Therapeutic Ligands Antagonize Estrogen Receptor Function by Impairing Its Mobility.

Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.

Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

USP7 small-molecule inhibitors interfere with ubiquitin binding.

The ubiquitin system regulates essential cellular processes in eukaryotes. Ubiquitin is ligated to substrate proteins as monomers or chains and the topology of ubiquitin modifications regulates substrate interactions with specific proteins. Thus ubiquitination directs a variety of substrate fates including proteasomal degradation. Deubiquitinase enzymes cleave ubiquitin from substrates and are implicated in disease; for example, ubiquitin-specific protease-7 (USP7) regulates stability of the p53 tumour suppressor and other proteins critical for tumour cell survival. However, developing selective deubiquitinase inhibitors has been challenging and no co-crystal structures have been solved with small-molecule inhibitors. Here, using nuclear magnetic resonance-based screening and structure-based design, we describe the development of selective USP7 inhibitors GNE-6640 and GNE-6776. These compounds induce tumour cell death and enhance cytotoxicity with chemotherapeutic agents and targeted compounds, including PIM kinase inhibitors. Structural studies reveal that GNE-6640 and GNE-6776 non-covalently target USP7 12 Å distant from the catalytic cysteine. The compounds attenuate ubiquitin binding and thus inhibit USP7 deubiquitinase activity. GNE-6640 and GNE-6776 interact with acidic residues that mediate hydrogen-bond interactions with the ubiquitin Lys48 side chain, suggesting that USP7 preferentially interacts with and cleaves ubiquitin moieties that have free Lys48 side chains. We investigated this idea by engineering di-ubiquitin chains containing differential proximal and distal isotopic labels and measuring USP7 binding by nuclear magnetic resonance. This preferential binding protracted the depolymerization kinetics of Lys48-linked ubiquitin chains relative to Lys63-linked chains. In summary, engineering compounds that inhibit USP7 activity by attenuating ubiquitin binding suggests opportunities for developing other deubiquitinase inhibitors and may be a strategy more broadly applicable to inhibiting proteins that require ubiquitin binding for full functional activity.

Why People Think They Might Hasten Their Death When Faced With Irremediable Health Conditions Compared to Why They Actually Do so.

This study surveys the differences of relatively healthy proponents of end-of-life choices and people with irremediable health conditions having already made the decision to hasten their deaths on what each group considers important in influencing a desire to hasten death. Psychosocial factors were more important than physical ones for both groups; but those contemplating what might influence them to hasten their deaths in the future thought pain and feeling ill would be much bigger factors than they turned out to be for those deciding to do so. Those having decided to hasten their deaths cited the lack of any further viable medical treatments and having to live in a nursing home as bigger factors. Identifying these psychosocial factors influencing a desire for a hastened death suggests that caregivers and medical providers may want to review what compassionate understanding and support looks like for people wanting to hasten their death.

Discovery of GNE-502 as an orally bioavailable and potent degrader for estrogen receptor positive breast cancer.

Fulvestrant is an FDA-approved drug with a dual mechanism of action (MOA), acting as a full antagonist and degrader of the estrogen receptor protein. A significant limitation of fulvestrant is the dosing regimen required for efficacy. Due to its high lipophilicity and poor pharmacokinetic profile, fulvestrant needs to be administered through intramuscular injections which leads to injection site soreness. This route of administration also limits the dose and target occupancy in patients. We envisioned a best-in-class molecule that would function with the same dual MOA as fulvestrant, but with improved physicochemical properties and would be orally bioavailable. Herein we report our progress toward that goal, resulting in a new lead GNE-502 which addressed some of the liabilities of our previously reported lead molecule GNE-149.

Oxidation of Cytochrome 605 Is the Rate-Limiting Step when Ferrimicrobium acidiphilum Respires Aerobically on Soluble Iron.

