Robustness of DNA looping across multiple cell divisions in individual bacteria.

DNA looping has emerged as a central paradigm of transcriptional regulation, as it is shared across many living systems. One core property of DNA looping-based regulation is its ability to greatly enhance repression or activation of genes with only a few copies of transcriptional regulators. However, this property based on a small number of proteins raises the question of the robustness of such a mechanism with respect to the large intracellular perturbations taking place during growth and division of the cell. Here we address the issue of sensitivity to variations of intracellular parameters of gene regulation by DNA looping. We use the lac system as a prototype to experimentally identify the key features of the robustness of DNA looping in growing Escherichia coli cells. Surprisingly, we observe time intervals of tight repression spanning across division events, which can sometimes exceed 10 generations. Remarkably, the distribution of such long time intervals exhibits memoryless statistics that is mostly insensitive to repressor concentration, cell division events, and the number of distinct loops accessible to the system. By contrast, gene regulation becomes highly sensitive to these perturbations when DNA looping is absent. Using stochastic simulations, we propose that the observed robustness to division emerges from the competition between fast, multiple rebinding events of repressors and slow initiation rate of the RNA polymerase. We argue that fast rebinding events are a direct consequence of DNA looping that ensures robust gene repression across a range of intracellular perturbations.

All-or-none amyloid disassembly via chaperone-triggered fibril unzipping favors clearance of α-synuclein toxic species.

α-synuclein aggregation is present in Parkinson's disease and other neuropathologies. Among the assemblies that populate the amyloid formation process, oligomers and short fibrils are the most cytotoxic. The human Hsc70-based disaggregase system can resolve α-synuclein fibrils, but its ability to target other toxic assemblies has not been studied. Here, we show that this chaperone system preferentially disaggregates toxic oligomers and short fibrils, while its activity against large, less toxic amyloids is severely impaired. Biochemical and kinetic characterization of the disassembly process reveals that this behavior is the result of an all-or-none abrupt solubilization of individual aggregates. High-speed atomic force microscopy explicitly shows that disassembly starts with the destabilization of the tips and rapidly progresses to completion through protofilament unzipping and depolymerization without accumulation of harmful oligomeric intermediates. Our data provide molecular insights into the selective processing of toxic amyloids, which is critical to identify potential therapeutic targets against increasingly prevalent neurodegenerative disorders.

Center of pressure limb path differences for the detection of lameness in dogs: a preliminary study.

BACKGROUND: The limb center of pressure (COP) path measures and quantifies the load distribution within a limb in a still or moving subject. Under this premise, the aim of this study was to test whether data derived from this parameter could detect the differences between sound and lame limbs in unilaterally lame dogs with elbow dysplasia. To accomplish this purpose, ten unilaterally lame dogs of similar conformation were walked over a pressure platform. Next, the COP path, in relation to the position of sound and lame limbs, was measured in a coordinate system over a standard paw template obtained by pedobarography during the whole support phase. To compare variables, force platform data (peak vertical force and vertical impulse) from the same animals were obtained. Sound and lame limb statokinesiograms were also obtained while the animals stood still. RESULTS: The statistical analysis clearly showed that COP in lame limbs start cranially and were shorter than sound limbs. In addition, the value of the COP excursion index was lower in lame limbs. Finally, the area of statokinesiograms was greater in lame limbs. CONCLUSION: This methodology based in limb COP characteristics serves to discriminate between sound and lame limbs in dogs with elbow dysplasia.

Pedobarography: a novel approach to test the efficacy of treatments for lameness; an experience with mavacoxib in dogs with elbow osteoarthritis.

BACKGROUND: Pedobarographic analyses detect pressure redistribution among limbs and within limbs in humans, equids and dogs. The main objective of this study was to assess the usefulness of a set of pedobarographic parameters for the detection of lameness, as well as for its suitability for assessing the effects of therapies against osteoarthritis in dogs. With this purpose, eleven large-breed lame dogs with unilateral osteoarthritis due to elbow dysplasia were evaluated using a pressure platform prior to (D0) and after 3 months (D90) of treatment with mavacoxib, a COX-2 selective NSAID. The obtained parameters were: pressure distribution between lame and sound limbs, as well as paw area, mean pressure, and peak pressure of both lame and sound limbs. RESULTS: The results showed statistical differences in all these parameters between lame and sound limbs at D0; however, at D90, differences were significantly decreased as result of the treatment, indicating a substantial functional recovery under the study design conditions. CONCLUSIONS: The provided data prove the suitability of this novel technique in canine models for the quantitative and objective assessment of lameness, but also for the evaluation of treatments for lameness caused by articular pain.

