Combining Scattering Experiments and Colloid Theory to Characterize Charge Effects in Concentrated Antibody Solutions.

Charges and their contribution to protein-protein interactions are essential for the key structural and dynamic properties of monoclonal antibody (mAb) solutions. In fact, they influence the apparent molecular weight, the static structure factor, the collective diffusion coefficient, or the relative viscosity, and their concentration dependence. Further, charges play an important role in the colloidal stability of mAbs. There exist standard experimental tools to characterize mAb net charges, such as the measurement of the electrophoretic mobility, the second virial coefficient, or the diffusion interaction parameter. However, the resulting values are difficult to directly relate to the actual overall net charge of the antibody and to theoretical predictions based on its known molecular structure. Here, we report the results of a systematic investigation of the solution properties of a charged IgG1 mAb as a function of concentration and ionic strength using a combination of electrophoretic measurements, static and dynamic light scattering, small-angle X-ray scattering, and tracer particle-based microrheology. We analyze and interpret the experimental results using established colloid theory and coarse-grained computer simulations. We discuss the potential and limits of colloidal models for the description of the interaction effects of charged mAbs, in particular pointing out the importance of incorporating shape and charge anisotropy when attempting to predict structural and dynamic solution properties at high concentrations.

Integrative Salt Selection and Formulation Optimization: Perspectives of Disproportionation and Microenvironmental pH Modulation.

We report a novel utilization of a pH modifier as a disproportionation retardant in a tablet formulation. The drug molecule of interest has significant bioavailability challenges that require solubility enhancement. In addition to limited salt/cocrystal options, disproportionation of the potential salt(s) was identified as a substantial risk. Using a combination of Raman spectroscopy with chemometrics and quantitative X-ray diffraction in specially designed stress testing, we investigated the disproportionation phenomena. The learnings and insight drawn from crystallography drove the selection of the maleate form as the target API. Inspired by the fumarate form's unique stability and solubility characteristics, we used fumaric acid as the microenvironmental pH modulator. Proof-of-concept experiments with high-risk (HCl) and moderate-risk (maleate) scenarios confirmed the synergistic advantage of fumaric acid, which interacts with the freebase released by disproportionation to form a more soluble species. The resultant hemifumarate helps maintain the solubility at an elevated level. This work demonstrates an innovative technique to mediate the solubility drop during the "parachute" phase of drug absorption using compendial excipients, and this approach can potentially serve as an effective risk-mitigating strategy for salt disproportionation.

Molecular basis of force-pCa relation in MYL2 cardiomyopathy mice: Role of the super-relaxed state of myosin.

In this study, we investigated the role of the super-relaxed (SRX) state of myosin in the structure-function relationship of sarcomeres in the hearts of mouse models of cardiomyopathy-bearing mutations in the human ventricular regulatory light chain (RLC, MYL2 gene). Skinned papillary muscles from hypertrophic (HCM-D166V) and dilated (DCM-D94A) cardiomyopathy models were subjected to small-angle X-ray diffraction simultaneously with isometric force measurements to obtain the interfilament lattice spacing and equatorial intensity ratios (I11/I10) together with the force-pCa relationship over a full range of [Ca2+] and at a sarcomere length of 2.1 µm. In parallel, we studied the effect of mutations on the ATP-dependent myosin energetic states. Compared with wild-type (WT) and DCM-D94A mice, HCM-D166V significantly increased the Ca2+ sensitivity of force and left shifted the I11/I10-pCa relationship, indicating an apparent movement of HCM-D166V cross-bridges closer to actin-containing thin filaments, thereby allowing for their premature Ca2+ activation. The HCM-D166V model also disrupted the SRX state and promoted an SRX-to-DRX (super-relaxed to disordered relaxed) transition that correlated with an HCM-linked phenotype of hypercontractility. While this dysregulation of SRX ↔ DRX equilibrium was consistent with repositioning of myosin motors closer to the thin filaments and with increased force-pCa dependence for HCM-D166V, the DCM-D94A model favored the energy-conserving SRX state, but the structure/function-pCa data were similar to WT. Our results suggest that the mutation-induced redistribution of myosin energetic states is one of the key mechanisms contributing to the development of complex clinical phenotypes associated with human HCM-D166V and DCM-D94A mutations.

