Second Data Release from the European Pulsar Timing Array: Challenging the Ultralight Dark Matter Paradigm.

Pulsar Timing Array experiments probe the presence of possible scalar or pseudoscalar ultralight dark matter particles through decade-long timing of an ensemble of galactic millisecond radio pulsars. With the second data release of the European Pulsar Timing Array, we focus on the most robust scenario, in which dark matter interacts only gravitationally with ordinary baryonic matter. Our results show that ultralight particles with masses 10^{-24.0} eV≲m≲10^{-23.3} eV cannot constitute 100% of the measured local dark matter density, but can have at most local density ρ≲0.3 GeV/cm^{3}.

Adsorption of brilliant green dye onto activated carbon prepared from cashew nut shell by KOH activation: Studies on equilibrium isotherm.

Activated carbon from cashew nut shell via a potassium hydroxide (KOH) at 600 °C in an N2 atmosphere and their characteristics using FT-IR, XRD, SEM with EDS, and BET analysis was investigated. The cashew nut shell activated carbon obtained by KOH activation with a CNS/KOH ratio of 1:1 at 600 °C (N2 atmosphere) for 2 h had the highest surface area (407.80 m2/g) as compared to other ratio samples. Amongst, CNS/KOH ratios of 1:1 sample are used for the adsorbent, they are effects of contact time, pH, adsorbent dose, and initial dye concentration on brilliant green (BG) removal efficiency were studied. Moreover, the Langmuir and Freundlich adsorption models consisted utilized to affirm the adsorption isotherms. They are, best fitting for BG experimental equilibrium data was achieved with the Langmuir isotherm, giving a maximum BG adsorption capacity of 243.90 mg/g.

High Tensile Strength of Engineered ß-Solenoid Fibrils via Sonication and Pulling.

We present estimates of ultimate tensile strength (UTS) for two engineered ß-solenoid protein mutant fibril structures (spruce budworm and Rhagium inquisitor antifreeze proteins) derived from sonication-based measurements and from force pulling molecular dynamics simulations, both in water. Sonication experiments generate limiting scissioned fibrils with a well-defined length-to-width correlation for the mutant spruce budworm protein and the resultant UTS estimate is 0.66 ± 0.08 GPa. For fibrils formed from engineered R. inquisitor antifreeze protein, depending upon geometry, we estimate UTSs of 3.5 ± 3.2-5.5 ± 5.1 GPa for proteins with interfacial disulfide bonds, and 1.6 ± 1.5-2.5 ± 2.3 GPa for the reduced form. The large error bars for the R. inquisitor structures are intrinsic to the broad distribution of limiting scission lengths. Simulations provide pulling velocity-dependent UTSs increasing from 0.2 to 1 GPa in the available speed range, and 1.5 GPa extrapolated to the speeds expected in the sonication experiments. Simulations yield low-velocity values for the Young's modulus of 6.0 GPa. Without protein optimization, these mechanical parameters are similar to those of spider silk and Kevlar, but in contrast to spider silk, these proteins have a precisely known sequence-structure relationship.

Theoretical modeling of multiprotein complexes by iSPOT: Integration of small-angle X-ray scattering, hydroxyl radical footprinting, and computational docking.

Structural determination of protein-protein complexes such as multidomain nuclear receptors has been challenging for high-resolution structural techniques. Here, we present a combined use of multiple biophysical methods, termed iSPOT, an integration of shape information from small-angle X-ray scattering (SAXS), protection factors probed by hydroxyl radical footprinting, and a large series of computationally docked conformations from rigid-body or molecular dynamics (MD) simulations. Specifically tested on two model systems, the power of iSPOT is demonstrated to accurately predict the structures of a large protein-protein complex (TGFß-FKBP12) and a multidomain nuclear receptor homodimer (HNF-4α), based on the structures of individual components of the complexes. Although neither SAXS nor footprinting alone can yield an unambiguous picture for each complex, the combination of both, seamlessly integrated in iSPOT, narrows down the best-fit structures that are about 3.2Å and 4.2Å in RMSD from their corresponding crystal structures, respectively. Furthermore, this proof-of-principle study based on the data synthetically derived from available crystal structures shows that the iSPOT-using either rigid-body or MD-based flexible docking-is capable of overcoming the shortcomings of standalone computational methods, especially for HNF-4α. By taking advantage of the integration of SAXS-based shape information and footprinting-based protection/accessibility as well as computational docking, this iSPOT platform is set to be a powerful approach towards accurate integrated modeling of many challenging multiprotein complexes.

