Structure of the space of folding protein sequences defined by large language models.

Proteins populate a manifold in the high-dimensional sequence space whose geometrical structure guides their natural evolution. Leveraging recently-developed structure prediction tools based on transformer models, we first examine the protein sequence landscape as defined by an effective energy that is a proxy of sequence foldability. This landscape shares characteristics with optimization challenges encountered in machine learning and constraint satisfaction problems. Our analysis reveals that natural proteins predominantly reside in wide, flat minima within this energy landscape. To investigate further, we employ statistical mechanics algorithms specifically designed to explore regions with high local entropy in relatively flat landscapes. Our findings indicate that these specialized algorithms can identify valleys with higher entropy compared to those found using traditional methods such as Monte Carlo Markov Chains. In a proof-of-concept case, we find that these highly entropic minima exhibit significant similarities to natural sequences, especially in critical key sites and local entropy. Additionally, evaluations through Molecular Dynamics suggests that the stability of these sequences closely resembles that of natural proteins. Our tool combines advancements in machine learning and statistical physics, providing new insights into the exploration of sequence landscapes where wide, flat minima coexist alongside a majority of narrower minima.

Fox eyes lift-A new protocol for eyebrow elevation with laser Fotona.

BACKGROUND: The eyelids play an important role in our appearance and are usually the first to show signs of age. The Fotona SP Spectro Systems consist of a range of noninvasive laser treatments that work together synergistically to tighten the collagen in four dimensions and provide long-lasting firmness to the face. The Fotona SP Spectro combines two wavelengths: Er:YAG (2940 nm) and Nd:YAG (1064 nm) with four distinct treatments: SmoothLiftingTM, FRAC3®, PIANO®, and SupErficialTM, allowing safe, painless, noninvasive, and no downtime rejuvenation. AIMS: To present a new protocol of treatment with Fotona SP Spectro for eyebrow elevation, which we call fox eyes lift (FEL), and compare it to the standard SmoothEye® (SE) protocol. METHODS: This is a prospective, interventional, split-face study. The sample consisted of 21 subjects (19 women) with a mean age of 50.1 ± 7.9 years who underwent two different protocols, that is, SE on one side and FEL on the other. The protocol used on each side was selected by drawing lots. Three sessions were held at 1-month intervals. Standardized photographic documentation was obtained before and 30 days after the end of treatment. Eyebrow position before and after complete treatment was quantified using ImageJ software. RESULTS: Statistical analysis by ANOVA showed a significant improvement in eyebrow position after treatment with both protocols, with a significantly greater effect of FEL (p = 0.0003 d = 0.95). CONCLUSION: Fox eyes lift is an efficient and safe technique providing significant improvement in the position of the eyebrow.

Use of 2-hydroxy-4-(methylthio)-butanoic acid to inhibit Salmonella and Listeria in raw meat for feline diets and palatability in domestic cats.

While the raw pet food market continues to grow, the risk of bacterial contamination in these types of diets is a major concern, with Salmonella enterica and Listeria monocytogenes being the most frequently associated pathogens in raw pet food product recalls. dl-Methionine is included in some commercial feline kibble and canned diets to improve protein quality; however, an alternative to this is a liquid methionine supplement, 2-hydroxy-4-(methylthio)-butanoic acid (HMTBa), which is also an organic acid. 2-Hydroxy-4-(methylthio)-butanoic acid has previously demonstrated similar efficacy to formic acid against pathogens in a liquid environment and may be a good candidate to inhibit S. enterica and L. monocytogenes in raw ground meat. First, the minimum inhibitory concentration and minimum bactericidal concentration of HMTBa against these pathogens under laboratory growth conditions were determined by measuring growth of pathogens over 36 h when exposed to 10 concentrations of HMTBa (0.10% to 1.00%) mixed with tryptic soy broth. 2-Hydroxy-4-(methylthio)-butanoic acid included at ≥0.50% was bactericidal to S. enterica and L. monocytogenes (P < 0.05). Next, five levels of HMTBa (0.50% to 1.25%) were included in raw ground meat mixtures inoculated with cocktails of S. enterica or L. monocytogenes, and contamination levels were determined at four timepoints: immediately, and after refrigerated storage (4 °C) at 24, 48, and 72 h after removal from freezer (24 h at -20 °C). 2-Hydroxy-4-(methylthio)-butanoic acid included as 1.25% of the meat mixture reduced S. enterica and L. monocytogenes compared with the control (P < 0.05); however, it did not result in total kill of either of these pathogens. Following this, feeding behaviors of seven domestic cats were assessed when offered a raw chicken diet treated with or without 1.25% HMTBa for 5 d each, after which a 2-d 2-choice preference test was conducted. Cats demonstrated a preference for raw diets without HMTBa, but still readily consumed diets with 1.25% HMTBa, suggesting that such a diet was still palatable to them.