An easily processable silver nanowires-dual-cellulose conductive paper for versatile flexible pressure sensors.

To date, flexible pressure sensors built on silver nanowires (AgNWs) have attracted tremendous attention, owing to their versatile applications in wearable, human-interactive, health-monitoring devices. Cellulose and its derivatives, which show great promise in serving flexible pressure sensors as the desired substrate due to their natural abundance, biocompatibility, easy processibility, and low costs. Herein, we reported a rational strategy to design a silver nanowires-dual-cellulose conductive paper. Its morphology, chemical and crystal structures, thermal stability, mechanical performances, and electrical properties were carefully studied. The results suggested that good tensile properties (tensile strength ≤8.10 MPa), high electrical conductivity (≤ 1.74 × 104 S·m-1) with long-term stability, and good adhesion stability (bending cycles over 500) were obtained. Furthermore, the use of such conductive paper as substrate for versatile flexible pressure sensors was demonstrated, which exhibited fast response (~ 0.48 s) and high sensitivity, in response to finger motion, voice recognition, and human pulse, etc.

The Influence of the Material Structure on the Mechanical Properties of Geopolymer Composites Reinforced with Short Fibers Obtained with Additive Technologies.

Additive manufacturing technologies have a lot of potential advantages for construction application, including increasing geometrical construction flexibility, reducing labor costs, and improving efficiency and safety, and they are in line with the sustainable development policy. However, the full exploitation of additive manufacturing technology for ceramic materials is currently limited. A promising solution in these ranges seems to be geopolymers reinforced by short fibers, but their application requires a better understanding of the behavior of this group of materials. The main objective of the article is to investigate the influence of the microstructure of the material on the mechanical properties of the two types of geopolymer composites (flax and carbon-reinforced) and to compare two methods of production of geopolymer composites (casting and 3D printing). As raw material for the matrix, fly ash from the Skawina coal power plant (located at: Skawina, Lesser Poland, Poland) was used. The provided research includes mechanical properties, microstructure investigations with the use of scanning electron microscope (SEM), confocal microscopy, and atomic force microscope (AFM), chemical and mineralogical (XRD-X-ray diffraction, and XRF-X-ray fluorescence), analysis of bonding in the materials (FT-IR), and nuclear magnetic resonance spectroscopy analysis (NMR). The best mechanical properties were reached for the sample made by simulating 3D printing process for the composite reinforced by flax fibers (48.7 MPa for the compressive strength and 9.4 MPa for flexural strength). The FT-IR, XRF and XRD results show similar composition of all investigated materials. NMR confirms the presence of SiO4 and AlO4 tetrahedrons in a three-dimensional structure that is crucial for geopolymer structure. The microscopy observations show a better coherence of the geopolymer made in additive technology to the reinforcement and equal fiber distribution for all investigated materials. The results show the samples made by the additive technology had comparable, or better, properties with those made by a traditional casting method.

Intriguing Cytotoxicity of the Street Dissociative Anesthetic Methoxphenidine: Unexpected Impurities Spotted.

The black market for new psychoactive substances has been constantly evolving and the substances that appear on this market cause a considerable number of issues, in extreme cases leading to human deaths. While monitoring the drug black market, we detected a sample of a dissociative anesthetic methoxphenidine, the salt of which contained an unusual anion in the form of bromo- and chloro-zincate complex. Concerning the unknown and potentially hazardous properties of this sample, we performed an in vitro cytotoxicity screening in cell lines of various origins (e.g., kidney, liver, bladder) which was compared with the toxicity results of the methoxphenidine standard prepared for this purpose. The street methoxphenidine sample exhibited markedly higher toxicity than the standard, which was probably caused by the anion impurity. Since it is not usual to analyze anions in salts of novel psychoactive substances, but such samples may be commonly available at the drug black market, we have developed a method for their identification with X-ray powder diffraction (XRPD), which also enabled us to distinguish between different polymorphs/solvates of methoxphenidine that were crystallized in the laboratory. XRPD offers additional data about samples, which may not be discovered by routine techniques, and in some cases, they may help to find out essential information.

