Influence of the linkage type between the polymer backbone and side groups on the surface segregation of methyl groups during film formation.

Although poly(vinyl acetate) (PVAc) differs from poly(methyl acrylate) (PMA) only in the reversed position of the ester group, a large difference in the concentration dependence of the casting solution on the corresponding surface structure of the cast films of PVAc, PMA and poly(methyl methacrylate) (PMMA) was observed. The hydrophobicity of both PMA and PMMA films increased with increasing concentration of the corresponding polymer solution, whereas cast PVAc films showed the reverse trend. The surface structure of the cast films prepared with different concentrations of the casting solution, characterized by sum frequency generation (SFG) vibrational spectra, showed that the order of the methylene groups increased while that of the acetyl methyl group decreased on the surface of cast PVAc film with increasing concentration of casting solution. However, the order of the ester methyl group increased and that of methylene groups did not change for cast PMA films with increasing concentration of casting solution. The cast PMMA film showed a reverse trend compared with the corresponding PMA film. It is apparent that well-ordered ester or acetyl methyl groups on the surface, which are oriented away from the polymer film, rather than methylene groups, play an important role in determining surface hydrophobicity, as the latter shield the OC[double bond, length as m-dash]O groups of PVAc, PMA and PMMA film surfaces from being exposed, resulting in low surface free energy. The reason for this difference is attributed to the relatively low energy for ester methyl group reorientation, an ester group structure nearer to the trans state and more regular local configuration of segments in concentrated solutions of PMA and PMMA compared to that of PVAc.

Stick-slip phenomenon in measurements of dynamic contact angles and surface viscoelasticity of poly(styrene-b-isoprene-b-styrene) triblock copolymers.

In this paper, a series of poly(styrene-b-isoprene-b-styrene) triblock copolymers (SIS), with different chemical components, was synthesized by anionic polymerization. The relationships between surface structures of these block copolymers and their stick-slip phenomena were investigated. There is a transition from stick-slip to a closely smooth motion for the SIS films with increasing PS content; the patterns almost vanish and the three-phase line appears to move overall smoothly on the film surface. The results show that the observed stick-slip pattern is strongly dependent on surface viscoelasticity. The jumping angle Δθ, which is defined as θ(1) - θ(2) (when a higher limit to θ(1) is obtained, the triple line "jumps" from θ(1) to θ(2) with increases in drop volume), was employed to scale the stick-slip behavior on various SIS film surfaces. Scanning force microscopy/atomic force microscopy (AFM) and sum frequency generation methods were used to investigate the surface structures of the films and the contributions of various possible factors to the observed stick-slip behavior. It was found that there is a linear relationship between jumping angle Δθ and the slope of the approach curve obtained from AFM force measurement. This means that the stick-slip behavior may be attributed mainly to surface viscoelasticity for SIS block copolymers. The measurement of jumping angle Δθ may be a valuable method for studying surface structure relaxation of polymer films.

Entropy-Enhanced Mechanochemical Activation for Thermal Degrafting of Surface-Tethered Dry Polystyrene Brushes.

Dry polymer brushes have attracted great attention because of their potential utility in regulating interface properties. However, it is still unknown whether dry polymer brushes will exhibit degrafting behavior as a result of thermal annealing. Herein, a study of the conformational entropy effect on thermal degrafting of dry polystyrene (PS) brushes is presented. For PS brushes with an initial grafting density (σpini) of 0.61 nm-2, degrafting behavior was observed at 393 K, and the equilibrium σp was approximately 0.14 nm-2 at 413 K. However, for brushes with σpini ≤ 0.14 nm-2, thermal degrafting was not observed even if the temperature was increased to 453 K. Furthermore, we found that the degrafting rate was faster for PS brushes with higher σpini and higher molecular weights when σpini > 0.14 nm-2. Our findings confirmed that degrafting is a mechanochemical activation process driven by tension imposed on bonds that anchor the chains to the surface, and the process is amplified by conformational entropy.

Enhanced Glass Transition Temperature of Thin Polystyrene Films Having an Underneath Cross-Linked Layer.

Due to the importance of the interface in the segmental dynamics of supported macromolecule ultrathin films, the glass transition temperature (Tg) of polystyrene (PS) ultrathin films upon solid substrates modified with a cross-linked PS (CLPS) layer has been investigated. The results showed that the Tg of the thin PS films on a silica surface with a ∼5 nm cross-linked layer increased with reducing film thickness. Meanwhile, the increase in Tg of the thin PS films became more pronounced with increasing the cross-linking density of the layer. For example, a 20 nm thick PS film supported on CLPS with 1.8 kDa of cross-linking degree exhibited a ∼35 and ∼50 K increase in Tg compared to its bulk and that on neat SiO2 substrate, respectively. Such a large Tg elevation for the ultrathin PS films was attributed to the interfacial aggregation states in which chains diffused through nanolevel voids formed in the cross-linked layer to the SiO2-Si surface. In such a situation, the chains were topologically constrained in the cross-linked layer with less mobility. These results offer us the opportunity to tailor interfacial effects by changing the degree of cross-linking, which has great potential application in many polymer nanocomposites.