Native state of natural proteins optimizes local entropy.

The differing ability of polypeptide conformations to act as the native state of proteins has long been rationalized in terms of differing kinetic accessibility or thermodynamic stability. Building on the successful applications of physical concepts and sampling algorithms recently introduced in the study of disordered systems, in particular artificial neural networks, we quantitatively explore how well a quantity known as the local entropy describes the native state of model proteins. In lattice models and all-atom representations of proteins, we are able to efficiently sample high local entropy states and to provide a proof of concept of enhanced stability and folding rate. Our methods are based on simple and general statistical-mechanics arguments, and thus we expect that they are of very general use.

Nutrient content changes from steaming or soaking timothy-alfalfa hay: effects on feed preferences and acute glycemic response in Standardbred racehorses1.

Soaking hay and steaming hay are strategies that are used to reduce respirable dust particles for horses but may result in variable nutrient losses, including nonstructural carbohydrates (NSC) and minerals. Since these losses have not been quantified in Canadian hay yet, the first aim of this study was to identify nutrient losses from first-cut timothy-alfalfa hay grown in southern Ontario, Canada, after soaking for 30 min or steaming for 60 min. It is uncertain whether horses prefer hay when it is dry, soaked, or steamed. To address this, 13 Standardbred racehorses were offered 2 of these hays side by side for 30 min on 6 consecutive occasions until all possible combinations had been offered. Quantity of hay eaten was determined and horses were video recorded during feedings to assess time spent eating and investigating hay. Additionally, consumption of feeds with differing NSC levels has been observed to influence glycemic response in horses; however, this has not been measured in horses consuming steamed hay before and the results from soaked hay studies have been inconclusive. As such, the final aim of this study was to examine acute glycemic response in horses after being fed dry, soaked, and steamed hays. Blood glucose was measured every 30 min from 9 Standardbred racehorses for 6 h following a meal of 0.5% of their body weight of treatment hay on a dry matter basis (DMB). Soaked, but not steamed, hay had lower concentrations of soluble protein, NSC, and potassium in contrast to the same dry hay (P < 0.05). Peak glucose, average blood glucose, total area under the curve, and time to peak did not differ among treatments (P > 0.05). We conclude that acute glycemic response of racehorses was not influenced by soaking or steaming hay. Horses also consumed less soaked hay (DMB) than dry or steamed hay (P < 0.05) and spent less time eating soaked hay than dry or steamed hay (P < 0.05).

Statistical mechanical properties of sequence space determine the efficiency of the various algorithms to predict interaction energies and native contacts from protein coevolution.

Studying evolutionary correlations in alignments of homologous sequences by means of an inverse Potts model has proven useful to obtain residue-residue contact energies and to predict contacts in proteins. The quality of the results depend much on several choices of the detailed model and on the algorithms used. We built, in a very controlled way, synthetic alignments with statistical properties similar to those of real proteins, and used them to assess the performance of different inversion algorithms and of their variants. Realistic synthetic alignments display typical features of low-temperature phases of disordered systems, a feature that affects the inversion algorithms. We showed that a Boltzmann-learning algorithm is computationally feasible and performs well in predicting the energy of native contacts. However, all algorithms, when applied to alignments of realistic size, suffer of false positives quite equally, making the quality of the prediction of native contacts with the different algorithm much system-dependent.