Dynamics of Ultrafast Phase Transitions in (001) Si on the Shock-Wave Front.

We demonstrate an ultrafast (<0.1 ps) reversible phase transition in silicon (Si) under ultrafast pressure loading using molecular dynamics. Si changes its structure from cubic diamond to ß-Sn on the shock-wave front. The phase transition occurs when the shock-wave pressure exceeds 11 GPa. Atomic volume, centrosymmetry, and the X-ray-diffraction spectrum were revealed as effective indicators of phase-transition dynamics. The latter, being registered in actual experimental conditions, constitutes a breakthrough in the path towards simple X-ray optical cross-correlation and pump-probe experiments.

Conformational trapping of an ABC transporter in polymer lipid nanoparticles.

ATP-binding cassette (ABC) proteins play important roles in cells as importers and exporters but as membrane proteins they are subject to well-known challenges of isolating pure and stable samples for study. One solution to this problem is to use styrene-maleic acid lipid particles (SMALPs). Styrene-maleic acid (SMA) can be added directly to membranes, forming stable nanoparticles incorporating membrane proteins and lipids. Here we use Sav1866, a well-characterised bacterial protein, as a proxy for ABC proteins in general. We show that stable and monodispersed Sav1866 can be purified at high yield using SMA. This protein can be used for biophysical characterisations showing that its overall structure is consistent with existing evidence. However, like other ABC proteins in SMALPs it does not hydrolyse ATP. The lack of ATPase activity in ABC-SMALPs may result from conformational trapping of the proteins in SMALPs. Undertaken in a controlled manner, conformational trapping is a useful tool to stabilise protein samples into a single conformation for structural studies. Due to their inability to hydrolyse ATP, the conformation of Sav1866-SMALPs cannot be altered using ATP and vanadate after purification. To achieve controlled trapping of Sav1866-SMALPs we show that Sav1866 in crude membranes can be incubated with ATP, magnesium and sodium orthovanadate. Subsequent solubilisation and purification with SMA produces a sample of Sav1866-SMALPs with enhanced stability, and in a single conformational state. This method may be generally applicable to vanadate-sensitive ABC proteins and overcomes a limitation of the SMALP system for the study of this protein family.

Atomic structure of Lanreotide nanotubes revealed by cryo-EM.

Functional and versatile nano- and microassemblies formed by biological molecules are found at all levels of life, from cell organelles to full organisms. Understanding the chemical and physicochemical determinants guiding the formation of these assemblies is crucial not only to understand the biological processes they carry out but also to mimic nature. Among the synthetic peptides forming well-defined nanostructures, the octapeptide Lanreotide has been considered one of the best characterized, in terms of both the atomic structure and its self-assembly process. In the present work, we determined the atomic structure of Lanreotide nanotubes at 2.5-Å resolution by cryoelectron microscopy (cryo-EM). Surprisingly, the asymmetric unit in the nanotube contains eight copies of the peptide, forming two tetramers. There are thus eight different environments for the peptide, and eight different conformations in the nanotube. The structure built from the cryo-EM map is strikingly different from the molecular model, largely based on X-ray fiber diffraction, proposed 20 y ago. Comparison of the nanotube with a crystal structure at 0.83-Å resolution of a Lanreotide derivative highlights the polymorphism for this peptide family. This work shows once again that higher-order assemblies formed by even well-characterized small peptides are very difficult to predict.

X-ray diffraction and in vivo studies reveal the quinary structure of Trypanosoma cruzi nucleoside diphosphate kinase 1: a novel helical oligomer structure.