Assessment of the Efficacy of Platelet-Rich Plasma in the Treatment of Traumatic Canine Fractures.

The role of platelet-rich plasma (PRP) in promoting the healing of bone fractures has not yet been clearly stated. The aim of this prospective clinical study was to evaluate the effectiveness of plasma rich in growth factors (PRGF, a PRP derivate) in the treatment of naturally-occurring bone fractures in dogs. With this objective, sixty-five dogs with radius/ulna or tibia/fibula bone fractures were randomly divided into two groups (PRGF and saline solution (SS) groups) and checked at days 0, 7, 14, 21, 28, 35, 42, 49, 56, 60, 63, 70, 120, and 180. All the fractures were treated with an external skeletal fixation, and pain was controlled with Carprofen. Healing was evaluated by physical examination, limb function, radiography, and by a Likert-type owner satisfaction questionnaire. A faster fracture healing was observed in the PRGF group, with statistically significant differences with respect to the SS group. Swelling at the fracture site was significantly greater at day 14 and 28 in animals injected with PRGF, and more pain on palpation was found in the area at day 28. The injection of PRGF in acute bone fractures accelerates bone healing.

Clearly Detectable, Kinetically Restricted Solid-Solid Phase Transition in cis-Ceramide Monolayers.

Sphingosine [(2 S,3 R,4 E)-2-amino-4-octadecene-1,3-diol] is the most common sphingoid base in mammals. Ceramides are N-acyl sphingosines. Numerous small variations on this canonical structure are known, including the 1-deoxy, the 4,5-dihydro, and many others. However, whenever there is a Δ4 double bond, it adopts the trans (or E) configuration. We synthesized a ceramide containing 4 Z-sphingosine and palmitic acid ( cis-pCer) and studied its behavior in the form of monolayers extended on an air-water interface. cis-pCer acted very differently from the trans isomer in that, upon lateral compression of the monolayer, a solid-solid transition was clearly observed at a mean molecular area ≤44 Å2·molecule-1, whose characteristics depended on the rate of compression. The solid-solid transition, as well as states of domain coexistence, could be imaged by atomic force microscopy and by Brewster-angle microscopy. Atomistic molecular dynamics simulations provided results compatible with the experimentally observed differences between the cis and trans isomers. The data can help in the exploration of other solid-solid transitions in lipids, both in vitro and in vivo, that have gone up to now undetected because of their less obvious change in surface properties along the transition, as compared to cis-pCer.

Assessment of static posturography and pedobarography for the detection of unilateral forelimb lameness in ponies.

BACKGROUND: Static posturography and pedobarography are based on the detection of postural imbalance and, consequently, the pressure redistribution between limbs in lame subjects. These techniques have proven to be useful for the detection of lameness in humans and dogs. The main objective of this study was to test the suitability of static posturography and pedobarography in diagnosing lameness in ponies. A pressure platform was used to obtain postural data (statokinesiograms, mean X and Y, length, LFS ratio, and mean velocity) from 10 sound ponies and 7 ponies with unilateral forelimb lameness. Static pedobarographic data (pressure distribution, mean pressure, and peak pressure) were also collected and compared with force plate data (peak vertical force and vertical impulse) obtained from the same animals at the walk. RESULTS: Significant differences were seen between lame and sound ponies for almost all evaluated parameters. With this sample size, differences between lame and sound limbs/groups were detected with a statistical power of 90%, except for mean X and Y. CONCLUSIONS: Static posturography and pedobarography provide a complementary approach for lameness detection in equids.

Posturography and dynamic pedobarography in lame dogs with elbow dysplasia and cranial cruciate ligament rupture.