Proteins that oxidize extracellular substrates in Gram-positive bacteria are poorly understood. Ferrimicrobium acidiphilum is an actinobacterium that respires aerobically on extracellular ferrous ions at pH 1.5. In situ absorbance measurements were conducted on turbid suspensions of intact Fm. acidiphilum using an integrating cavity absorption meter designed for that purpose. Initial velocity kinetic studies monitored the appearance of product ferric ions in the presence of catalytic quantities of cells. Cell-catalyzed iron oxidation obeyed the Michaelis-Menten equation with Km and Vmax values of 71 µM and 0.29 fmol/min/cell, respectively. Limited-turnover kinetic studies were conducted with higher concentrations of cells to detect and monitor changes in the absorbance properties of cellular redox proteins when the cells were exposed to limited quantities of soluble reduced iron. A single a-type cytochrome with reduced absorbance peaks at 448 and 605 nm was the only redox-active chromophore that was visible as the cells respired aerobically on iron. The reduced cytochrome 605 exhibited mathematical and correlational properties that were consistent with the hypothesis that oxidation of the cytochrome constituted the rate-limiting step in the aerobic respiratory process, with a turnover number of 35 ± 2 s-1 Genomic and proteomic analyses showed that Fm. acidiphilum could and did express only two a-type heme copper terminal oxidases. Cytochrome 605 was associated with the terminal oxidase gene that is located between nucleotides 31,090 and 33,039, inclusive, in the annotated circular genome of this bacterium.IMPORTANCE The identities and functions of proteins involved in aerobic respiration on extracellular ferrous ions at acidic pH are poorly understood in the four phyla of Gram-positive eukaryotes and archaea where such activities occur. In situ absorbance measurements were conducted on Fm. acidiphilum as it respired on extracellular iron using an integrating cavity absorption meter that permitted accurate optical measurements in turbid suspensions of the intact bacterium under physiological conditions. The significance of these measurements is that they permitted a direct spectrophotometric examination of the extents and rates of biological electron transfer events in situ under noninvasive physiological conditions without disrupting the complexity of the live cellular environment. One thing is certain: one way to understand how a protein functions in an intact organism is to actually observe that protein as it functions in the intact organism. This paper provides an example of just such an observation.

Antibody-mediated delivery of chimeric protein degraders which target estrogen receptor alpha (ERα).

Chimeric molecules which effect intracellular degradation of target proteins via E3 ligase-mediated ubiquitination (e.g., PROTACs) are currently of high interest in medicinal chemistry. However, these entities are relatively large compounds that often possess molecular characteristics which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. Accordingly, we explored whether conjugation of chimeric degraders to monoclonal antibodies using technologies originally developed for cytotoxic payloads might provide alternate delivery options for these novel agents. In this report we describe the construction of several degrader-antibody conjugates comprised of two distinct ERα-targeting degrader entities and three independent ADC linker modalities. We subsequently demonstrate the antigen-dependent delivery to MCF7-neo/HER2 cells of the degrader payloads that are incorporated into these conjugates. We also provide evidence for efficient intracellular degrader release from one of the employed linkers. In addition, preliminary data are described which suggest that reasonably favorable in vivo stability properties are associated with the linkers utilized to construct the degrader conjugates.

Unexpected equivalent potency of a constrained chromene enantiomeric pair rationalized by co-crystal structures in complex with estrogen receptor alpha.

Despite tremendous progress made in the understanding of the ERα signaling pathway and the approval of many therapeutic agents, ER+ breast cancer continues to be a leading cause of cancer death in women. We set out to discover compounds with a dual mechanism of action in which they not only compete with estradiol for binding with ERα, but also can induce the degradation of the ERα protein itself. We were attracted to the constrained chromenes containing a tetracyclic benzopyranobenzoxepine scaffold, which were reported as potent selective estrogen receptor modulators (SERMs). Incorporation of a fluoromethyl azetidine side chain yielded highly potent and efficacious selective estrogen receptor degraders (SERDs), such as 16aa and surprisingly, also its enantiomeric pair 16ab. Co-crystal structures of the enantiomeric pair 16aa and 16ab in complex with ERα revealed default (mimics the A-D rings of endogenous ligand estradiol) and core-flipped binding modes, rationalizing the equivalent potency observed for these enantiomers in the ERα degradation and MCF-7 anti-proliferation assays.