Trypanosoma cruzi is a flagellated protozoan parasite that causes Chagas disease, which represents a serious health problem in the Americas. Nucleoside diphosphate kinases (NDPKs) are key enzymes that are implicated in cellular energy management. TcNDPK1 is the canonical isoform in the T. cruzi parasite. TcNDPK1 has a cytosolic, perinuclear and nuclear distribution. It is also found in non-membrane-bound filaments adjacent to the nucleus. In the present work, X-ray diffraction and in vivo studies of TcNDPK1 are described. The structure reveals a novel, multi-hexameric, left-handed helical oligomer structure. The results of directed mutagenesis studies led to the conclusion that the microscopic TcNDPK1 granules observed in vivo in T. cruzi parasites are made up by the association of TcNDPK1 oligomers. In the absence of experimental data, analysis of the interactions in the X-ray structure of the TcNDPK1 oligomer suggests the probable assembly and disassembly steps: dimerization, assembly of the hexamer as a trimer of dimers, hexamer association to generate the left-handed helical oligomer structure and finally oligomer association in a parallel manner to form the microscopic TcNDPK1 filaments that are observed in vivo in T. cruzi parasites. Oligomer disassembly takes place on the binding of substrate in the active site of TcNDPK1, leading to dissociation of the hexamers. This study constitutes the first report of such a protein arrangement, which has never previously been seen for any protein or NDPK. Further studies are needed to determine its physiological role. However, it may suggest a paradigm for protein storage reflecting the complex mechanism of action of TcNDPK1.

Two states of a light-sensitive membrane protein captured at room temperature using thin-film sample mounts.

Room-temperature diffraction methods are highly desirable for dynamic studies of biological macromolecules, since they allow high-resolution structural data to be collected as proteins undergo conformational changes. For crystals grown in lipidic cubic phase (LCP), an extruder is commonly used to pass a stream of microcrystals through the X-ray beam; however, the sample quantities required for this method may be difficult to produce for many membrane proteins. A more sample-efficient environment was created using two layers of low X-ray transmittance polymer films to mount crystals of the archaerhodopsin-3 (AR3) photoreceptor and room-temperature diffraction data were acquired. By using transparent and opaque polymer films, two structures, one corresponding to the desensitized, dark-adapted (DA) state and the other to the ground or light-adapted (LA) state, were solved to better than 1.9 Šresolution. All of the key structural features of AR3 were resolved, including the retinal chromophore, which is present as the 13-cis isomer in the DA state and as the all-trans isomer in the LA state. The film-sandwich sample environment enables diffraction data to be recorded at room temperature in both illuminated and dark conditions, which more closely approximate those in vivo. This simple approach is applicable to a wide range of membrane proteins crystallized in LCP and light-sensitive samples in general at synchrotron and laboratory X-ray sources.

Recent advances in small-angle scattering and its expanding impact in structural biology.

Applications of small-angle scattering (SAS) in structural biology have benefited from continuing developments in instrumentation, tools for data analysis, modeling capabilities, standards for data and model presentation, and data archiving. The interplay of these capabilities has enabled SAS to contribute to advances in structural biology as the field pushes the boundaries in studies of biomolecular complexes and assemblies as large as whole cells, membrane proteins in lipid environments, and dynamic systems on time scales ranging from femtoseconds to hours. This review covers some of the important advances in biomolecular SAS capabilities for structural biology focused on over the last 5 years and presents highlights of recent applications that demonstrate how the technique is exploring new territories.

Biofabrication of ZnO nanoparticles using Acacia arabica leaf extract and their antibiofilm and antioxidant potential against foodborne pathogens.

