Dimeric Aggregates Research Articles

Number and brightness analysis to study spatio-temporal distribution of the angiotensin II AT1 and the endothelin-1 ETA receptors: Influence of ligand binding

The angiotensin II AT1 and the endothelin 1 ETA receptors play a crucial role in the pathogenesis of cardiovascular diseases like hypertension, heart failure, stroke, pulmonary hypertension, and cardiac hypertrophy. Both receptors are members of the rhodopsion-like superfamily of G protein-coupled receptors which can exist as monomers, dimers, and higher order aggregates.Recently, oligomerization of these two receptors have been described by several biophysical methods based mainly on luminescence and fluorescence energy transfer. Since this oligomerization can occur either spontaneously or it can be induced by ligand-binding, the aim of this work was to address whether the oligomerization of these receptors occurs upon ligand-binding. For this purpose the Number and Brightness analysis, a method that allows the identification, localization, and quantification of protein aggregates in the plasma membrane of a single cell, was used. An advantage of this method is that it is not limited to certain dyes specially required for Fluorescence Resonance Energy Transfer measurements.Our results showed that stably transfected angiotensin II AT1 receptors and transiently transfected endothelin 1 ETA receptors, were found as monomeric, dimeric, and tetrameric receptor aggregates. Interestingly, the binding of antihypertensive agents like losartan and BQ123, earlier suggested to be inverse agonists, significantly increased the proportion of monomers and reduced the occurrence of dimers on the cell membrane; while the kown endothelin 1 ETA antagonist sitaxentan did not influence the aggregation state of these receptors.

A Self-Assembled Unit Comprising 12 Squaraine Dyes Built Up from Two Star-Shaped Hexasquarainyl-Benzene Molecules.

We describe the aggregate formation and optical properties of a star-shaped hexaarylbenzene with six squaraine chromophores (=hexasquarainyl benzene). Comprehensive concentration-dependence studies in acetone/CHCl3 mixtures reveal a strong propensity to form discrete dimeric aggregates with a high binding constant in excess of 106 m-1 . In this context, a large hypsochromic shift of almost 2700 cm-1 was found in the absorption spectrum, indicating H-type exciton coupling. The aggregate band is characterised by a very small band width of only 560 cm-1 , probably caused by exchange narrowing. Both experimental and computational methods were used to elucidate the supramolecular aggregate structure, which is assumed to consist of two stacked hexasquarainyl benzene monomers.

Intermolecular Hydrogen Bond in Acetic Acid Solutions. Raman Spectra and ab initio Calculations

C=O vibrational bands of various aggregated formations of pure acetic acid in the Raman spectra have different values of the depolarization ratio. Ab initio calculations using the Gaussian 98 W program in the Hartree-Fock approximation with the basic set of Gaussian functions RHF 6-31G++(d, p) show that this is how it should be. Thus, the formation of aggregates from molecules leads not only to a change in the dipole moment of the molecules, but also to a change in the bond polarizability tensor. Calculations according to this program showed that one of the hydrogen atoms of CH3 group of acetonitrile can participate in the intermolecular hydrogen bond. This can lead to the formation of closed acetonitrile-acetic acid dimers. However, a comparison of the calculated and experimental data shows that in the case of acid-acetonitrile molecules, the H-bond is formed in the direction of the elongation –О-Н…N of acetonitrile molecules. The activity of one of the hydrogen atoms of the CH3 group of acetonitrile leads to the formation of closed dimeric aggregates in pure acetonitrile with a shift of the СN band of acetonitrile towards lower frequencies. The low-frequency asymmetry of СN acetonitrile band in the Raman spectra is associated with the presence of such aggregates in liquid acetonitrile.

Mechanism of Pseudomonas aeruginosa Small Protease (PASP), a Corneal Virulence Factor.