Discovery of a C-8 hydroxychromene as a potent degrader of estrogen receptor alpha with improved rat oral exposure over GDC-0927.

Phenolic groups are responsible for the high clearance and low oral bioavailability of the estrogen receptor alpha (ERα) clinical candidate GDC-0927. An exhaustive search for a backup molecule with improved pharmacokinetic (PK) properties identified several metabolically stable analogs, although in general at the expense of the desired potency and degradation efficiency. C-8 hydroxychromene 30 is the first example of a phenol-containing chromene that not only maintained excellent potency but also exhibited 10-fold higher oral exposure in rats. The improved in vivo clearance in rat was hypothesized to be the result of C-8 hydroxy group being sterically protected from glucuronide conjugation. The excellent potency underscores the possibility of replacing the presumed indispensable phenolic group at C-6 or C-7 of the chromene core. Co-crystal structures were obtained to highlight the change in key interactions and rationalize the retained potency.

Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy.

In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10-11 mM of the surfactant n-tetradecyl-ß-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan's GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan's REES values are high (9.3-18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4-5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan's excited state dipole moment, and "solvent" reorientation around Laurdan's chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan's lifetime.

Termination of re-entrant atrial tachycardia via optogenetic stimulation with optimized spatial targeting: insights from computational models.

KEY POINTS: Optogenetics has emerged as a potential alternative to electrotherapy for treating heart rhythm disorders, but its applicability for terminating atrial arrhythmias remains largely unexplored. We used computational models reconstructed from clinical MRI scans of fibrotic patient atria to explore the feasibility of optogenetic termination of atrial tachycardia (AT), comparing two different illumination strategies: distributed vs. targeted. We show that targeted optogenetic stimulation based on automated, non-invasive flow-network analysis of patient-specific re-entry morphology may be a reliable approach for identifying the optimal illumination target in each individual (i.e. the critical AT isthmus). The above-described approach yields very high success rates (up to 100%) and requires dramatically less input power than distributed illumination We conclude that simulations in patient-specific models show that targeted light pulses lasting longer than the AT cycle length can efficiently and reliably terminate AT if the human atria can be successfully light-sensitized via gene delivery of ChR2. ABSTRACT: Optogenetics has emerged as a potential alternative to electrotherapy for treating arrhythmia, but feasibility studies have been limited to ventricular defibrillation via epicardial light application. Here, we assess the efficacy of optogenetic atrial tachycardia (AT) termination in human hearts using a strategy that targets for illumination specific regions identified in an automated manner. In three patient-specific models reconstructed from late gadolinium-enhanced MRI scans, we simulated channelrhodopsin-2 (ChR2) expression via gene delivery. In all three models, we attempted to terminate re-entrant AT (induced via rapid pacing) via optogenetic stimulation. We compared two strategies: (1) distributed illumination of the endocardium by multi-optrode grids (number of optrodes, Nopt  = 64, 128, 256) and (2) targeted illumination of the critical isthmus, which was identified via analysis of simulated activation patterns using an algorithm based on flow networks. The illuminated area and input power were smaller for the targeted approach (19-57.8 mm2 ; 0.6-1.8 W) compared to the sparsest distributed arrays (Nopt  = 64; 124.9 ± 6.3 mm2 ; 3.9 ± 0.2 W). AT termination rates for distributed illumination were low, ranging from <5% for short pulses (1/10 ms long) to ∼20% for longer stimuli (100/1000 ms). When we attempted to terminate the same AT episodes with targeted illumination, outcomes were similar for short pulses (1/10 ms long: 0% success) but improved for longer stimuli (100 ms: 54% success; 1000 ms: 90% success). We conclude that simulations in patient-specific models show that light pulses lasting longer than the AT cycle length can efficiently and reliably terminate AT in atria light-sensitized via gene delivery. We show that targeted optogenetic stimulation based on analysis of AT morphology may be a reliable approach for defibrillation and requires less power than distributed illumination.