Emergence of multidrug resistant pathogens is increasing globally at an alarming rate with a need to discover novel and effective methods to cope infections due to these pathogens. Green nanoparticles have gained attention to be used as efficient therapeutic agents because of their safety and reliability. In the present study, we prepared zinc oxide nanoparticles (ZnO NPs) from aqueous leaf extract of Acacia arabica. The nanoparticles produced were characterized through UV-Visible spectroscopy, scanning electron microscopy, and X-ray diffraction. In vitro antibacterial susceptibility testing against foodborne pathogens was done by agar well diffusion, growth kinetics and broth microdilution assays. Effect of ZnO NPs on biofilm formation (both qualitatively and quantitatively) and exopolysaccharide (EPS) production was also determined. Antioxidant potential of green synthesized nanoparticles was detected by DPPH radical scavenging assay. The cytotoxicity studies of nanoparticles were also performed against HeLa cell lines. The results revealed that diameter of zones of inhibition against foodborne pathogens was found to be 16-30 nm, whereas the values of MIC and MBC ranged between 31.25-62.5 µg/ml. Growth kinetics revealed nanoparticles bactericidal potential after 3 hours incubation at 2 × MIC for E. coli while for S. aureus and S. enterica reached after 2 hours of incubation at 2 × MIC, 4 × MIC, and 8 × MIC. 32.5-71.0% inhibition was observed for biofilm formation. Almost 50.6-65.1% (wet weight) and 44.6-57.8% (dry weight) of EPS production was decreased after treatment with sub-inhibitory concentrations of nanoparticles. Radical scavenging potential of nanoparticles increased in a dose dependent manner and value ranged from 19.25 to 73.15%. Whereas cytotoxicity studies revealed non-toxic nature of nanoparticles at the concentrations tested. The present study suggests that green synthesized ZnO NPs can substitute chemical drugs against antibiotic resistant foodborne pathogens.

N-terminal phosphorylation regulates the activity of glycogen synthase kinase 3 from Plasmodium falciparum.

As the decline of malaria cases stalled over the last five years, novel targets in Plasmodium falciparum are necessary for the development of new drugs. Glycogen Synthase Kinase (PfGSK3) has been identified as a potential target, since its selective inhibitors were shown to disrupt the parasitès life cycle. In the uncanonical N-terminal region of the parasite enzyme, we identified several autophosphorylation sites and probed their role in activity regulation of PfGSK3. By combining molecular modeling with experimental small-angle X-ray scattering data, we show that increased PfGSK3 activity is promoted by conformational changes in the PfGSK3 N-terminus, triggered by N-terminal phosphorylation. Our work provides novel insights into the structure and regulation of the malarial PfGSK3.

[Detection and Analysis of Drug Crystals in Medical Transdermal Patches by Using X-ray Diffraction Measurement].

The crystallization of active pharmaceutical ingredients (APIs) in matrix-type transdermal patches has implications for the rate of drug absorption through the skin and patch adhesion strength. Therefore, the presence or absence and the degree of API crystallinity must be controlled to guarantee the quality of patches. In this study, the utility of laboratory-level X-ray diffractometers for the detection and analysis of crystalline APIs in transdermal patches was investigated using medical patches of tulobuterol and isosorbide dinitrate. Several matrix-type patches employ a controlled drug delivery system containing intentionally crystallized API. Both benchtop and high-resolution laboratory X-ray diffractometers can detect several characteristic peaks of the APIs in these patches even if the patches are wrapped in an outer bag, although a benchtop model provides peak heights one-seventh to one-fifth that of a high-resolution instrument. An isosorbide dinitrate patch containing an unintentionally crystallized spot was wrapped in an outer bag, followed by measurements using both X-ray diffractometers. For both instruments, several isosorbide dinitrate-derived peaks were detected only at the crystallized spot, although the signal-to-noise ratio was poorer for the benchtop model. These results show that a high-resolution X-ray diffractometer is advantageous for high-detection sensitivity and offers a high degree of freedom of the measurement position on the sample. It was concluded that a laboratory-level high-resolution X-ray diffractometer can be used to examine the crystalline state of APIs in patches inside an unopened outer bag.

Improved water dispersion and bioavailability of coenzyme Q10 by bacterial cellulose nanofibers.

The purpose of this study was to investigate the potential of bacterial cellulose nanofiber suspension (BCNs) as stabilizer in anti-solvent precipitation and its effect on improving bioavailability of coenzyme Q10. Bacterial cellulose (BC) was hydrolyzed by sulfuric acid followed by the oxidation with hydrogen peroxide to prepare BCNs. The suspension of BCNs-loaded CoQ10 (CoQ10-BCNs) were prepared by antisolvent precipitation. The zeta potential of CoQ10-BCNs was about -36.01 mV. The properties of CoQ10, BCNs and CoQ10-BCNs were studied by scanning electron microscopy, transmission electron microscope, Fourier-transform infrared spectroscopy, X-ray diffraction, differential scanning calorimetry and thermo gravimetric analysis. The crystallinity of CoQ10 decreased in CoQ10-BCNs compared with the raw CoQ10, and CoQ10-BCNs have good physicochemical stability. In oral bioavailability studies, the area under curve (AUC) of CoQ10-BCNs was about 3.62 times higher than the raw CoQ10 in rats.