BACKGROUND: The usefulness of studying posture and its modifications due to locomotor deficiencies of multiple origins has been widely proven in humans. To assess its suitability in the canine species, static posturography and dynamic pedobarography were performed on lame dogs affected with unilateral elbow dysplasia and cranial cruciate ligament rupture by using a pressure platform. With this objective, statokinesiograms and stabilograms, the percentage of pressure distribution between limbs, paw area, mean pressure, and peak pressure, were obtained from lame and sound dogs. These data were compared with Peak Vertical Force values originated from a force platform in the same recording sessions. RESULTS: Significant differences were found in the parameters mentioned above between sound and lame dogs and limbs. CONCLUSIONS: Posturography and pedobarography are useful and reliable for the monitoring of fore and hindlimb lameness in dogs, providing a new set of parameters for lameness detection.

Combined plasma rich in growth factors and adipose-derived mesenchymal stem cells promotes the cutaneous wound healing in rabbits.

BACKGROUND: The use of Plasma Rich in Growth Factors (PRGF) and Adipose Derived Mesenchymal Stem Cells (ASCs) are today extensively studied in the field of regenerative medicine. In recent years, human and veterinary medicine prefer to avoid using traumatic techniques and choose low or non-invasive procedures. The objective of this study was to evaluate the efficacy of PRGF, ASCs and the combination of both in wound healing of full-thickness skin defects in rabbits. With this purpose, a total of 144 rabbits were used for this study. The animals were divided in three study groups of 48 rabbits each depending on the administered treatment: PRGF, ASCs, and PGRF+ASCs. Two wounds of 8 mm of diameter and separated from each other by 20 mm were created on the back of each rabbit: the first was treated with saline solution, and the second with the treatment assigned for each group. Macroscopic and microscopic evolution of wounds was assessed at 1, 2, 3, 5, 7 and 10 days post-surgery. With this aim, 8 animals from each treatment group and at each study time were euthanized to collect wounds for histopathological study. RESULTS: Wounds treated with PRGF, ASCs and PRGF+ASCs showed significant higher wound healing and epithelialization rates, more natural aesthetic appearance, significant lower inflammatory response, significant higher collagen deposition and angiogenesis compared with control wounds. The combined treatment PRGF+ASCs showed a significant faster cutaneous wound healing process. CONCLUSIONS: The combined treatment PRGF+ASCs showed the best results, suggesting this is the best choice to enhance wound healing and improve aesthetic results in acute wounds.

Effect of intraarticular inoculation of mesenchymal stem cells in dogs with hip osteoarthritis by means of objective force platform gait analysis: concordance with numeric subjective scoring scales.

BACKGROUND: Subjective pain assessment scales have been widely used for assessing lameness in response to pain, but the accuracy of these scales has been questioned. To assess scale accuracy, 10 lame, presa Canario dogs with osteoarthritis (OA) associated with bilateral hip dysplasia were first treated with mesenchymal stem cells. Then, potential lameness improvement was analyzed using two pain scales (Bioarth and visual analog scale). These data were compared with similar data collected using a force platform with the same animals during a period of 6 months after treatment. RESULTS: The F test for intraclass correlation showed that concordance in pain/lameness scores between the 2 measuring methodologies was not significant (P value ≥ 0.9213; 95 % confidence interval, -0.56, 0.11). Although subjective pain assessment showed improvement after 6 months, force platform data demonstrated those same animals had returned to the initial lameness state. CONCLUSION: Use of pain assessment scales to measure lameness associated with OA did not have great accuracy and concordance when compared with quantitative force platform gait analysis.

Assessment of the effect of intraarticular injection of autologous adipose-derived mesenchymal stem cells in osteoarthritic dogs using a double blinded force platform analysis.

