Conformational fluctuations and phases in fused in sarcoma (FUS) low-complexity domain.

The well-known phenomenon of phase separation in synthetic polymers and proteins has become a major topic in biophysics because it has been invoked as a mechanism of compartment formation in cells, without the need for membranes. Most of the coacervates (or condensates) are composed of Intrinsically Disordered Proteins (IDPs) or regions that are structureless, often in interaction with RNA and DNA. One of the more intriguing IDPs is the 526-residue RNA-binding protein, Fused in Sarcoma (FUS), whose monomer conformations and condensates exhibit unusual behavior that are sensitive to solution conditions. By focussing principally on the N-terminus low-complexity domain (FUS-LC comprising residues 1-214) and other truncations, we rationalize the findings of solid-state NMR experiments, which show that FUS-LC adopts a non-polymorphic fibril structure (core-1) involving residues 39-95, flanked by fuzzy coats on both the N- and C-terminal ends. An alternate structure (core-2), whose free energy is comparable to core-1, emerges only in the truncated construct (residues 110-214). Both core-1 and core-2 fibrils are stabilized by a Tyrosine ladder as well as hydrophilic interactions. The morphologies (gels, fibrils, and glass-like) adopted by FUS seem to vary greatly, depending on the experimental conditions. The effect of phosphorylation is site-specific. Simulations show that phosphorylation of residues within the fibril has a greater destabilization effect than residues that are outside the fibril region, which accords well with experiments. Many of the peculiarities associated with FUS may also be shared by other IDPs, such as TDP43 and hnRNPA2. We outline a number of problems for which there is no clear molecular explanation.

More than 10,000 pre-Columbian earthworks are still hidden throughout Amazonia.

Indigenous societies are known to have occupied the Amazon basin for more than 12,000 years, but the scale of their influence on Amazonian forests remains uncertain. We report the discovery, using LIDAR (light detection and ranging) information from across the basin, of 24 previously undetected pre-Columbian earthworks beneath the forest canopy. Modeled distribution and abundance of large-scale archaeological sites across Amazonia suggest that between 10,272 and 23,648 sites remain to be discovered and that most will be found in the southwest. We also identified 53 domesticated tree species significantly associated with earthwork occurrence probability, likely suggesting past management practices. Closed-canopy forests across Amazonia are likely to contain thousands of undiscovered archaeological sites around which pre-Columbian societies actively modified forests, a discovery that opens opportunities for better understanding the magnitude of ancient human influence on Amazonia and its current state.

Allosteric communication between ACE2 active site and binding interface with SARS-CoV-2.

SARS-CoV-2, the virus causing COVID-19, initiates cell invasion by deploying a receptor binding domain (RBD) to recognize the host transmembrane peptidase angiotensin-converting enzyme 2 (ACE2). Numerous experimental and theoretical studies have adopted high-throughput and structure-guided approaches to (i) understand how the RBD recognizes ACE2, (ii) rationalize, and (iii) predict the effect of viral mutations on the binding affinity. Here, we investigate the allosteric signal triggered by the dissociation of the ACE2-RBD complex. To this end, we construct an Elastic Network Model (ENM), and we use the Structural Perturbation Method (SPM). Our key result is that complex dissociation opens the ACE2 substrate-binding cleft located away from the interface and that fluctuations of the ACE2 binding cleft are facilitated by RBD binding. These and other observations provide a structural and dynamical basis for the influence of SARS-CoV-2 on ACE2 enzymatic activity. In addition, we identify a conserved glycine (G502 in SARS-CoV-2) as a key participant in complex disassembly.

Optimization of the first extraction protocol for metabolomic studies of Brucella abortus.

