Restoring vision and rebuilding the retina by Müller glial cell reprogramming.

Müller glia are non-neuronal support cells that play a vital role in the homeostasis of the eye. Their radial-oriented processes span the width of the retina and respond to injury through a cellular response that can be detrimental or protective depending on the context. In some species, protective responses include the expression of stem cell-like genes which help to fuel new neuron formation and even restoration of vision. In many lower vertebrates including fish and amphibians, this response is well documented, however, in mammals it is severely limited. The remarkable plasticity of cellular reprogramming in lower vertebrates has inspired studies in mammals for repairing the retina and restoring sight, and recent studies suggest that mammals are also capable of regeneration, albeit to a lesser degree. Endogenous regeneration, whereby new retinal neurons are created from existing support cells, offers an exciting alternative approach to existing tissue transplant, gene therapy, and neural prosthetic approaches being explored in parallel. This review will highlight the role of Müller glia during retinal injury and repair. In the end, prospects for advancing retinal regeneration research will be considered.

Human retinal ganglion cell neurons generated by synchronous BMP inhibition and transcription factor mediated reprogramming.

In optic neuropathies, including glaucoma, retinal ganglion cells (RGCs) die. Cell transplantation and endogenous regeneration offer strategies for retinal repair, however, developmental programs required for this to succeed are incompletely understood. To address this, we explored cellular reprogramming with transcription factor (TF) regulators of RGC development which were integrated into human pluripotent stem cells (PSCs) as inducible gene cassettes. When the pioneer factor NEUROG2 was combined with RGC-expressed TFs (ATOH7, ISL1, and POU4F2) some conversion was observed and when pre-patterned by BMP inhibition, RGC-like induced neurons (RGC-iNs) were generated with high efficiency in just under a week. These exhibited transcriptional profiles that were reminiscent of RGCs and exhibited electrophysiological properties, including AMPA-mediated synaptic transmission. Additionally, we demonstrated that small molecule inhibitors of DLK/LZK and GCK-IV can block neuronal death in two pharmacological axon injury models. Combining developmental patterning with RGC-specific TFs thus provided valuable insight into strategies for cell replacement and neuroprotection.

Chromatin Accessibility and Transcriptional Differences in Human Stem Cell-Derived Early-Stage Retinal Organoids.

Retinogenesis involves the specification of retinal cell types during early vertebrate development. While model organisms have been critical for determining the role of dynamic chromatin and cell-type specific transcriptional networks during this process, an enhanced understanding of the developing human retina has been more elusive due to the requirement for human fetal tissue. Pluripotent stem cell (PSC) derived retinal organoids offer an experimentally accessible solution for investigating the developing human retina. To investigate cellular and molecular changes in developing early retinal organoids, we developed SIX6-GFP and VSX2-tdTomato (or VSX2-h2b-mRuby3) dual fluorescent reporters. When differentiated as 3D organoids these expressed GFP at day 15 and tdTomato (or mRuby3) at day 25, respectively. This enabled us to explore transcriptional and chromatin related changes using RNA-seq and ATAC-seq from pluripotency through early retina specification. Pathway analysis of developing organoids revealed a stepwise loss of pluripotency, while optic vesicle and retina pathways became progressively more prevalent. Correlating gene transcription with chromatin accessibility in early eye field development showed that retinal cells underwent a clear change in chromatin landscape, as well as gene expression profiles. While each dataset alone provided valuable information, considering both in parallel provided an informative glimpse into the molecular nature eye development.

GNNQQNY--investigation of early steps during amyloid formation.

Protein aggregation has been implicated in the pathology of several neurodegenerative diseases, and a better understanding of how it proceeds is essential for the development of therapeutic strategies. Recently, the amyloidogenic heptapeptide GNNQQNY has emerged as a molecule of choice for fundamental studies of protein aggregation. A number of experimental and computational studies have examined the structure of the GNNQQNY aggregate. Less work, however, has been aimed at understanding its aggregation pathway. In this study, we present a detailed computational analysis of such a pathway. To that end, transition path sampling Monte Carlo simulations are used to examine the dimerization process. A statistical analysis of the reaction pathways shows that the dimerization reaction proceeds via a zipping mechanism, initiated with the formation of distinct contacts at the third residue (N). Asparagine residues are found to play a key role in the early stages of aggregation. And, contrary to previous belief, it is also shown that the tyrosine terminal group is not required to stabilize the dimer. In fact, an asparagine residue leads to faster aggregation of the peptide.