PurposePseudomonas aeruginosa is the leading cause of contact lens–associated bacterial keratitis. Secreted bacterial proteases have a key role in keratitis, including the P. aeruginosa small protease (PASP), a proven corneal virulence factor. We investigated the mechanism of PASP and its importance to corneal toxicity.MethodsPASP, a serine protease, was tested for activity on various substrates. The catalytic triad of PASP was sought by bioinformatic analysis and site-directed mutagenesis. All mutant constructs were expressed in a P. aeruginosa PASP-deficient strain; the resulting proteins were purified using ion-exchange, gel filtration, or affinity chromatography; and the proteolytic activity was assessed by gelatin zymography and a fluorometric assay. The purified PASP proteins with single amino acid changes were injected into rabbit corneas to determine their pathological effects.ResultsPASP substrates were cleaved at arginine or lysine residues. Alanine substitution of PASP residues Asp-29, His-34, or Ser-47 eliminated protease activity, whereas PASP with substitution for Ser-59 (control) retained activity. Computer modeling and Western blot analysis indicated that formation of a catalytic triad required dimer formation, and zymography demonstrated the protease activity of the homodimer, but not the monomer. PASP with the Ser-47 mutation, but not with the control mutation, lacked corneal toxicity, indicating the importance of protease activity.ConclusionsPASP is a secreted serine protease that can cleave proteins at arginine or lysine residues and PASP activity requires dimer or larger aggregates to create a functional active site. Most importantly, proteolytic PASP molecules demonstrated highly significant toxicity for the rabbit cornea.

Validity of density-functional-theory-based molecular modeling for UV/visible spectroscopy and rationale of panchromatic PbI64−(MeNH3+)4-structured molecular solar cells

Density-functional-theory-based molecular modeling verifies that perovskite solar cells (PSC) are composed of semiconducting and panchromatic layers constructed by van der Waals and Coulomb interactions (vdW&Clmb) between PbI64−(MeNH3+)4-derived species. Firstly we validate that DFT-based UV/vis spectral analysis is a useful approach for vdW&Clmb based on assignments of the fine-structured UV/vis spectrum of a benzene solution. The UV/vis spectral analysis of aggregated PbI64−(MeNH3+)4 proves that dimeric aggregates of [PbI64−(MeNH3+)4]2 have a panchromatic UV/vis spectrum of λ max ranging from 417 to 959 nm. Further analysis proves that the strong vdW&Clmb of PbI64−(MeNH3+)4 leads to unidirectional electron transport at the structured components such as the dimer of [PbI64−(MeNH3+)4]2, nc-TiO2/PbI64−(MeNH3+)4, and PbI64−(MeNH3+)4/spiro-OMeTAD. PbI64−(MeNH3+)4-structured solar cells should have a photoelectron diffusion length enhanced by the alignment of the frontier molecular orbitals in the structured PbI64−(MeNH3+)4 components, which supports remarkable short-circuit photocurrent, open-circuit voltage, and fill factor for the molecular-orbital-connected solar cell of HOTi9O18H/[PbI64−(MeNH3+)4]n/spiro-OMeTAD under solar-light irradiance.

Interaction of a dimeric carbocyanine dye aggregate with bovine serum albumin in non-aggregated and aggregated forms.

The interaction of fluorescent dyes with serum proteins has garnered significant interest owing to potential for non-covalent labeling and imaging applications. In this work, dimeric benzothiazole-based trimethine cyanine dyes are synthesized and their interaction with bovine serum albumin studied. The dimeric cyanine dyes mainly exist as H-dimers and H-aggregates in aqueous solution. A combination of absorbance, fluorescence, circular dichroism spectroscopy and atomic force and fluorescence microscopy indicate the formation of dye-BSA complexes. Binding of one of the dimeric dyes on BSA with a Ka of 1.49×105M-1 results in disruption of dye self-aggregates and unfolding of the dyes into the monomeric or open conformation. Fluorescence enhancement experienced by the dimeric dyes upon interaction with BSA is superior to that registered by Thioflavin T. Surfactant SDS has been used to further tune the self-aggregation of the dimeric dye resulting in a 200-fold fluorescence enhancement in presence of BSA. Interaction of a dimeric dye with BSA under conditions favoring protein aggregation is found to result in faster dye binding and the resulting fluorescence enhancement is easily visualized by fluorescence microscopy. The interaction of a dimeric cyanine dye aggregate with BSA is promising for non-covalent labeling applications in sharp contrast to the monomeric dye counterpart.