Brucellosis is a zoonosis prevalent worldwide and very recurrent in less developed or developing regions. This zoonosis affects livestock, generating high financial losses to producers, in addition to transmitting diseases to humans through meat consumption or handling contaminated products and animals. In this study, five extraction methods for Brucella abortus intracellular metabolites, using different solvent compositions and cell membrane disruption procedures, were evaluated. Derivatized extracts were analyzed by GC-HRMS. Raw data were processed in XCMS Online and the results were evaluated through multivariate statistical analysis using the MetaboAnalyst platform. The identification of the extracted metabolites was performed by the Unknowns software using the NIST 17.L library. The extraction performance of each method was evaluated for thirteen representative metabolites, comprising four different chemical classes. Most of these compounds are reported in the cell membrane composition of Gram-negative bacteria. The method based on extraction with methanol/chloroform/water presented the best performance in the evaluation of the extracted compounds and in the statistical results. Therefore, this method was selected for extracting intracellular metabolites from cultures of Brucella abortus for untargeted metabolomics analysis.

Entropic contribution of ACE2 glycans to RBD binding.

The spike protein of the SARS-CoV-2 virus (the causative agent of COVID-19) recognizes the host cell by binding to the peptidase domain (PD) of the extracellular receptor angiotensin-converting enzyme 2 (ACE2). A variety of carbohydrates could be attached to the six asparagines in the PD, resulting in a heterogeneous population of ACE2 glycoforms. Experiments have shown that the binding affinity of glycosylated and deglycosylated ACE2 to the virus is virtually identical. In most cases, the reduction in glycan size correlates with stronger binding, which suggests that volume exclusion, and hence entropic forces, determine the binding affinity. Here, we quantitatively test the entropy-based hypothesis by developing a lattice model for the complex between ACE2 and the SARS-CoV-2 spike protein receptor-binding domain (RBD). Glycans are treated as branched polymers with only volume exclusion, which we justify using all-atom molecular dynamics simulations in explicit water. We show that the experimentally measured changes in the ACE2-RBD dissociation constants for a variety of engineered ACE2 glycoforms are in reasonable agreement with our theory, thus supporting our hypothesis. However, a quantitative recovery of all the experimental data could require weak attractive interactions.

Vitis vinifera Red Globe grape: In natura investigations on skin pigmentation using phase-resolved photoacoustic and TDDFT methods.

In this work we aimed to investigate the source of the Red Globe grape skin pigmentation. To achieve this goal, we used the phase-resolved photoacoustic technique to investigate the sample in natura and to access the phase-dependent absorbing entities. In addition, we used the time-dependent density functional theory (TDDFT) to contrast with our experimental spectroscopic results. We measured the absorption spectrum of the Red Globe grape in natura using the photoacoustic technique and recovered the main source pigmentation spectrum using the phase-resolved technique. Finally, using the TDDFT, we qualitatively analyzed the physical sources of the grape pigmentation and we found significant evidence that the main biomolecules responsible for the coloration of the grape are the cyanidin-3-O-glucoside and the peonidin-3-O-glucoside.

Conformational Fluctuations and Phases in Fused in Sarcoma (FUS) Low-Complexity Domain.

The well known phenomenon of phase separation in synthetic polymers and proteins has become a major topic in biophysics because it has been invoked as a mechanism of compartment formation in cells, without the need for membranes. Most of the coacervates (or condensates) are composed of Intrinsically Disordered Proteins (IDPs) or regions that are structureless, often in interaction with RNA and DNA. One of the more intriguing IDPs is the 526-residue RNA binding protein, Fused In Sarcoma (FUS), whose monomer conformations and condensates exhibit unusual behavior that are sensitive to solution conditions. By focussing principally on the N-terminus low complexity domain (FUS-LC comprising residues 1-214) and other truncations, we rationalize the findings of solid state NMR experiments, which show that FUS-LC adopts a non-polymorphic fibril (core-1) involving residues 39-95, flanked by fuzzy coats on both the N- and C- terminal ends. An alternate structure (core-2), whose free energy is comparable to core-1, emerges only in the truncated construct (residues 110-214). Both core-1 and core-2 fibrils are stabilized by a Tyrosine ladder as well as hydrophilic interactions. The morphologies (gels, fibrils, and glass-like behavior) adopted by FUS seem to vary greatly, depending on the experimental conditions. The effect of phosphorylation is site specific and affects the stability of the fibril depending on the sites that are phosphorylated. Many of the peculiarities associated with FUS may also be shared by other IDPs, such as TDP43 and hnRNPA2. We outline a number of problems for which there is no clear molecular understanding.