Integrating experiment, theory and simulation to determine the structure and dynamics of mammalian chromosomes.

Eukaryotic chromosomes are complex polymers, which largely exceed in size most biomolecules that are usually modelled in computational studies and whose molecular interactions are to a large extent unknown. Since the folding of the chromatin fiber in the cell nucleus is tightly linked to biological function and gene expression in particular, characterizing the conformational and dynamical properties of chromosomes has become crucial in order to better understand how genes are regulated. In parallel with the development of experimental techniques allowing to measure physical contacts within chromosomes inside the cell nucleus, a large variety of physical models to study the structure and mechanisms of chromosome folding have recently emerged. Such models can be roughly divided into two classes, based on whether they adopt specific hypotheses on the interaction mechanism within chromosomes, or learn those interactions on the available experimental data using the principle of maximum entropy. All of them have played a key role in interpreting experimental data and advancing our understanding the folding principles of the chromatin fiber.

Modelling genome-wide topological associating domains in mouse embryonic stem cells.

Chromosome conformation capture (3C)-based techniques such as chromosome conformation capture carbon copy (5C) and Hi-C revealed that the folding of mammalian chromosomes is highly hierarchical. A fundamental structural unit in the hierarchy is represented by topologically associating domains (TADs), sub-megabase regions of the genome within which the chromatin fibre preferentially interacts. 3C-based methods provide the mean contact probabilities between chromosomal loci, averaged over a large number of cells, and do not give immediate access to the single-cell conformations of the chromatin fibre. However, coarse-grained polymer models based on 5C data can be used to extract the single-cell conformations of single TADs. Here, we extend this approach to analyse around 2500 TADs in murine embryonic stem cells based on high-resolution Hi-C data. This allowed to predict the cell-to-cell variability in single contacts within genome-wide TADs and correlations between them. Based on these results, we predict that TADs are more similar to ideal chains than to globules in terms of their physical size and three-dimensional shape distribution. Furthermore, we show that their physical size and the degree of structural anisotropy of single TADs are correlated with the level of transcriptional activity of the genes that it harbours. Finally, we show that a large number of multiplets of genomic loci co-localize more often than expected by random, and these loci are particularly enriched in promoters, enhancers and CTCF-bound sites. These results provide the first genome-wide structural reconstruction of TADs using polymeric models obeying the laws of thermodynamics and reveal important universal trends in the correlation between chromosome structure and transcription.

Thermodynamic and structural effect of urea and guanidine chloride on the helical and on a hairpin fragment of GB1 from molecular simulations.

With the help of molecular-dynamics simulations, we studied the effect of urea and guanidine chloride on the thermodynamic and structural properties of the helical fragment of protein GB1, comparing them with those of its second beta hairpin. We showed that the helical fragment in different solvents populates an ensemble of states that is more complex than that of the hairpin, and thus the associated experimental observables (circular-dichroism spectra, secondary chemical shifts, m values), that we back-calculated from the simulations and compared with the actual data, are more difficult to interpret. We observed that in the case of both peptides, urea binds tightly to their backbone, while guanidine exerts its denaturing effect in a more subtle way, strongly affecting the electrostatic properties of the solution. This difference can have consequences in the way denaturation experiments are interpreted. Proteins 2017; 85:753-763. © 2016 Wiley Periodicals, Inc.

Looping probability of random heteropolymers helps to understand the scaling properties of biopolymers.

Random heteropolymers are a minimal description of biopolymers and can provide a theoretical framework to the investigate the formation of loops in biophysical experiments. The looping probability as a function of polymer length was observed to display in some biopolymers, like chromosomes in cell nuclei or long RNA chains, anomalous scaling exponents. Combining a two-state model with self-adjusting simulated-tempering calculations, we calculate numerically the looping properties of several realizations of the random interactions within the chain. We find a continuous set of exponents upon varying the temperature, which arises from finite-size effects and is amplified by the disorder of the interactions. We suggest that this could provide a simple explanation for the anomalous scaling exponents found in experiments. In addition, our results have important implications notably for the study of chromosome folding as they show that scaling exponents cannot be the sole criteria for testing hypothesis-driven models of chromosome architecture.

