

All-atom lipid bilayer self-assembly with the AMBER and CHARMM lipid force fields.

This communication reports the first example of spontaneous lipid bilayer formation in unbiased all-atom molecular dynamics (MD) simulations. Using two different lipid force fields we show simulations started from random mixtures of lipids and water in which four different types of phospholipids self-assemble into organized bilayers in under 1 microsecond.

Lipid14: The Amber Lipid Force Field.

The AMBER lipid force field has been updated to create Lipid14, allowing tensionless simulation of a number of lipid types with the AMBER MD package. The modular nature of this force field allows numerous combinations of head and tail groups to create different lipid types, enabling the easy insertion of new lipid species. The Lennard-Jones and torsion parameters of both the head and tail groups have been revised and updated partial charges calculated. The force field has been validated by simulating bilayers of six different lipid types for a total of 0.5 µs each without applying a surface tension; with favorable comparison to experiment for properties such as area per lipid, volume per lipid, bilayer thickness, NMR order parameters, scattering data, and lipid lateral diffusion. As the derivation of this force field is consistent with the AMBER development philosophy, Lipid14 is compatible with the AMBER protein, nucleic acid, carbohydrate, and small molecule force fields.

LIPID11: a modular framework for lipid simulations using amber.

Accurate simulation of complex lipid bilayers has long been a goal in condensed phase molecular dynamics (MD). Structure and function of membrane-bound proteins are highly dependent on the lipid bilayer environment and are challenging to study through experimental methods. Within Amber, there has been limited focus on lipid simulations, although some success has been seen with the use of the General Amber Force Field (GAFF). However, to date there are no dedicated Amber lipid force fields. In this paper we describe a new charge derivation strategy for lipids consistent with the Amber RESP approach and a new atom and residue naming and type convention. In the first instance, we have combined this approach with GAFF parameters. The result is LIPID11, a flexible, modular framework for the simulation of lipids that is fully compatible with the existing Amber force fields. The charge derivation procedure, capping strategy, and nomenclature for LIPID11, along with preliminary simulation results and a discussion of the planned long-term parameter development are presented here. Our findings suggest that LIPID11 is a modular framework feasible for phospholipids and a flexible starting point for the development of a comprehensive, Amber-compatible lipid force field.