Information flow, gating, and energetics in dimeric molecular motors.

Molecular motors, kinesin and myosin, are dimeric consisting of two linked identical monomeric globular proteins. Fueled by the free energy generated by ATP hydrolysis, they walk on polar tracks (microtubule or filamentous actin) processively, which means that only one head detaches and executes a mechanical step while the other stays bound to the track. One motor head must regulate the chemical state of the other, referred to as "gating", a concept that is still not fully understood. Inspired by experiments, showing that only a fraction of the energy from ATP hydrolysis is used to advance the kinesin motors against load, we demonstrate that the rest of the energy is associated with chemical transitions in the two heads. The coordinated chemical transitions involve communication between the two heads - a feature that characterizes gating. We develop a general framework, based on information theory and stochastic thermodynamics, and establish that gating could be quantified in terms of information flow between the motor heads. Applications to kinesin-1 and Myosin V show that information flow, with positive cooperativity, at external resistive loads less than a critical value, Fc. When force exceeds Fc, effective information flow ceases. Interestingly, Fc, which is independent of the input energy generated through ATP hydrolysis, coincides with the force at which the probability of backward steps starts to increase. Our findings suggest that transport efficiency is optimal only at forces less than Fc, which implies that these motors must operate at low loads under in vivo conditions.

Technique for Darkening of Extracted Teeth Simulating Pulpal Necrosis Discoloration.

Background: The use of discolored teeth is required to test whitening products, and it is difficult to obtain them, given their scarcity. Objective: To present a technique for in vitro darkening of extracted teeth simulating pulpal necrosis discoloration. Materials and Methods: Hemolysates I and II from human blood were subjected or not to laser irradiation (442 nm) for 1 h. The concentration of oxyhemoglobin (O2Hb) was analyzed by ultraviolet/visible spectroscopy, and the conversion of O2Hb to methemoglobin (MetHb) by transmission spectroscopy was assessed immediately and after 3 and 40 days. For darkening evaluation, bovine incisors were divided into two groups (n = 25), and their pulp chambers were filled with hemolysate solution II (HSII) and hemolysate II solution + laser (HSII+L). After storage in artificial saliva for 40 days at 37°C, color changes were measured by a colorimeter and ΔE was compared with the NBS parameters. Data were analyzed using a mixed linear model (α=5%). Results: HSII+L presented the lowest O2Hb and higher MetHb. The conversion of O2Hb to MetHb in HSII+L was 42% higher than in HSII. Both groups were effective in darkening the teeth, according to the NBS. Darkening stabilized from day 35. HSII promoted a marked color difference. Conclusion: The proposed technique was effective in darkening the extracted teeth simulating necrosis discoloration for in vitro models.

Effects of Gold Nanoparticles on the Stepping Trajectories of Kinesin.

A substantial increase in the temporal resolution of the stepping of dimeric molecular motors is possible by tracking the position of a large gold nanoparticle (GNP) attached to a labeled site on one of the heads. This technique was employed to measure the stepping trajectories of conventional kinesin (Kin1) using the time-dependent position of the GNP as a proxy. The trajectories revealed that the detached head always passes to the right of the head that is tightly bound to the microtubule (MT) during a step. In interpreting the results of such experiments, it is assumed that the GNP does not significantly alter the diffusive motion of the detached head. We used coarse-grained simulations of a system consisting of the MT-Kin1 complex with and without attached GNP to investigate how the stepping trajectories are affected. The two significant findings are: (1) The GNP does not faithfully track the position of the stepping head, and (2) the rightward bias is typically exaggerated by the GNP. Both these findings depend on the precise residue position to which the GNP is attached. Surprisingly, the stepping trajectories of kinesin are not significantly affected if, in addition to the GNP, a 1 µm diameter cargo is attached to the coiled coil. Our simulations suggest the effects of the large probe have to be considered when inferring the stepping mechanisms using GNP tracking experiments.