Aggregation of slipped-cofacial phthalocyanine J-type dimers: Spectroscopic and AFM study

Direct metallation of 2-hydroxyphthalocyanine J-type slipped-cofacial dimeric ligand by Mg, Zn, Cu, Ni and Co salts has been carried out to obtain corresponding metal complexes selectively without any noticeable dissociation or polymerization of the starting ligand. Integrated analysis of aggregation properties in the synthesized series has been conducted with the involvement of AFM microscopy, UV/Vis spectroscopy and theoretical assessment. As a result, a nonlinear relationship between absorption and concentration was found, with aggregation beginning to appear at concentrations above 3.3 × 10−5 mol L−1 with predominant formation of trimers from the dimeric molecules in THF solutions.

On the Binding Free Energy and Molecular Origin of Sickle Cell Hemoglobin Aggregation.

Protein aggregation is associated with various diseases, including Alzheimer and Parkinson as well as sickle cell disease (SCD). From a molecular point of view, protein aggregation depends on a complex balance of electrostatic and hydrophobic interactions mediated by water. An impressive manifestation of the importance of this balance concerns the human hemoglobin (HbA) mutant, HbS (sickle cell Hb), where a single substitution at the 6th position of HbA β-chains, from glutamic acid to valine, causes the polymerization of deoxygenated HbS (deoxy-HbS), responsible for SCD. HbS polymerization is believed to occur via a double nucleation mechanism initiated by the formation of HbS fibers (homogeneous nucleation), followed by fiber growth. Furthermore, it was proposed that homogeneous nucleation proceeds through a two-step mechanism, where metastable dense clusters play the role of nucleation precursors. Thus, hindering or delaying the formation of such precursors could represent a potential SCD therapeutic route. Here, we study, through molecular dynamics, the binding free energy and protein-protein contacts involved in the deoxy-HbS dimer aggregation and stabilization process. A binding free energy of ∼-14.0 ± 1 kcal/mol is estimated from a one-dimensional potential of mean force. Analysis of protein-protein interactions shows that both electrostatic and van der Waals interactions play an important role on the aggregation of HbS. With respect to the former, our results indicate that aggregation is largely favored by the formation of salt bridges (SB), mostly, Lys-Glu, Lys-Asp, and Heme-Lys SB, which outweigh electrostatic repulsions involving similar residues. Thus, our results suggest that a potential antisickling drug could be one with the ability to weaken or hinder the formation of a few SB between carboxylate and ammonium groups.

Twisted Linker Effect on Naphthalene Diimide-Based Dimer Electron Acceptors for Non-fullerene Organic Solar Cells.

Naphthalene diimide (NDI) dimers, NDI-Ph-NDI with a phenyl linker and NDI-Xy-NDI with a xylene linker, are designed and synthesized. The influence of the xylene and phenyl linkers on optical properties, electrochemical properties, morphology, and device performance is systematically investigated. Non-fullerene organic solar cells (OSCs) with NDI-Ph-NDI show poor device efficiency due to aggregation of polymer chains and/or NDI dimers caused by the highly planar structure of NDI-Ph-NDI. Although NDI-Xy-NDI is a non-planar structure, uniform surface morphology and weak bimolecular recombination lead to high power conversion efficiencies of 3.11%, which is the highest value in non-fullerene OSCs with NDI small molecules.