Solution structures of rat amylin peptide: simulation, theory, and experiment.

Amyloid deposits of amylin in the pancreas are an important characteristic feature found in patients with Type-2 diabetes. The aggregate has been considered important in the disease pathology and has been studied extensively. However, the secondary structures of the individual peptide have not been clearly identified. In this work, we present detailed solution structures of rat amylin using a combination of Monte Carlo and molecular dynamics simulations. A new Monte Carlo method is presented to determine the free energy of distinct biomolecular conformations. Both folded and random-coil conformations of rat amylin are observed in water and their relative stability is examined in detail. The former contains an alpha-helical segment comprised of residues 7-17. We find that at room temperature the folded structure is more stable, whereas at higher temperatures the random-coil structure predominates. From the configurations and weights we calculate the alpha-carbon NMR chemical shifts, with results that are in reasonable agreement with experiments of others. We also calculate the infrared spectrum in the amide I stretch regime, and the results are in fair agreement with the experimental line shape presented herein.

Hydrodynamic effects on the translocation rate of a polymer through a pore.

The translocation of large DNA molecules through narrow pores has been examined in the context of multiscale simulations that include a full coupling of fluctuating hydrodynamic interactions, boundary effects, and molecular conformation. The actual rate constants for this process are determined for the first time, and it is shown that hydrodynamic interactions can lead to translocation rates that vary by multiple orders of magnitude when molecular weights are only changed by a factor of 10, in stark contrast to predictions from widely used free draining calculations.

Folding of polyglutamine chains.

Long polyglutamine chains have been associated with a number of neurodegenerative diseases. These include Huntington's disease, where expanded polyglutamine (PolyQ) sequences longer than 36 residues are correlated with the onset of symptoms. In this paper we study the folding pathway of a 54-residue PolyQ chain into a beta-helical structure. Transition path sampling Monte Carlo simulations are used to generate unbiased reactive pathways between unfolded configurations and the folded beta-helical structure of the polyglutamine chain. The folding process is examined in both explicit water and an implicit solvent. Both models reveal that the formation of a few critical contacts is necessary and sufficient for the molecule to fold. Once the primary contacts are formed, the fate of the protein is sealed and it is largely committed to fold. We find that, consistent with emerging hypotheses about PolyQ aggregation, a stable beta-helical structure could serve as the nucleus for subsequent polymerization of amyloid fibrils. Our results indicate that PolyQ sequences shorter than 36 residues cannot form that nucleus, and it is also shown that specific mutations inferred from an analysis of the simulated folding pathway exacerbate its stability.

Density of states-based molecular simulations.

One of the central problems in statistical mechanics is that of finding the density of states of a system. Knowledge of the density of states of a system is equivalent to knowledge of its fundamental equation, from which all thermodynamic quantities can be obtained. Over the past several years molecular simulations have made considerable strides in their ability to determine the density of states of complex fluids and materials. In this review we discuss some of the more promising approaches proposed in the recent literature along with their advantages and limitations.

Improved transition path sampling methods for simulation of rare events.

The free energy surfaces of a wide variety of systems encountered in physics, chemistry, and biology are characterized by the existence of deep minima separated by numerous barriers. One of the central aims of recent research in computational chemistry and physics has been to determine how transitions occur between deep local minima on rugged free energy landscapes, and transition path sampling (TPS) Monte-Carlo methods have emerged as an effective means for numerical investigation of such transitions. Many of the shortcomings of TPS-like approaches generally stem from their high computational demands. Two new algorithms are presented in this work that improve the efficiency of TPS simulations. The first algorithm uses biased shooting moves to render the sampling of reactive trajectories more efficient. The second algorithm is shown to substantially improve the accuracy of the transition state ensemble by introducing a subset of local transition path simulations in the transition state. The system considered in this work consists of a two-dimensional rough energy surface that is representative of numerous systems encountered in applications. When taken together, these algorithms provide gains in efficiency of over two orders of magnitude when compared to traditional TPS simulations.