Studies of the early stages of the dynamic setting process of chemically activated restorative glass-ionomer cements.

OBJECTIVE: To evaluate the early stages of the setting process of chemically activated restorative glass-ionomer cements (GICs). MATERIAL AND METHODS: Five GICs were evaluated (n = 5): Equia Forte (GC), Equia Forte HT (GC), Ketac Universal (3M ESPE), Maxxion R (FGM) and Riva Self Cure (SDI) by Thermography, Fourier Transform Infrared Attenuated Total Reflectance Spectroscopy (FTIR-ATR) and Gillmore needle indentation mechanical testing. The FTIR-ATR spectra showed the formation of metal carboxylates within the cements and enabled the stabilization time (ST) to be determined and the thermographic camera measured the temperature field images in the sample. Data were statistically analyzed by ANOVA and Tukey-Kramer (α = 5%). RESULTS: The Gillmore needle test showed that the order of hardening was opposite to the order of ST values determined by FTIR. The results with the thermographic camera showed two stages of temperature variation, which coincided with the evolution of specific infrared bands. The exception was Maxxion R, which showed only a single step change in temperature. CONCLUSION: The early stages of the GIC setting reaction show temperature changes, both endothermic and exothermic, at specific times, confirming the occurrence of individual chemical reactions. The early setting involves reactions other than carboxylate formation.Significance: This study gives further detail of the early stages of the setting of GICs, and past research regarding the setting reaction of GIC.

The comparative measurement of body segment parameters using dual energy X-ray absorptiometry between sexes.

BACKGROUND: The study aimed to determine the proportion of the body segments in relation to the total body mass in healthy people, as well as analyze the composition of each segment and compare these results between sexes. METHODS: A total of 60 young adults (30 men and 30 women) were subjected to a full-body scan by dual energy X-rays absorptiometry (DXA) under standardized conditions. The regions of interest (ROI) were determined by a single trained evaluator. The body was divided into 16 segments to obtain values of total mass, lean mass (LM), fat mass, bone mineral content (BMC), lean mass percentage (%LM) and fat mass percentage (%FM) of each body segment represented by the 16 ROI. RESULTS: Men presented higher absolute mass in the upper limbs (Δ=32.87%; P<0.05). The proportion of the lower limbs (Δ=6.83%; P<0.05) and trunk (Δ=5.07%; P<0.05) of men is higher than women. In addition, males have more LM in the upper limbs (Δ=42.19%; P<0.05) and trunk (Δ=26.46%; P<0.001), and more BMC in the trunk (Δ=18.78%; P<0.05) and forearms (Δ=32.21%; P<0.05). They also present higher %LM (Δ=6.48%; P<0.001) and lower %FM (Δ=54.43%; P<0.001) than women in the forearms. CONCLUSIONS: The different body segments represent a different percentage of the total body mass in men than in women, as well as men present more LM and BMC in the trunk and upper limbs.

Impact of aortic angle on transcatheter aortic valve implantation outcome with Evolut-R, Portico, and Acurate-NEO.