Coherent random lasing controlled by Brownian motion of the active scatterer

The stability of the scattering loop is fundamental for coherent random lasing in a dynamic scattering system. In this work, fluorescence of DPP (N, N-di [3-(isobutyl polyhedral oligomeric silsesquioxanes) propyl] perylene diimide) is scattered to produce RL and we realize the transition from incoherent RL to coherent RL by controlling the Brownian motion of the scatterers (dimer aggregates of DPP) and the stability of scattering loop. To produce coherent random lasers, the loop needs to maintain a stable state within the loop-stable time, which can be determined through controlled Brownian motion of scatterers in the scattering system. The result shows that the loop-stable time is within 5.83 × 10−5 s to 1.61 × 10−4 s based on the transition from coherent to incoherent random lasing. The time range could be tuned by finely controlling the viscosity of the solution. This work not only develops a method to predict the loop-stable time, but also develops the study between Brownian motion and random lasers, which opens the road to a variety of novel interdisciplinary investigations involving modern statistical mechanics and disordered photonics.

Antibody engineering to improve manufacturability

Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies. To overcome the manufacturability issues, LC shuffling was performed with the original HC to select antibodies with unique LCs which retained acceptable binding affinities. Transient CHO-K1 expression evaluation of the new LCs co-expressed with the original HC identified antibodies with high expression at a 1:1 or 2:1 LC:HC; the expression levels of these new antibodies were comparable to those of other well-expressed antibodies. Expression of these new antibodies in stably transfected CHO-K1 cells confirmed these results. In addition, antibodies containing the new LCs had very low levels of high molecular weight aggregates and HC dimer. These results demonstrate that certain antibody manufacturability issues can be attributed to LC and that engineering antibodies by pairing HCs with alternate LCs can improve antibody expression and product quality while maintaining or improving affinity.

DIMERIC MOLECULAR AGGREGATION MOTIF IN CRYSTAL OF 2,7-DIETHOXY-1-(4-NITROBENZOYL)NAPHTHALENE: CORRELATION OF SINGLE MOLECULAR STRUCTURE, MOLECULAR ACCUMULATION STRUCTURE AND NON-COVALENT-BONDING INTERACTIONS

Crystal structure of 2,7-diethoxy-1-(4-nitrobenzoyl)naphthalene, C 21 H 19 NO 5 , is reported and discussed on the characteristics of the spatial organization of single molecule and molecular aggregation as contrasted with a homologous compound. The molecular structures of these compounds differ only in the kind of alkoxy group of 2,7-positions of naphthalene rings, i.e. , ethoxy groups for title compound and methoxy ones for homologue. In single molecular crystal structure, the 4-nitrobenzoyl groups of these molecules are attached non-coplanarly to the naphthalene ring. The congested situation makes bonds connecting naphthalene ring and carbonyl group fixed to turn stereogenetic, which allow the independent existence of atrope stereoisomers of ( R )- and ( S )-enantiomeric conformer molecules, contrary to their solute state where the fast interconversion disturbs the distinction of the enantiomeric molecules. The two pairs of the enantiomeric molecules are related by two-fold helical axis in the asymmetric unit of P 2 1 /n space group for title compound and P 2 1 /c for homologous compound, exhibiting the number of molecules ( Z ) is four for both compounds. In crystal of title compound, ( R )- and ( S )-enantiomers are connected to each other by pi…pi stacking interaction and two types of C–H…O=N non-classical hydrogen bonds, (sp 2 )C–H…O=N and (sp 3 )C–H…O=N non-classical hydrogen bonds along b -axis, forming centrosymmetric dimeric molecular aggregates. The dimeric units are stacked into columnar structure by (sp 2 )C–H…O=C non-classical hydrogen bonds between molecular unit of identical enantiomeric configuration along a -axis. The columns are also connected by (sp 2 )C–H…OEt non-classical hydrogen bonds between molecular unit of identical enantiomeric configuration along c -axis to give sheet-like aggregate composed of molecules of same enantiomeric configuration spreading on ac -plane. The sheets are piled up through (sp 3 )C–H…pi non-classical hydrogen bonds between opposite enantiomeric molecular units of next dimeric aggregates along b -axis. In crystal of the homologous compound, 2,7-dimethoxy-1-(4-nitrobenzoyl)naphthalene, centrosymmetric dimeric aggregate resulted from the association of ( R )- and ( S )-enantiomers via pi…pi stacking interactions are also observed. The centrosymmetric dimeric aggregates are unidirectionally lined by two kinds of non-classical hydrogen bonds between molecular unit of identical enantiomeric configuration, (sp 3 )C–H…O=C and (sp 3 )C–H…pi non-classical hydrogen bonds, giving columnar structure. The columns are accumulated giving a wavy sheet structure composed of stripes of respective enantiomeric configuration oriented alternatively and anti-parallelly through weak (sp 2 )C–H…pi non-classical hydrogen bonds. The difference of higher ordered structure between title compound and homologue is plausibly explained according to one CH 2 margin in 2,7-dialkoxy groups, i.e. , elongation of the least length in alkyl group of title compound compared to homologue brings about the sterically significant hindrance among dimeric aggregates that makes rather anisotropic intermolecular non-covalent bonding interactions resulting in accumulating sheet structure. For 1-monoaroylnaphthalene compounds, the most stabilized single molecular structure on condition that the sufficiently effective intermolecular interaction is absent is proposed the perpendicular alignment of naphthalene and benzene rings to prevent the steric repulsion of two aromatic rings. The smaller spatial volume of methyl group in homologue molecule is plausibly able to be merged without large alternation in the single molecular structure most stabilized. Contrarily, the additional methylene unit at 2,7-alkoxy group probably requires the rather large perturbation from the supposed structure most stabilized.