Conjugation of Indoles to Antibodies through a Novel Self-Immolating Linker.

A novel strategy to attach indole-containing payloads to antibodies through a carbamate moiety and a self-immolating, disulfide-based linker is described. This new strategy was employed to connect a selective estrogen receptor down-regulator (SERD) to various antibodies in a site-selective manner. The resulting conjugates displayed potent, antigen-dependent down-regulation of estrogen receptor levels in MCF7-neo/HER2 and MCF7-hB7H4 cells. They also exhibited similar antigen-dependent modulation of the estrogen receptor in tumors when administered intravenously to mice bearing MCF7-neo/HER2 tumor xenografts. The indole-carbamate moiety present in the new linker was stable in whole blood from various species and also exhibited good in vivo stability properties in mice.

Genus-Wide Comparative Genomics of Malassezia Delineates Its Phylogeny, Physiology, and Niche Adaptation on Human Skin.

Malassezia is a unique lipophilic genus in class Malasseziomycetes in Ustilaginomycotina, (Basidiomycota, fungi) that otherwise consists almost exclusively of plant pathogens. Malassezia are typically isolated from warm-blooded animals, are dominant members of the human skin mycobiome and are associated with common skin disorders. To characterize the genetic basis of the unique phenotypes of Malassezia spp., we sequenced the genomes of all 14 accepted species and used comparative genomics against a broad panel of fungal genomes to comprehensively identify distinct features that define the Malassezia gene repertoire: gene gain and loss; selection signatures; and lineage-specific gene family expansions. Our analysis revealed key gene gain events (64) with a single gene conserved across all Malassezia but absent in all other sequenced Basidiomycota. These likely horizontally transferred genes provide intriguing gain-of-function events and prime candidates to explain the emergence of Malassezia. A larger set of genes (741) were lost, with enrichment for glycosyl hydrolases and carbohydrate metabolism, concordant with adaptation to skin's carbohydrate-deficient environment. Gene family analysis revealed extensive turnover and underlined the importance of secretory lipases, phospholipases, aspartyl proteases, and other peptidases. Combining genomic analysis with a re-evaluation of culture characteristics, we establish the likely lipid-dependence of all Malassezia. Our phylogenetic analysis sheds new light on the relationship between Malassezia and other members of Ustilaginomycotina, as well as phylogenetic lineages within the genus. Overall, our study provides a unique genomic resource for understanding Malassezia niche-specificity and potential virulence, as well as their abundance and distribution in the environment and on human skin.

The Multicenter Aerobic Iron Respiratory Chain of Acidithiobacillus ferrooxidans Functions as an Ensemble with a Single Macroscopic Rate Constant.

Electron transfer reactions among three prominent colored proteins in intact cells of Acidithiobacillus ferrooxidans were monitored using an integrating cavity absorption meter that permitted the acquisition of accurate absorbance data in suspensions of cells that scattered light. The concentrations of proteins in the periplasmic space were estimated to be 350 and 25 mg/ml for rusticyanin and cytochrome c, respectively; cytochrome a was present as one molecule for every 91 nm(2) in the cytoplasmic membrane. All three proteins were rapidly reduced to the same relative extent when suspensions of live bacteria were mixed with different concentrations of ferrous ions at pH 1.5. The subsequent molecular oxygen-dependent oxidation of the multicenter respiratory chain occurred with a single macroscopic rate constant, regardless of the proteins' in vitro redox potentials or their putative positions in the aerobic iron respiratory chain. The crowded electron transport proteins in the periplasm of the organism constituted an electron conductive medium where the network of protein interactions functioned in a concerted fashion as a single ensemble with a standard reduction potential of 650 mV. The appearance of product ferric ions was correlated with the reduction levels of the periplasmic electron transfer proteins; the limiting first-order catalytic rate constant for aerobic respiration on iron was 7,400 s(-1). The ability to conduct direct spectrophotometric studies under noninvasive physiological conditions represents a new and powerful approach to examine the extent and rates of biological events in situ without disrupting the complexity of the live cellular environment.