BACKGROUND: Regenerative medicine using Mesenchymal Stem Cells (MSC) alone or combined with Plasma Rich in Growth Factors (PRGF) is a rapidly growing area of clinical research and is currently also being used to treat osteoarthritis (OA). Force platform analysis has been consistently used to verify and quantify the efficacy of different therapeutic strategies for the treatment of OA in dogs including MSC associated to PRGF, but never with AD-MSC alone. The aim of this study was to use a force platform to measure the efficacy of intraarticular ADMSC administration for limb function improvement in dogs with severe OA. RESULTS: Ten lame dogs with severe hip OA and a control group of 5 sound dogs were used for this study. Results were statistically analyzed to detect a significant increase in peak vertical force (PVF) and vertical impulse (VI) in treated dogs. Mean values of PVF and VI were significantly improved within the first three months post-treatment in the OA group, increasing 9% and 2.5% body weight, respectively, at day 30. After this, the effect seems to decrease reaching initial values. CONCLUSION: Intraarticular ADMSC therapy objectively improved limb function in dogs with hip OA. The duration of maximal effect was less than 3 months.

Communication: system-size scaling of Boltzmann and alternate Gibbs entropies.

It has recurrently been proposed that the Boltzmann textbook definition of entropy S(E) = k ln Ω(E) in terms of the number of microstates Ω(E) with energy E should be replaced by the expression S(G)(E) = k ln Σ(E' < E)Ω(E') examined by Gibbs. Here, we show that SG either is equivalent to S in the macroscopic limit or becomes independent of the energy exponentially fast as the system size increases. The resulting exponential scaling makes the realistic use of SG unfeasible and leads in general to temperatures that are inconsistent with the notions of hot and cold.

The unreasonable effectiveness of equilibrium gene regulation through the cell cycle.

Systems like the prototypical lac operon can reliably hold repression of transcription upon DNA replication across cell cycles with just 10 repressor molecules per cell and behave as if they were at equilibrium. The origin of this phenomenology is still an unresolved question. Here, we develop a general theory to analyze strong perturbations in quasi-equilibrium systems and use it to quantify the effects of DNA replication in gene regulation. We find a scaling law linking actual with predicted equilibrium transcription via a single kinetic parameter. We show that even the lac operon functions beyond the physical limits of naive regulation through compensatory mechanisms that suppress non-equilibrium effects. Synthetic systems without adjuvant activators, such as the cAMP receptor protein (CRP), lack this reliability. Our results provide a rationale for the function of CRP, beyond just being a tunable activator, as a mitigator of cell cycle perturbations.

Systems biophysics of gene expression.

Gene expression is a process central to any form of life. It involves multiple temporal and functional scales that extend from specific protein-DNA interactions to the coordinated regulation of multiple genes in response to intracellular and extracellular changes. This diversity in scales poses fundamental challenges to the use of traditional approaches to fully understand even the simplest gene expression systems. Recent advances in computational systems biophysics have provided promising avenues to reliably integrate the molecular detail of biophysical process into the system behavior. Here, we review recent advances in the description of gene regulation as a system of biophysical processes that extend from specific protein-DNA interactions to the combinatorial assembly of nucleoprotein complexes. There is now basic mechanistic understanding on how promoters controlled by multiple, local and distal, DNA binding sites for transcription factors can actively control transcriptional noise, cell-to-cell variability, and other properties of gene regulation, including precision and flexibility of the transcriptional responses.

Controlled, blinded force platform analysis of the effect of intraarticular injection of autologous adipose-derived mesenchymal stem cells associated to PRGF-Endoret in osteoarthritic dogs.

BACKGROUND: Adipose-derived mesenchymal stem cell (ADMSC) therapy in regenerative medicine is a rapidly growing area of research and is currently also being used to treat osteoarthritis (OA). Force platform analysis has been consistently used to verify the efficacy of different therapeutic strategies for the treatment of OA in dogs, but never with AD-MSC.The aim of this study was to use a force platform to measure the efficacy of intraarticular ADMSC administration for limb function improvement in dogs with severe OA. RESULTS: Eight lame dogs with severe hip OA and a control group of 5 sound dogs were used for this study. Results were statistically analyzed to detect a significant increase in peak vertical force (PVF) and vertical impulse (VI) in treated dogs. Mean values of PVF and VI were significantly improved after treatment of the OA groups, reaching 53.02% and 14.84% of body weight, respectively, at day 180, compared with only 43.56% and 12.16% at day 0. CONCLUSION: This study objectively demonstrated that intraarticular ADMSC therapy resulted in reduced lameness due to OA.