2,2'-Bipyridin-1'-ium 1-oxide bromide monohydrate.

The title compound 2,2'-bipyridin-1'-ium 1-oxide bromide crystallizes as a monohydrate, C10H9N2+·Br-·H2O. Structural disorder is observed due to the fact that protonation, as well as oxidation, of the N atoms of 2,2'-bi-pyridine occurs at either of the N atoms. The disorder extends to the remainder of the cation, with a refined occupancy rate of 0.717 (4) for the major moiety. An intra-molecular N-H⋯O hydrogen bond forces the bi-pyridine unit into an s-cis conformation. Each pair of neighbouring 2,2'-bipyridin-1'-ium ions forms a dimeric aggregate by hydrogen bonds between their respective N-O and the N-H functions. These dimers and hydrogen-bonding inter-actions with bromide ions and the water mol-ecule give rise to a complex supra-molecular arrangement.

Preparation and SERS performance of Au NP/paper strips based on inkjet printing and seed mediated growth: The effect of silver ions

Surface-enhanced Raman scattering (SERS) has been widely used in biomedical sensing with the advantages of high sensitivity and label-free. However, the fabrication of SERS substrates with good Raman activity, reproducibility, and low cost is still under development in practical applications. This paper presents a practicable method for fabricating Au NP/paper strips by using inkjet printing and seed mediated growth. Small gold seed synthesized by borohydride reduction was used as ink and printed on the filter paper. The printed gold seed grew in situ in the growth solution and formed the gold nanoparticle (Au NP)/paper strips. The fabricated paper strip was characterized by diffuse reflectance spectroscopy and scanning electron microscopy (SEM). The diffuse reflectance spectra indicated that the Au NP/paper strips had two local surface plasmon resonance (LSPR) peaks: the short one at around 540 nm and the long one located in the range of 640–840 nm. And the long LSPR peak firstly shifted to red then to blue with the increased concentrations of silver ions in growth solution. From the SEM images, the shape of grown Au NPs was diverse, including sphere, rod, ellipsoid, dimer, trimer, and big aggregates. We thought the short peak came from the LSPR of nanospheres and the transvers LSPR of rod and ellipsoid like particles, while the long peak mainly came from the plasmonic coupling of dimer along the inter-particle axis. The obtained Au NP/paper strip with the long peak located around 650 nm had the highest SERS activity, which could be attributed to the plasmon resonance induced local field enhancement and nanogap effect. Also, the SERS performance results indicated the printed SERS strips exhibited satisfied uniformity and stability, demonstrating the potential of Au NP/paper strip in real-world applications.