CF3+ and CF2H+: new reagents for n-alkane determination in chemical ionisation reaction mass spectrometry.

Alkanes provide a particular analytical challenge to commonly used chemical ionisation methods such as proton-transfer from water owing to their basicity. It is demonstrated that the fluorocarbon ions CF3+ and CF2H+, generated from CF4, as reagents provide an effective means of detecting light n-alkanes in the range C2-C6 using direct chemical ionisation mass spectrometry. The present work assesses the applicability of the reagents in Chemical Ionisation Mass Spectrometric (CI-TOF-MS) environments with factors such as high moisture content, operating pressures of 1-10 Torr, accelerating electric fields (E/N) and long-lived intermediate complex formation. Of the commonly used chemical ionisation reagents, H3O+ and NO+ only react with hexane and higher while O2+ reacts with all the target samples, but creates significant fragmentation. By contrast, CF3+ and CF2H+ acting together were found to produce little or no fragmentation. In dry conditions with E/N = 100 Td or higher the relative intensity of CF2H+ to CF3+ was mostly less than 1% but always less than 3%, making CF3+ the main reagent ion. Using O2+ in a parallel series of experiments, a substantially greater degree of fragmentation was observed. The detection sensitivities of the alkanes with CF3+ and CF2H+, while relatively low, were found to be better than those observed with O2+. Experiments using alkane mixtures in the ppm range have shown the ionisation technique based on CF3+ and CF2H+ to be particularly useful for measurements of alkane/air mixtures found in polluted environments. As a demonstration of the technique's effectiveness in complex mixtures, the detection of n-alkanes in a smoker's breath is demonstrated.

Observations of the release of non-methane hydrocarbons from fractured shale.

The organic content of shale has become of commercial interest as a source of hydrocarbons, owing to the development of hydraulic fracturing ("fracking"). While the main focus is on the extraction of methane, shale also contains significant amounts of non-methane hydrocarbons (NMHCs). We describe the first real-time observations of the release of NMHCs from a fractured shale. Samples from the Bowland-Hodder formation (England) were analyzed under different conditions using mass spectrometry, with the objective of understanding the dynamic process of gas release upon fracturing of the shale. A wide range of NMHCs (alkanes, cycloalkanes, aromatics, and bicyclic hydrocarbons) are released at parts per million or parts per billion level with temperature- and humidity-dependent release rates, which can be rationalized in terms of the physicochemical characteristics of different hydrocarbon classes. Our results indicate that higher energy inputs (i.e., temperatures) significantly increase the amount of NMHCs released from shale, while humidity tends to suppress it; additionally, a large fraction of the gas is released within the first hour after the shale has been fractured. These findings suggest that other hydrocarbons of commercial interest may be extracted from shale and open the possibility to optimize the "fracking" process, improving gas yields and reducing environmental impacts.

A cellular model for sporadic ALS using patient-derived induced pluripotent stem cells.

Development of therapeutics for genetically complex neurodegenerative diseases such as sporadic amyotrophic lateral sclerosis (ALS) has largely been hampered by lack of relevant disease models. Reprogramming of sporadic ALS patients' fibroblasts into induced pluripotent stem cells (iPSC) and differentiation into affected neurons that show a disease phenotype could provide a cellular model for disease mechanism studies and drug discovery. Here we report the reprogramming to pluripotency of fibroblasts from a large cohort of healthy controls and ALS patients and their differentiation into motor neurons. We demonstrate that motor neurons derived from three sALS patients show de novo TDP-43 aggregation and that the aggregates recapitulate pathology in postmortem tissue from one of the same patients from which the iPSC were derived. We configured a high-content chemical screen using the TDP-43 aggregate endpoint both in lower motor neurons and upper motor neuron like cells and identified FDA-approved small molecule modulators including Digoxin demonstrating the feasibility of patient-derived iPSC-based disease modeling for drug screening.