In Silico Measurements of Twist and Bend Moduli for ß-Solenoid Protein Self-Assembly Units.

We compute potentials of mean force for bend and twist deformations via force pulling and umbrella sampling experiments for four ß-solenoid proteins (BSPs) that show promise in nanotechnology applications. In all cases, we find quasi-Hooke's law behavior until the point of rupture. Bending moduli show modest anisotropy for two-sided and three-sided BSPs, and little anisotropy for a four-sided BSP. There is a slight clockwise/counterclockwise asymmetry in the twist potential of mean force, showing greater stiffness when the applied twist follows the intrinsic twist. When we extrapolate to beam theory appropriate for amyloid fibrils of the BSPs, we find bend/twist moduli which are somewhat smaller than those in the literature for other amyloid fibrils. Twist persistence lengths are on the order of a micron, and bend persistence lengths are several microns. Provided the intrinsic twist can be reversed, these results support the usage of BSPs in biomaterials applications.

Quantitative mapping of protein structure by hydroxyl radical footprinting-mediated structural mass spectrometry: a protection factor analysis.

Measurements from hydroxyl radical footprinting (HRF) provide rich information about the solvent accessibility of amino acid side chains of a protein. Traditional HRF data analyses focus on comparing the difference in the modification/footprinting rate of a specific site to infer structural changes across two protein states, e.g., between a free and ligand-bound state. However, the rate information itself is not fully used for the purpose of comparing different protein sites within a protein on an absolute scale. To provide such a cross-site comparison, we present a new, to our knowledge, data analysis algorithm to convert the measured footprinting rate constant to a protection factor (PF) by taking into account the known intrinsic reactivity of amino acid side chain. To examine the extent to which PFs can be used for structural interpretation, this PF analysis is applied to three model systems where radiolytic footprinting data are reported in the literature. By visualizing structures colored with the PF values for individual peptides, a rational view of the structural features of various protein sites regarding their solvent accessibility is revealed, where high-PF regions are buried and low-PF regions are more exposed to the solvent. Furthermore, a detailed analysis correlating solvent accessibility and local structural contacts for gelsolin shows a statistically significant agreement between PF values and various structure measures, demonstrating that the PFs derived from this PF analysis readily explain fundamental HRF rate measurements. We also tested this PF analysis on alternative, chemical-based HRF data, showing improved correlations of structural properties of a model protein barstar compared to examining HRF rate data alone. Together, this PF analysis not only permits a novel, to our knowledge, approach of mapping protein structures by using footprinting data, but also elevates the use of HRF measurements from a qualitative, cross-state comparison to a quantitative, cross-site assessment of protein structures in the context of individual conformational states of interest.

Cross-talk between the ligand- and DNA-binding domains of estrogen receptor.

Estrogen receptor alpha (ERα) is a hormone-responsive transcription factor that contains several discrete functional domains, including a ligand-binding domain (LBD) and a DNA-binding domain (DBD). Despite a wealth of knowledge about the behaviors of individual domains, the molecular mechanisms of cross-talk between LBD and DBD during signal transduction from hormone to DNA-binding of ERα remain elusive. Here, we apply a multiscale approach combining coarse-grained (CG) and atomistically detailed simulations to characterize this cross-talk mechanism via an investigation of the ERα conformational landscape. First, a CG model of ERα is built based on crystal structures of individual LBDs and DBDs, with more emphasis on their interdomain interactions. Second, molecular dynamics simulations are implemented and enhanced sampling is achieved via the "push-pull-release" strategy in the search for different LBD-DBD orientations. Third, multiple energetically stable ERα conformations are identified on the landscape. A key finding is that estradiol-bound LBDs utilize the well-described activation helix H12 to pack and stabilize LBD-DBD interactions. Our results suggest that the estradiol-bound LBDs can serve as a scaffold to position and stabilize the DBD-DNA complex, consistent with experimental observations of enhanced DNA binding with the LBD. Final assessment using atomic-level simulations shows that these CG-predicted models are significantly stable within a 15-ns simulation window and that specific pairs of lysine residues in close proximity at the domain interfaces could serve as candidate sites for chemical cross-linking studies. Together, these simulation results provide a molecular view of the role of ERα domain interactions in response to hormone binding.