Control of gene expression by modulated self-assembly.

Numerous transcription factors self-assemble into different order oligomeric species in a way that is actively regulated by the cell. Until now, no general functional role has been identified for this widespread process. Here, we capture the effects of modulated self-assembly in gene expression with a novel quantitative framework. We show that this mechanism provides precision and flexibility, two seemingly antagonistic properties, to the sensing of diverse cellular signals by systems that share common elements present in transcription factors like p53, NF-κB, STATs, Oct and RXR. Applied to the nuclear hormone receptor RXR, this framework accurately reproduces a broad range of classical, previously unexplained, sets of gene expression data and corroborates the existence of a precise functional regime with flexible properties that can be controlled both at a genome-wide scale and at the individual promoter level.

Dynamics-informed deconvolutional neural networks for super-resolution identification of regime changes in epidemiological time series.

The ability to infer the timing and amplitude of perturbations in epidemiological systems from their stochastically spread low-resolution outcomes is crucial for multiple applications. However, the general problem of connecting epidemiological curves with the underlying incidence lacks the highly effective methodology present in other inverse problems, such as super-resolution and dehazing from computer vision. Here, we develop an unsupervised physics-informed convolutional neural network approach in reverse to connect death records with incidence that allows the identification of regime changes at single-day resolution. Applied to COVID-19 data with proper regularization and model-selection criteria, the approach can identify the implementation and removal of lockdowns and other nonpharmaceutical interventions (NPIs) with 0.93-day accuracy over the time span of a year.

Multi-landmark alignment of genomic signals reveals conserved expression patterns across transcription start sites.

The prevalent one-dimensional alignment of genomic signals to a reference landmark is a cornerstone of current methods to study transcription and its DNA-dependent processes but it is prone to mask potential relations among multiple DNA elements. We developed a systematic approach to align genomic signals to multiple locations simultaneously by expanding the dimensionality of the genomic-coordinate space. We analyzed transcription in human and uncovered a complex dependence on the relative position of neighboring transcription start sites (TSSs) that is consistently conserved among cell types. The dependence ranges from enhancement to suppression of transcription depending on the relative distances to the TSSs, their intragenic position, and the transcriptional activity of the gene. Our results reveal a conserved hierarchy of alternative TSS usage within a previously unrecognized level of genomic organization and provide a general methodology to analyze complex functional relationships among multiple types of DNA elements.

Far-from-equilibrium processes without net thermal exchange via energy sorting.

Many important processes at the microscale require far-from-equilibrium conditions to occur, as in the functioning of mesoscopic bioreactors, nanoscopic rotors, and nanoscale mass conveyors. Achieving such conditions, however, is typically based on energy inputs that strongly affect the thermal properties of the environment and the controllability of the system itself. Here, we present a general class of far-from-equilibrium processes that suppress the net thermal exchange with the environment by maintaining the Maxwell-Boltzmann velocity distribution intact. This new phenomenon, referred to as ghost equilibrium, results from the statistical cancellation of superheated and subcooled nonequilibrated degrees of freedom that are autonomously generated through a microscale energy sorting process. We provide general conditions to observe this phenomenon and study its implications for manipulating energy at the microscale. The results are applied explicitly to two mechanistically different cases, an ensemble of rotational dipoles and a gas of trapped particles, which encompass a great variety of common situations involving both rotational and translational degrees of freedom.

Trafficking coordinate description of intracellular transport control of signaling networks.

Many cellular networks rely on the regulated transport of their components to transduce extracellular information into precise intracellular signals. The dynamics of these networks is typically described in terms of compartmentalized chemical reactions. There are many important situations, however, in which the properties of the compartments change continuously in a way that cannot naturally be described by chemical reactions. Here, we develop an approach based on transport along a trafficking coordinate to precisely describe these processes and we apply it explicitly to the TGF-ß signal transduction network, which plays a fundamental role in many diseases and cellular processes. The results of this newly introduced approach accurately capture the distinct TGF-ß signaling dynamics of cells with and without cancerous backgrounds and provide an avenue to predict the effects of chemical perturbations in a way that closely recapitulates the observed cellular behavior.