Pentavalent phosphorus as a unique phosphorus donor in POCl3 homodimer and POCl3-H2O heterodimer: matrix isolation infrared spectroscopic and computational studies.

Phosphorus, an important element among the pnicogen group, opens up avenues for experimental and computational explorations of its interaction in a variety of compounds. Although experimental proof of trivalent phosphorus bonding is limited and is growing with time, phosphorus bonding with pentavalent phosphorus has been a long sought after interaction both computationally and experimentally. In the present work, for the first time, we have provided unambiguous experimental evidence for the pentavalent phosphorus bonding interaction by exploiting a phosphoryl chloride (POCl3) prototype under isolated conditions at low temperatures. The POCl3 dimer and higher aggregates can be set as a unique example possessing pentavalent phosphorus bonding with a competing halogen bonding interaction. The POCl3-H2O heterodimer is another interesting system, stabilized through multiple phosphorus and hydrogen bonded interactions. Using matrix isolation infrared spectroscopy, the POCl3 homodimer and POCl3-H2O heterodimer were characterized and the structures were elucidated by employing ab initio and DFT methods. The multifaceted interactions in the POCl3 paradigm were investigated by using Natural Bond Orbital, Energy Decomposition and Electrostatic Potential Mapping analyses.

Aggregation and metal-complexation behaviour of THPP porphyrin in ethanol/water solutions as function of pH

The effect of pH change on 5,10,15,20-Tetrakis(4-hydroxyphenyl)-21H,23H-porphine (THPP) with its aggregation as function of water-ethanol mixture was studied with UV–vis, fluorescence, Raman and computational analysis. In neutral pH, THPP was present as free-base and, increasing the water amount, aggregation occurred with the formation of H- and J-aggregates. The aggregation constant and the concentration of dimers were calculated, other information about the dimer aggregation were evaluated by computational study.In acidic pH, by the insertions of two hydrogens in the porphyrin rings, the porphyrin changed its geometry with a ring deformation confirmed by red-shifted spectrum and quenching in fluorescence; at this low pH, increasing the water amount, the acidic form (THPPH2)2+ resulted more stable due to a polar environment with stronger interaction by hydrogen bonding.In basic pH, reached by NH4OH, THPP porphyrin was able to react with alkali metals in order to form sitting-atop complex (M2THPP) confirmed by the typical absorption spectrum of metallo-porphyrin, Raman spectroscopy and by computational analysis.

Influence of cavitation and high shear stress on HSA aggregation behavior.

Neither the influence of high shear rates nor the impact of cavitation on protein aggregation is fully understood. The effect of cavitation bubble collapse‐derived hydroxyl radicals on the aggregation behavior of human serum albumin (HSA) was investigated. Radicals were generated by pumping through a micro‐orifice, ultra‐sonication, or chemically by Fenton's reaction. The amount of radicals produced by the two mechanical methods (0.12 and 11.25 nmol/(L min)) was not enough to change the protein integrity. In contrast, Fenton's reaction resulted in 382 nmol/(L min) of radicals, inducing protein aggregation. However, the micro‐orifice promoted the formation of soluble dimeric HSA aggregates. A validated computational fluid dynamic model of the orifice revealed a maximum and average shear rate on the order of 108 s−1 and 1.2 × 106 s−1, respectively. Although these values are among the highest ever reported in the literature, dimer formation did not occur when we used the same flow rate but suppressed cavitation. Therefore, aggregation is most likely caused by the increased surface area due to cavitation‐mediated bubble growth, not by hydroxyl radical release or shear stress as often reported.

Photophysics of Zinc Porphyrin Aggregates in Dilute Water-Ethanol Solutions.