Potency-Enhanced Peptidomimetic VHL Ligands with Improved Oral Bioavailability.

The von Hippel-Lindau (VHL) protein plays a pivotal role in regulating the hypoxic stress response and has been extensively studied and utilized in the targeted protein degradation field, particularly in the context of bivalent degraders. In this study, we present a comprehensive peptidomimetic structure-activity relationship (SAR) approach, combined with cellular NanoBRET target engagement assays to enhance the existing VHL ligands. Through systematic modifications of the molecule, we identified the 1,2,3-triazole group as an optimal substitute of the left-hand side amide bond that yields 10-fold higher binding activity. Moreover, incorporating conformationally constrained alterations on the methylthiazole benzylamine moiety led to the development of highly potent VHL ligands with picomolar binding affinity and significantly improved oral bioavailability. We anticipate that our optimized VHL ligand, GNE7599, will serve as a valuable tool compound for investigating the VHL pathway and advancing the field of targeted protein degradation.

Mechanistic inquiry into the role of tissue remodeling in fibrotic lesions in human atrial fibrillation.

Atrial fibrillation (AF), the most common arrhythmia in humans, is initiated when triggered activity from the pulmonary veins propagates into atrial tissue and degrades into reentrant activity. Although experimental and clinical findings show a correlation between atrial fibrosis and AF, the causal relationship between the two remains elusive. This study used an array of 3D computational models with different representations of fibrosis based on a patient-specific atrial geometry with accurate fibrotic distribution to determine the mechanisms by which fibrosis underlies the degradation of a pulmonary vein ectopic beat into AF. Fibrotic lesions in models were represented with combinations of: gap junction remodeling; collagen deposition; and myofibroblast proliferation with electrotonic or paracrine effects on neighboring myocytes. The study found that the occurrence of gap junction remodeling and the subsequent conduction slowing in the fibrotic lesions was a necessary but not sufficient condition for AF development, whereas myofibroblast proliferation and the subsequent electrophysiological effect on neighboring myocytes within the fibrotic lesions was the sufficient condition necessary for reentry formation. Collagen did not alter the arrhythmogenic outcome resulting from the other fibrosis components. Reentrant circuits formed throughout the noncontiguous fibrotic lesions, without anchoring to a specific fibrotic lesion.

Multi-scale models of whole cells: progress and challenges.

Whole-cell modeling is "the ultimate goal" of computational systems biology and "a grand challenge for 21st century" (Tomita, Trends in Biotechnology, 2001, 19(6), 205-10). These complex, highly detailed models account for the activity of every molecule in a cell and serve as comprehensive knowledgebases for the modeled system. Their scope and utility far surpass those of other systems models. In fact, whole-cell models (WCMs) are an amalgam of several types of "system" models. The models are simulated using a hybrid modeling method where the appropriate mathematical methods for each biological process are used to simulate their behavior. Given the complexity of the models, the process of developing and curating these models is labor-intensive and to date only a handful of these models have been developed. While whole-cell models provide valuable and novel biological insights, and to date have identified some novel biological phenomena, their most important contribution has been to highlight the discrepancy between available data and observations that are used for the parametrization and validation of complex biological models. Another realization has been that current whole-cell modeling simulators are slow and to run models that mimic more complex (e.g., multi-cellular) biosystems, those need to be executed in an accelerated fashion on high-performance computing platforms. In this manuscript, we review the progress of whole-cell modeling to date and discuss some of the ways that they can be improved.