Fast-SAXS-pro: a unified approach to computing SAXS profiles of DNA, RNA, protein, and their complexes.

A generalized method, termed Fast-SAXS-pro, for computing small angle x-ray scattering (SAXS) profiles of proteins, nucleic acids, and their complexes is presented. First, effective coarse-grained structure factors of DNA nucleotides are derived using a simplified two-particle-per-nucleotide representation. Second, SAXS data of a 18-bp double-stranded DNA are measured and used for the calibration of the scattering contribution from excess electron density in the DNA solvation layer. Additional test on a 25-bp DNA duplex validates this SAXS computational method and suggests that DNA has a different contribution from its hydration surface to the total scattering compared to RNA and protein. To account for such a difference, a sigmoidal function is implemented for the treatment of non-uniform electron density across the surface of a protein/nucleic-acid complex. This treatment allows differential scattering from the solvation layer surrounding protein/nucleic-acid complexes. Finally, the applications of this Fast-SAXS-pro method are demonstrated for protein/DNA and protein/RNA complexes.

2-Phenyl-ethanaminium 4-hy-droxy-benzoate.

In the title salt, C8H12N(+)·C7H5O3 (-), the cation is disordered over two orientations with site occupancies of 0.565 (7) and 0.435 (7). In the anion, the carboxyl-ate group makes the dihedral angle of 4.19 (18)° with the benzene ring. In the crystal, the ions are connected by N-H⋯O and O-H⋯O hydrogen bonds, forming a three-dimensional network.

4-Methyl-pyridinium 4-hy-droxy-benzoate.

In the crystal structure of the title salt, C(6)H(8)N(+)·C(7)H(5)O(3) (-), the anions and cations are linked by classical N-H⋯O hydrogen bonds. The anions are connected by pairs of C-H⋯O hydrogen bonds into inversion dimers and further linked by classical O-H⋯O hydrogen bonds. Weak π-π inter-actions [centroid-centroid distances = 3.740 (3) and 3.855 (3) Å] also occur. The dihedral angle between the CO(2) (-) group and the benzene ring to which it is attached is 20.95 (8)°.

Coarse-grained simulations of protein-protein association: an energy landscape perspective.

Understanding protein-protein association is crucial in revealing the molecular basis of many biological processes. Here, we describe a theoretical simulation pipeline to study protein-protein association from an energy landscape perspective. First, a coarse-grained model is implemented and its applications are demonstrated via molecular dynamics simulations for several protein complexes. Second, an enhanced search method is used to efficiently sample a broad range of protein conformations. Third, multiple conformations are identified and clustered from simulation data and further projected on a three-dimensional globe specifying protein orientations and interacting energies. Results from several complexes indicate that the crystal-like conformation is favorable on the energy landscape even if the landscape is relatively rugged with metastable conformations. A closer examination on molecular forces shows that the formation of associated protein complexes can be primarily electrostatics-driven, hydrophobics-driven, or a combination of both in stabilizing specific binding interfaces. Taken together, these results suggest that the coarse-grained simulations and analyses provide an alternative toolset to study protein-protein association occurring in functional biomolecular complexes.

4-Nitro-anilinium p-toluene-sulfonate.

In the cation of the title salt, C(6)H(7)N(2)O(2) (+)·C(7)H(7)O(3)S(-), the benzene ring makes a dihedral angle of 10.2 (2)° with the nitro group. In the crystal, the cations and anions are linked by weak N-H⋯O hydrogen bonds, forming a layer parallel to the ac plane. A weak C-H⋯O inter-action and π-π inter-actions [centroid-centroid distances of 3.738 (3) and 3.748 (3) Å] also observed within the layer.

1-Methyl-4-(4-methylstyryl)pyridinium 4-methylbenzenesulfonate.

In the title salt, C15H16N(+)·C7H7O3S(-), the dihedral angle between the pyridine and benzene rings of the cation is 5.98 (18)°. In the crystal, adjacent anions and cations are linked by weak non-classical C-H⋯O hydrogen bonds and π-π inter-actions, with a centroid-centroid distance of 3.749 (2) Å.

Enhanced photocatalytic degradation of organic pollutants by Ag-TiO2 loaded cassava stem activated carbon under sunlight irradiation.