OBJECTIVES: To investigate paravalvular leak (PVL) and devices success rates according to aortic angle (AA) in patients undergoing transcatheter aortic valve implantation (TAVI) with three new-generation self-expanding devices. BACKGROUND: The impact of aortic angle (AA) on TAVI device success and PVL rates is controversial. METHODS: This retrospective study included 392 patients submitted to TAVI for severe aortic stenosis with Portico, Evolut-R and Acurate-NEO, and available AA measurements at computed tomography (CT) angiography. AA was calculated from the implantation projection and was defined as the angle between the plane of aortic annulus and an ideal horizontal plane. Aorta was defined horizontal if AA>57° (75th percentile). RESULTS: In the horizontal group, the rates of moderate/severe PVL was higher in the Evolut-R group (20.8%), which was also characterized by a lower implant compared to that of Acurate-NEO, whereas device success was comparable among the three devices. AA was a significant predictor of moderate/severe PVLs (AUC 0.72, p = .002) only in the Evolut-R population. On multivariate analysis, calcium volume 850HU, bicuspid aortic valve, and implantation depth at the level of left coronary cusp were independent predictors of moderate/severe PVL. On univariate analysis in the horizontal aorta population, implantation depth was confirmed among the most significant predictors of moderate/severe PVL. CONCLUSIONS: Despite comparable device success rates, horizontal aorta represented a technical challenge only in the Evolut-R subgroup, which showed higher rates of moderate/severe PVL than Portico and Acurate-NEO, and was associated with a low implant.

The correlation of physicochemical properties of edible vegetable oils by chemometric analysis of spectroscopic data.

This work aimed to investigate and compare the composition and the physicochemical properties of 18 different sources of edible vegetable oils. A systematic study on the correlation between composition and physical properties was performed using Fourier Transform Infrared (FTIR) Spectroscopy and fatty acid chromatographic analysis. Principal component analysis of FTIR spectra is performed to classify edible oils concerning their physical properties. The results demonstrate the potentiality of the method associated with multivariate statistics analysis as powerful, fast, and non-destructive tools for characterization and quality control of edible vegetable oils.

Role of Long-range Allosteric Communication in Determining the Stability and Disassembly of SARS-COV-2 in Complex with ACE2.

Severe acute respiratory syndrome (SARS) and novel coronavirus disease (COVID-19) are caused by two closely related beta-coronaviruses, SARS-CoV and SARS-CoV-2, respectively. The envelopes surrounding these viruses are decorated with spike proteins, whose receptor binding domains (RBDs) initiate invasion by binding to the human angiotensin-converting enzyme 2 (ACE2). Subtle changes at the interface with ACE2 seem to be responsible for the enhanced affinity for the receptor of the SARS-CoV-2 RBD compared to SARS-CoV RBD. Here, we use Elastic Network Models (ENMs) to study the response of the viral RBDs and ACE2 upon dissassembly of the complexes. We identify a dominant detachment mode, in which the RBD rotates away from the surface of ACE2, while the receptor undergoes a conformational transition which stretches the active-site cleft. Using the Structural Perturbation Method, we determine the network of residues, referred to as the Allostery Wiring Diagram (AWD), which drives the large-scale motion activated by the detachment of the complex. The AWD for SARS-CoV and SARS-CoV-2 are remarkably similar, showing a network that spans the interface of the complex and reaches the active site of ACE2, thus establishing an allosteric connection between RBD binding and receptor catalytic function. Informed in part by the AWD, we used Molecular Dynamics simulations to probe the effect of interfacial mutations in which SARS-CoV-2 residues are replaced by their SARS-CoV counterparts. We focused on a conserved glycine (G502 in SARS-CoV-2, G488 in SARS-CoV) because it belongs to a region that initiates the dissociation of the complex along the dominant detachment mode, and is prominent in the AWD. Molecular Dynamics simulations of SARS-CoV-2 wild-type and G502P mutant show that the affinity for the human receptor of the mutant is drastically diminished. Our results suggest that in addition to residues that are in direct contact with the interface those involved in long range allosteric communication are also a determinant of the stability of the RBD-ACE2 complex.

True absolute determination of photoluminescence quantum yields by coupling multiwavelength thermal lens and photoluminescence spectroscopy.