Preparation and structural properties of starch phosphate modified by alkaline phosphatase.

In this study, a method for the synthesis of starch phosphate using the transferase properties of alkaline phosphatase was explored. Maize starch was treated with a pyrophosphate solution containing alkaline phosphatase and catalytic ions under pH 8 at 37 °C. The synthesis of starch phosphate was evaluated and compared with untreated and treated starch controls. The phosphorus content of the samples increased up to 8500% with the catalytic ion concentration, whereas the peak viscosity by up to 41.4% decreased. The crystallinity and enthalpy of the phosphorylated samples were reduced by up to 26.8% and 23.3%, respectively; however, no significant was observed by Fourier-transform infrared spectrometer. The roughness of the starch surface and the distribution of elemental phosphorus were observed by scanning electron microscopy and energy dispersive Spectrometry. X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry results further indicated the grafting of the phosphate radical.

Functionalization of cellulose fibers alongside growth of 2D LDH platelets through urea hydrolysis inspired Taro wettability.

Here, Cotton fabric was functionalized via hybrid coating including 2D MgAl LDH (layered double hydroxide) and SA (stearic acid). The urea hydrolysis was employed for construction of vertically aligned LDH on surface of cellulose fibers under hydrothermal condition. The in situ formation mechanism of LDH on cotton surface was nucleation, growth, and interaction with activated cellulose chains. The partial cellulose ionization in the alkaline solution led to nucleophilic behavior towards electron deficient atom. The effect of different ageing, synthesis temperature, and amount of SA were investigated and optimized at 100 °C for 24 h with 0.05 M. The superhydrophobic surface architecture of treated cotton with hierarchical micro/nanostructure was inspired from the Taro leaf structure with continuous contact line presented WCA of 154 ° and CAH of 9 °. The Cotton@LDH@SA exhibited efficient oil/water separation after several washes (>90%) with good stain resistant. Also, the physico-mechanical properties were studied.

Dissolution of cellulose into supercritical water and its dissolving state followed by structure formation from the solution system.

Cellulose was treated with supercritical water at 668 K and 25 MPa for 0.04 s in this study. The cellulose/water system was transparent at room temperature for a while after supercritical water treatment before a precipitate gradually appeared over several hours. The precipitation process was monitored by synchrotron X-ray scattering. The scattering functions of fractal systems and flat-like structures were utilized to explain the experimentally observed small-angle scattering profiles. Immediately after supercritical water treatment, the cellulose appeared to dissolve with a fractal dimension D of approximately 1, indicating that the cellulose molecules were rigid, followed by aggregation into a 5-nm-thick flat-like structure. The flat-like structure was determined to be similar to the molecular sheets observed during the early stages of precipitation in the cellulose/aqueous sodium hydroxide and cellulose/aqueous lithium hydroxide/urea systems. Resultant regenerated cellulose had high crystallinity, large crystal size, and a low degree of polymerization.

Kinetics of denaturation and renaturation processes of double-stranded helical polysaccharide, xanthan in aqueous sodium chloride.

Circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements were made for three xanthan samples, a double helical polysaccharide, in 5 or 10 mM aqueous NaCl after rapid temperature change to investigate the kinetics of the conformational change between the ordered and disordered states. After the rapid heating, the CD signal mainly reflecting the carbonyl groups on the side chains quickly changed (<150 s) while the scattering intensity from SAXS around q (magnitude of the scattering vector) = 1 nm-1 changed more gradually, reflecting the main-chain conformation. The difference between CD and SAXS implies us the intermediate conformation which can be regarded as a loose double helix. The SAXS profile in the rapid cooling process showed that the loose double helical structure was constructed within 150 s, but the CD signal slowly changed with around 2 days to recover the native tight double helix.