Thermodynamically-Weighted Conformational Ensemble of Cyclic RGD Peptidomimetics from NOE Data.

In the case of flexible molecules, the standard approach of transforming NOE intensities into spatial restraints and of building conformational models minimizing these restraints greatly neglects the richness of molecular conformations. Making use of NOE intensities measured in triplicate and of an iterative molecular-dynamics scheme, we optimized a force field to generate a set of conformations whose ensemble is compatible with the experimental data, and is weighted according to the Boltzmann distribution. This scheme is applied to two cyclic peptidomimetic ligands of integrins. Their difference in binding affinity is recapitulated in terms of their difference in conformational fluctuations.

Complete coverage of space favors modularity of the grid system in the brain.

Grid cells in the entorhinal cortex fire when animals that are exploring a certain region of space occupy the vertices of a triangular grid that spans the environment. Different neurons feature triangular grids that differ in their properties of periodicity, orientation, and ellipticity. Taken together, these grids allow the animal to maintain an internal, mental representation of physical space. Experiments show that grid cells are modular, i.e., there are groups of neurons which have grids with similar periodicity, orientation, and ellipticity. We use statistical physics methods to derive a relation between variability of the properties of the grids within a module and the range of space that can be covered completely (i.e., without gaps) by the grid system with high probability. Larger variability shrinks the range of representation, providing a functional rationale for the experimentally observed comodularity of grid cell periodicity, orientation, and ellipticity. We obtain a scaling relation between the number of neurons and the period of a module, given the variability and coverage range. Specifically, we predict how many more neurons are required at smaller grid scales than at larger ones.

A many-body term improves the accuracy of effective potentials based on protein coevolutionary data.

The study of correlated mutations in alignments of homologous proteins proved to be successful not only in the prediction of their native conformation but also in the development of a two-body effective potential between pairs of amino acids. In the present work, we extend the effective potential, introducing a many-body term based on the same theoretical framework, making use of a principle of maximum entropy. The extended potential performs better than the two-body one in predicting the energetic effect of 308 mutations in 14 proteins (including membrane proteins). The average value of the parameters of the many-body term correlates with the degree of hydrophobicity of the corresponding residues, suggesting that this term partly reflects the effect of the solvent.

Conformational dependence of the circular dichroism spectra of single amino acids from plane-waves-based density functional theory calculations.

We study the conformational dependence of circular dichroism (CD) spectra of amino acid molecules by means of an efficient ab initio DFT approach which is free from the typical gauge invariance issues arising with the use of localized basis sets and/or real-space grids. We analyze the dependence of the chiroptical spectra on the backbone dihedrals in the specific case of alanine and consider the role of side chain degrees of freedom at the examples of leucine, phenylalanine, and serine, whose side chains have different physicochemical properties. The results allow one to identify the most diagnostic regions of the CD spectra and to critically compare the conformations which match the experimental CD data with conformations extracted from the rotamer library. The inclusion of a solvation shell of explicit water molecules and its effect on the CD spectrum are analyzed at the example of alanine.

The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular dynamics simulations.

We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long-lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Refolding of mechanically denatured HIV-1-PR monomers is also a multiple-pathway process. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, and provide information regarding possible structural features of the PF state.

The dynamics of genetic control in the cell: the good and bad of being late.

The expression of genes in the cell is controlled by a complex interaction network involving proteins, RNA and DNA. The molecular events associated with the nodes of such a network take place on a variety of time scales, and thus cannot be regarded as instantaneous. In many cases, the cell is robust with respect to the delay in gene expression control, behaving as if it were instantaneous. However, there are specific cases in which delay gives rise to temporal oscillations. This is the case, for example, of the expression of tumour-suppressor protein p53, of protein Hes1, involved in the differentiation of stem cells, of NFkB and Wnt, in which case delay arises implicitly from the structure of the associated network. By means of delay rate equations, we study the kinetics of small regulatory networks, emphasizing the role of delay in an evolutionary context. These models suggest that oscillations are a typical outcome of the dynamics of regulatory networks, and evolution has to work to avoid them when not required (and not vice versa).