Dimeric and multimeric aggregates of a model metalloporphyrin, zinc tetraphenylporphyrin (ZnTPP), have been produced in a controlled manner by incrementally increasing the water content of dilute aqueous ethanol solutions. Steady state absorption, fluorescence emission, and fluorescence excitation spectra have been measured to identify the aggregates present as a function of solvent composition. The dynamics of the excited states of the aggregates produced initially by excitation in the Soret region have been measured by ultrafast fluorescence upconversion techniques. Only the monomer produces measurable emission from S2 with a picosecond lifetime; all Soret-excited aggregates, including the dimer, decay radiationlessly on a femtosecond time scale. The S1 state is the only significant product of the radiationless decay of the S2 state of the excited monomer, and the aggregates also produce substantial quantum yields of S1 fluorescence when initially excited in the Soret region. The resulting fluorescent aggregates all decay on a subnanosecond time scale, likely by a mechanism that involves dissociation of the excited monomer from the excitonic multimer. The ZnTPP dimers excited at their ground state geometries in the Soret region exhibit a dynamic behavior that is quite different from those produced following noncoherent triplet-triplet annihilation under the same conditions. The important implications of these observations in determining the aggregation conditions promoting efficient photon upconversion by excitonic annihilation in a variety of media are thoroughly discussed.

A new monoclinic polymorph of N-(3-methyl-phen-yl)eth-oxy-carbo-thio-amide: crystal structure and Hirshfeld surface analysis.

The title compound, C10H13NOS, is a second monoclinic polymorph (space group P21/c, Z' = 2) of the previously reported C2/c (Z = 1) polymorph [Tadbuppa & Tiekink (2005 ▸). Z. Kristallogr. New Cryst. Struct. 220, 395-396]. Two independent mol-ecules comprise the asymmetric unit of the new polymorph and each of these exists as a thioamide-thione tautomer. In each molecule, the central CNOS chromophore is strictly planar [r.m.s. deviations = 0.0003 and 0.0015 Å] and forms dihedral angles of 6.17 (5) and 20.78 (5)° with the N-bound 3-tolyl rings, thereby representing the major difference between the mol-ecules. The thione-S and thio-amide-N-H atoms are syn in each mol-ecule and this facilitates the formation of an eight-membered thio-amide {⋯SCNH}2 synthon between them; the dimeric aggregates are consolidated by pairwise 3-tolyl-C-H⋯S inter-actions. In the extended structure, supra-molecular layers parallel to (102) are formed via a combination of 3-tolyl-C-H⋯π(3-tol-yl) and weak π-π inter-actions [inter-centroid distance between 3-tolyl rings = 3.8535 (12) Å]. An analysis of the Hirshfeld surfaces calculated for both polymorphs reveals the near equivalence of one of the independent mol-ecules of the P21/c form to that in the C2/c form.

Electron Microscopy Visualization of Vitronectin Adsorbed on COOH and NH2 Functionalized Surfaces: Distinctive Spatial Alignment and Regulated Cellular Responses

AbstractAdsorption of proteins associating with their conformational changes plays crucial roles in regulating biomaterial–cell interactions and consequent tissue responses to implanted biomaterials. This study reports direct visualization of typical serum protein, vitronectin, one of the key adhesive proteins that participate in mediating cell behaviors, upon adsorption on typically designed surfaces. Carbon films with their surfaces being plasma grafted functional groups COOH and NH2 are used as the model substrata for this study. Negative‐staining electron microscopy technique is employed for visualizing the adsorbed protein and 2D image classification is made and interpreted. Results show that adsorbed vitronectin tends to form multimer aggregate on the COOH‐grafted surfaces, exposing extensively its cell‐binding RGD (arginine‐glycine‐aspartic acid) motif for enhanced cell adhesion. The adsorbed vitronectin on the NH2‐grafted surface forms dimer aggregate with the binding sites being enwrapped. The COOH‐grafting triggers enhanced expressions of ITGA5, ITGAV, ITGB1, and ITGB3 of the adhered cells and this is likely attributed to the special spatial alignment of vitronectin upon adsorption. The conformational information of adsorbed vitronectin gained from the single particle electron microscopy analyses would shed light on design and construction of appropriate biomaterials surfaces for desired cellular behaviors.