Photoluminescence quantum yields denote a critical variable to characterise a fluorophore and its potential performance. Their determination, by means of methodologies employing reference standard materials, inevitably leads to large uncertainties. In response to this, herein we report for the first time an innovative and elegant methodology, whereby the use of neat solvent/reference material required by thermal lens approaches is eliminated by coupling it to photoluminescence spectroscopy, allowing for the discrimination between materials with similar photoluminescence quantum yields. To achieve this, both radiative and non-radiative transitions are simultaneously measured using a photoluminescence spectrometer coupled to a multiwavelength thermal lens spectroscopy setup in a mode-mismatched dual-beam configuration, respectively. The absorption factor independent ratio of the thermal lens and photoluminescence signals can then be used to determine the fluorescence quantum yield both accurately and precisely. We validated our reported method using rhodamine 6G and further applied it to three novel structurally related diketopyrrolopyrrole based materials, which, in contrast to results obtained by other methods, unveiled significant differences in their photoluminescence quantum yields.

Processivity and Velocity for Motors Stepping on Periodic Tracks.

Processive molecular motors enable cargo transportation by assembling into dimers capable of taking several consecutive steps along a cytoskeletal filament. In the well-accepted hand-over-hand stepping mechanism, the trailing motor detaches from the track and binds the filament again in the leading position. This requires fuel consumption in the form of ATP hydrolysis and coordination of the catalytic cycles between the leading and the trailing heads. Alternate stepping pathways also exist, including inchworm-like movements, backward steps, and foot stomps. Whether all the pathways are coupled to ATP hydrolysis remains to be determined. Here, to establish the principles governing the dynamics of processive movement, we present a theoretical framework that includes all of the alternative stepping mechanisms. Our theory bridges the gap between the elemental rates describing the biochemical and structural transitions in each head and the experimentally measurable quantities such as velocity, processivity, and probability of backward stepping. Our results, obtained under the assumption that the track is periodic and infinite, provide expressions that hold regardless of the topology of the network connecting the intermediate states, and are therefore capable of describing the function of any molecular motor. We apply the theory to myosin VI, a motor that takes frequent backward steps and moves forward with a combination of hand-over-hand and inchworm-like steps. Our model quantitatively reproduces various observables of myosin VI motility reported by four experimental groups. The theory is used to predict the gating mechanism, the pathway for backward stepping, and the energy consumption as a function of ATP concentration.

Fragile-to-strong crossover, growing length scales, and dynamic heterogeneity in Wigner glasses.

Colloidal particles, which are ubiquitous, have become ideal testing grounds for the structural glass transition theories. In these systems glassy behavior arises as the density of the particles is increased. Thus, soft colloidal particles with varying degree of softness capture diverse glass-forming properties, observed normally in molecular glasses. Brownian dynamics simulations for a binary mixture of micron-sized charged colloidal suspensions show that tuning the softness of the interaction potential, achievable by changing the monovalent salt concentration results in a continuous transition from fragile to strong behavior. Remarkably, this is found in a system where the well characterized interaction potential between the colloidal particles is isotropic. We also show that the predictions of the random first-order transition (RFOT) theory quantitatively describes the universal features such as the growing correlation length, ξ∼(ϕ_{K}/ϕ-1)^{-ν} with ν=2/3 where ϕ_{K}, the analog of the Kauzmann temperature, depends on the salt concentration. As anticipated by the RFOT predictions, we establish a causal relationship between the growing correlation length and a steep increase in the relaxation time and dynamic heterogeneity as the system is compressed. The broad range of fragility observed in Wigner glasses is used to draw analogies with molecular and polymer glasses. The large variations in the fragility are normally found only when the temperature dependence of the viscosity is examined for a large class of diverse glass-forming materials. In sharp contrast, this is vividly illustrated in a single system that can be experimentally probed. Our work also shows that the RFOT predictions are accurate in describing the dynamics over the entire density range, regardless of the fragility of the glasses.