Thermodynamics of strongly allosteric inhibition: a model study of HIV-1 protease.

Protein inhibitors that shift the thermodynamic equilibrium towards a denatured state escape, in general, the straightforward framework of competitive or allosteric inhibitors. The equilibrium properties of peptides which compete with the folding, or more precisely destabilize the native state, of the human immunodeficiency virus (HIV)-1 protease monomer are studied within a structure-based model. The effect of peptides that disrupt the hydrophobic core of the protein can still be summarized in terms of an inhibition constant, which depends on the thermal stability of the protein. The state of the protein denatured by such a peptide is more structured than its intrinsic denatured state, but displays the same degree of compactness. Peptides that target less buried regions of the protein are less efficient and display a more complex thermodynamics that cannot be captured in a simple way.

Equilibrium properties of realistic random heteropolymers and their relevance for globular and naturally unfolded proteins.

Random heteropolymers do not display the typical equilibrium properties of globular proteins, but are the starting point to understand the physics of proteins and, in particular, to describe their non-native states. So far, they have been studied with mean-field models in the thermodynamic limit, or with computer simulations of very small chains on lattice. After describing a self-adjusting parallel-tempering technique to sample efficiently the low-energy states of frustrated systems without the need of tuning the system-dependent parameters of the algorithm, we apply it to random heteropolymers moving in continuous space. We show that if the mean interaction between monomers is negative, the usual description through the random-energy model is nearly correct, provided that it is extended to account for noncompact conformations. If the mean interaction is positive, such a simple description breaks out and the system behaves in a way more similar to Ising spin glasses. The former case is a model for the denatured state of globular proteins, the latter of naturally unfolded proteins, whose equilibrium properties thus result as qualitatively different.

Labial salivary gland transplantation for severe dry eye due to chemical burns and Stevens-Johnson syndrome.

PURPOSE: Salivary gland transplantation has been a promising alternative for the treatment of dry eye syndrome. In this article, we describe the results of an autotransplant procedure of labial salivary glands in the upper conjunctival fornix of patients with severe dry eye. METHODS: A total of 14 eyes from 14 patients presenting with Stevens-Johnson syndrome and chemical burns were prospectively analyzed after surgery (average follow-up of 14 months). We evaluated their underlying symptoms, visual acuity, biomicroscopy, Schirmer's test, break-up time, and need for lubricants before and after transplantation. RESULTS: All patients expressed improvement in their ocular discomfort. Nine eyes showed a slight best-corrected visual acuity improvement, while the vision of the remainder stayed stable. Corneal staining, present in all patients before surgery, was persistent in only four patients, but in a reduced area. Schirmer's test and break-up time showed significant increase in all patients (p < 0.05). In 71% of the patients, the use of lubricants was reduced. CONCLUSION: Labial salivary gland transplantation can improve the life quality of patients with compromised ocular surfaces who suffer from severe dry eye syndrome.

The molecular evolution of HIV-1 protease simulated at atomic detail.

Progress in understanding protein folding allows to simulate, with atomic detail, the evolution of amino-acid sequences folding to a given native conformation. A particularly attractive example is the HIV-1 protease, main target of therapies to fight AIDS, which under drug pressure is able to develop resistance within few months from the starting of therapy. By comparing the results of simulations of the evolution of the protease with the corresponding proteomic data, one can approximately determine the value of the associated evolution pressure under which the enzyme has become and, as a consequence, map out the energy landscape in sequence space of the HIV-1 protease. It is found that there are several families of sequences folding to the native conformations of the enzyme. Each of these families are characterized by different sets of highly conserved ("hot") amino acids which play a critical role in the folding and stability of the protease. There are two main possibilities for the virus to move from one family to a different one: (a) in a single generation, through the concerted mutations of the hot amino acids, a highly unlikely event, (b) through a folding path (if it exists), again a very improbable event. In fact, the number of generations needed by the virus to change stepwise its sequence from one family to another is astronomically large. These results point to the "hot" segments of the protease as promising targets for a nonconventional inhibition strategy, likely not to create resistance.