Improved density of states Monte Carlo method based on recycling of rejected states.

In this paper a new algorithm is presented that improves the efficiency of Wang and Landau algorithm or density of states (DOS) Monte Carlo simulations by employing rejected states. The algorithm is shown to have a performance superior to that of the original Wang-Landau [F. Wang and D. P. Landau, Phys. Rev. Lett. 86, 2050 (2001)] algorithm and the more recent configurational temperature DOS algorithm. The performance of the method is illustrated in the context of results for the Lennard-Jones fluid.

Order-parameter-based Monte Carlo simulation of crystallization.

A Monte Carlo simulation method is presented for simulation of phase transitions, with emphasis on the study of crystallization. The method relies on a random walk in order parameter Phi(q(N)) space to calculate a free energy profile between the two coexisting phases. The energy and volume data generated over the course of the simulation are subsequently reweighed to identify the precise conditions for phase coexistence. The usefulness of the method is demonstrated in the context of crystallization of a purely repulsive Lennard-Jones system. A systematic analysis of precritical and critical nuclei as a function of supercooling reveals a gradual change from a bcc to a fcc structure inside the crystalline nucleus as it grows at large degrees of supercooling. The method is generally applicable and is expected to find applications in systems for which two or more coexisting phases can be distinguished through one or more order parameters.

Optimal allocation of replicas in parallel tempering simulations.

We have studied the efficiency of parallel tempering simulations for a variety of systems including a coarse-grained protein, an atomistic model polypeptide, and the Lennard-Jones fluid. A scheme is proposed for the optimal allocation of temperatures in these simulations. The method is compared to the existing empirical approaches used for this purpose. Accuracy associated with the computed thermodynamic quantities such as specific heat is also computed and their dependence on the trial-exchange acceptance rate is reported.

Interactions of liquid crystal-forming molecules with phospholipid bilayers studied by molecular dynamics simulations.

Recent experiments have shown that liquid crystals can be used to image mammalian cell membranes and to amplify structural reorganization in phospholipid-laden liquid crystal-aqueous interfaces. In this work, molecular dynamics simulations were employed to explore the interactions between commonly used liquid crystal-forming molecules and phospholipid bilayers. In particular, umbrella sampling was used to obtain the potential of mean force of 4-cyano-4'-pentylbiphenyl (5CB) and 4'-(3,4-difluor-phenyl)-4-pentyl-bicylohexyl (5CF) molecules partitioning into a dipalmitoylphosphatidylcholine bilayer. In addition, results of simulations are presented for systems consisting of a fully hydrated bilayer with 5CB or 5CF molecules at the lowest (4.5 mol %) and highest (20 mol %) concentrations used in recent laboratory experiments. It is found that mesogens preferentially partition from the aqueous phase into the membrane; the potential of mean force exhibits highly favorable free energy differences for partitioning (-18 k(B)T for 5CB and -26 k(B)T for 5CF). The location and orientation of mesogens associated with the most stable free energies in umbrella sampling simulations of dilute systems were found to be consistent with those observed in liquid-crystal-rich bilayers. It is found that the presence of mesogens in the bilayer enhances the order of lipid acyl tails, and changes the spatial and orientational arrangement of lipid headgroup atoms. These effects are more pronounced at higher liquid-crystal concentrations. In comparing the behavior of 5CB and 5CF, a stronger spatial correlation (i.e., possibly leading to aggregation) is observed between 5CB molecules within a bilayer than between 5CF molecules. Also, the range of molecular orientations and positions along the bilayer normal is larger for 5CB molecules. At the same time, 5CF molecules were found to bind more strongly to lipid headgroups, thereby slowing the lateral motion of lipid molecules.