Ag-doped TiO2 and Ag-doped TiO2 loaded cassava stem activated carbon (Ag: TiO2/CSAC) were prepared by sol-gel method and are labelled as AT and AT/CSAC respectively. XRD results confirmed that the anatase-TiO2 and crystalline size are decreased (12.37 nm) through the silver doping and cassava stem activated carbon loading. UV-Vis showed that the AT/CSAC makes a red shift from the absorption edge compared to pure and AT samples and then the band gap is reduced (2.81 eV). The increased surface area (238.51 m2/g) of the AT/CSAC sample through the Ag and CSAC, respectively. The consequences point out that the highest photodegradation efficiency (98.08%) of the TiO2 upon silver doping and cassava stem activated carbon loading samples were brilliant green (BG) under sunlight irradiation.

Role of hydration force in the self-assembly of collagens and amyloid steric zipper filaments.

In protein self-assembly, types of surfaces determine the force between them. Yet the extent to which the surrounding water contributes to this force remains as a fundamental question. Here we study three self-assembling filament systems that respectively have hydrated (collagen), dry nonpolar, and dry polar (amyloid) interfaces. Using molecular dynamics simulations, we calculate and compare local hydration maps and hydration forces. We find that the primary hydration shells are formed all over the surface, regardless of the types of the underlying amino acids. The weakly oscillating hydration force arises from coalescence and depletion of hydration shells as two filaments approach, whereas local water diffusion, orientation, or hydrogen-bonding events have no direct effect. Hydration forces between hydrated, polar, and nonpolar interfaces differ in the amplitude and phase of the oscillation relative to the equilibrium surface separation. Therefore, water-mediated interactions between these protein surfaces, ranging in character from "hydrophobic" to "hydrophilic", have a common molecular origin based on the robustly formed hydration shells, which is likely applicable to a broad range of biomolecular assemblies whose interfacial geometry is similar in length scale to those of the present study.

An open-label, randomized prospective study to evaluate the efficacy and safety of Carica papaya leaf extract for thrombocytopenia associated with dengue fever in pediatric subjects.

OBJECTIVE: Thrombocytopenia in dengue fever (DF) is a well-known complication in both adults and pediatric subjects. Management of DF primarily includes symptomatic and intensive supportive care. There are studies available on the efficacy and safety of Carica papaya leaf extract (CPLE) in adult patients with DF. However, there are no published studies available on the efficacy and safety of CPLE in the pediatric age group. Hence, this study was conducted. METHODOLOGY: A prospective, open-label, randomized controlled study was conducted in subjects aged between 1 and 12 years having thrombocytopenia associated with DF (NS-1 antigen positive) or dengue hemorrhagic fever (DHF) grades I and II. All participants were randomized into the intervention group (n =147, CPLE syrup + standard therapy) and the control group (n=147, received only standard therapy). All subjects were followed up daily for 5 days with monitoring of blood counts. RESULTS: A total of 285 subjects were finally evaluated for efficacy, and nine dropped out (seven in the control group and two in the intervention group). However, all 294 subjects were evaluated for safety. CPLE (Caripill) syrup increased the platelet count significantly compared to the control group (P<0.05). In the intervention group, the platelet count increased from day 3 onward: platelet count on day 3 (mean platelet count =89,739.31, P=0.030), day 4 (mean platelet count =120,788.96, P=0.019), and day 5 (mean platelet count =168,922.75 P=0.023). Two children complained of nausea in the intervention group. Overall, Caripill syrup was well tolerated. CONCLUSION: CPLE syrup significantly increases the platelet count in pediatric DF patients and is well tolerated.

Region-specific role of water in collagen unwinding and assembly.

Conformational stability of the collagen triple helix affects its turnover and determines tissue homeostasis. Although it is known that the presence of imino acids (prolines or hydroxyprolines) confer stability to the molecule, little is known regarding the stability of the imino-poor region lacking imino acids, which plays a key role in collagen cleavage. In particular, there have been continuing debates about the role of water in collagen stability. We addressed these issues using molecular dynamics simulations on 30-residue long collagen triple helices, including a structure that has a biologically relevant 9-residue imino-poor region from type III collagen (PDB ID: 1BKV). A torsional map approach was used to characterize the conformational motion of the molecule that differ between imino-rich and imino-poor regions. At temperatures 300 K and above, unwinding initiates at a common cleavage site, the glycine-isoleucine bond in the imino-poor region. This provides a linkage between previous observations that unwinding of the imino-poor region is a requirement for collagenase cleavage, and that isolated collagen molecules are unstable at body temperature. We found that unwinding of the imino-poor region is controlled by dynamic water bridges between backbone atoms with average lifetimes on the order of a few picoseconds, as the degree of unwinding strongly correlated with the loss of water bridges, and unwinding could be either prevented or enhanced, respectively by enforcing or forbidding water bridge formation. While individual water bridges were short-lived in the imino-poor region, the hydration shell surrounding the entire molecule was stable even at 330 K. The diameter of the hydrated collagen including the first hydration shell was about 14 A, in good agreement with the experimentally measured inter-collagen distances. These results elucidate the general role of water in collagen turnover: water not only affects collagen cleavage by controlling its torsional motion, but it also forms a larger-scale lubrication layer mediating collagen self-assembly.

Site-directed mutant libraries for isolating minimal mutations yielding functional changes.

Powerful, facile new ways to create libraries of site-directed mutants are demonstrated. These include: (1) one-pot-PCR, (2) multi-pot-PCR, and (3) split-mix-PCR. One-pot-PCR uses mutant oligonucleotides to generate megaprimers in situ, and it was used to randomly incorporate 28 mutations in a gabT gene in a single reaction. In more difficult cases, multi-pot-PCR can be employed: mutant megaprimers are synthesized individually, then combined in a single mutagenesis PCR. This method was used to incorporate 14 out of 15 mutations in a pabB gene. Split-mix-PCR is a conceptually novel method for creation of site-directed mutant libraries. Separate PCRs for each mutant primer are performed, followed by pooling the products of the individual reactions. The pooled mixture is re-aliquoted into individual mutant oligonucleotide PCRs. These steps are repeated for each cycle. Split-mix-PCR results in a nearly random distribution of mutation sites, and a distribution of number-of-mutations per gene that is computable and narrow. Split-mix-PCR was applied to the directed evolution of aminodeoxychorismate synthase into anthranilate synthase, and easily allowed the determination of the fewest mutations required for introduction of novel activity.

Sequence analysis of infectious bursal disease virus isolates from India: phylogenetic relationships.

Prevalence of infectious bursal disease (IBD) among chickens in different parts of Tamil Nadu, India, has been studied by collection of bursal samples from suspected flocks and by performing reverse transcription-polymerase chain reaction (RT-PCR) for amplification of a specific product of 474 bp from the variable region of the VP2 gene. Among 53 bursal samples examined by RT-PCR, 40 showed a positive reaction. The amplified products were subjected to nucleotide sequencing and the obtained sequences were compared with those of IBD virus (IBDV) vaccine strain Georgia, the classical virulent strain 52/70 and the very virulent Japanese OKYM strain. Nucleotide homology data indicated that all the Tamil Nadu isolates showed homology ranging from 91 to 99.6% among themselves. When compared with the very virulent Japanese OKYM strain, four isolates grouped with that strain. Majority of the isolates clustered with the very the virulent OKYM strain as evident from phylogenetic analysis performed using the MEGA program. Comparison of the deduced amino acid sequences of IBDV isolates with those of the vaccine strain Georgia, the classical virulent strain 52/70 and the very virulent strain OKYM also revealed the presence of conserved serine-rich heptapeptide sequence in most of the isolates. Results of this study indicate that majority of the IBDV isolates are very virulent, which is evident from heavy mortality that has been reported in few flocks of poultry in spite of regular vaccination.

A Newfound Cancer-Activating Mutation Reshapes the Energy Landscape of Estrogen-Binding Domain.

The ligand-binding domain (LBD) of an estrogen receptor undergoes a large conformational switching from an inactive to active state in response to hormone stimuli. Very recently, a novel D538G mutant has been identified to be active in advanced breast cancer tumors. Here, we ask if molecular simulations can provide insight on its mechanistic impact on the receptor's activation status. It has been challenging for ab initio modeling to identify two distinct conformations of a single amino acid sequence as large as that of the LBD. Using a coarse-grained (CG) model, we are able to correctly reproduce this LBD conformational switching. Furthermore, we found that the D538G mutation reshapes the energy landscape by stabilizing both active and inactive conformations, but preferring the active by 1.5 kcal/mol. This observation is consistent with the concept of a mutation-shifting landscape and provides a structural explanation for the oncogenic D538G mutation at the detailed conformational level.