Enhanced immunogenicity of the tuberculosis subunit Rv0572c vaccine delivered in DMT liposome adjuvant as a BCG-booster.

COVID-19 has affected the progress made in the prevention and treatment of tuberculosis (TB); hence, the mortality of tuberculosis has risen. Different strategies-based novel TB vaccine candidates have been developed. This study identifies strategies to overcome the limitations of Bacille Calmette-Guérin (BCG) in preventing latent infection and reactivation of TB. The latency antigen Rv0572c was selected based on the mechanism of interaction between Mycobacterium tuberculosis and its host. The rRv0572c protein was used to stimulate whole blood samples derived from patients with clinically diagnosed active TB (ATBs) or latent TB infections (LTBIs) and healthy control (HCs) donors, confirming that this protein can be recognized by T cells in patients with TB, especially LTBIs. C57BL/6 mice were used to investigate the immunogenicity of the rRv0572c protein emulsified in the liposome adjuvant dimethyldioctadecylammonium [DDA], monophosphoryl lipid A [MPLA], trehalose-6, 6'-dibehenate [TDB] (DMT). The results demonstrated that rRv0572c/DMT could boost BCG-primed mice to induce antigen-specific CD4+ T cell production and generate functional T cells dominated by antigen-specific CD8+ T cells. The rRv0572c/DMT vaccine could also trigger limited Th2 humoral immune responses. These findings suggest that rRv0572c/DMT is a potential subunit vaccine candidate that can be used as a booster vaccine for BCG.

Decoupling Role of Film Thickness and Interfacial Effect on Polymer Thin Film Dynamics.

The film thickness and substrate interface are the two most common parameters to tune the dynamics of supported thin films. Here, we investigated the glass transition temperature (Tg) and thermal expansion of thin poly(methyl methacrylate) (PMMA) films with various thicknesses and different interfacial effects. We showed that, although the Tg of the thin films can be modulated equivalently by the two factors, their ability to change the expansivity (ß) is quite different; that is, ß increases notably with a reduction in the thickness, while it is insensitive to perturbations at the interface. We attribute the deviation in modulating ß by the thickness and the interfacial effect to the disparate abilities to change the free volume content in the film by a free surface and substrate interface. This leads to a situation where thin films with dissimilar thicknesses and interfacial properties can have the same Tg but very different ß values, suggesting that Tg alone cannot unequivocally quantify thin film dynamics.

Generation of a pHSPA6 gene-based multifunctional live cell sensor.

Biosensors utilizing intact live cells can report responses to certain stimuli rapidly and sensitively and have attracted a great deal of attention. The expression pattern of HSPA6, a little studied HSPA family member, has contributed to the development of multifunctional and intelligent whole-cell sensors. Herein, a new pHSPA6-based EGFP fluorescent reporter cell line was designed and developed via a CRISPR/Cas9-mediated knock-in strategy. The fluorescent reporter cell line has a precise EGFP integration site and gene copy number, and no selectable marker genes were introduced during the selection processes. Stimulation experiments with HSPA6-specific stressors indicated that EGFP fluorescent reporter cells could rapidly and effectively convert stress signals into EGFP fluorescent signals. Furthermore, cell proliferation and gene expression pattern analysis showed that the fluorescent reporter cells grew well and that both the integrated EGFP gene and the pHSPA6 gene were expressed rapidly and sensitively in response to stimulation. This study provides a new strategy for the construction of a cell model for HSPA6 expression/interaction and an intelligent live cell sensor, which can potentially be applied to numerous fields, such as those focusing on cellular models of HSPA6 signaling cascades, biomaterials, food security, environmental assessment, and drug screening.

Solvent effect on the film formation and the stability of the surface properties of poly(methyl methacrylate) end-capped with fluorinated units.

The surface structure and stability (the resistance to surface reconstruction) of end-capped poly(methyl methacrylate) films were greatly affected by the solvents used for film preparation. Films of end-capped PMMA with about four 2-perfluorooctylethyl methacrylate units cast with benzotrifluoride solution exhibited excellent stability and resistance to polar environments compared with those cast with cyclohexanone and toluene solutions. The observed difference in stability between these fluorinated surfaces is attributed to their surface microstructures formed during the film formation processes, which are closely related to the associative behavior of the end-capped PMMA in the solution. A relatively perfect close-packed and well-ordered structure of the perfluoroalkyl side chains at the surface of the PMMA(857)-ec-FMA(3.3) film was formed when the film was cast with benzotrifluoride solution, in which only unimers existed. This study indicates that such a solvent effect may be used to promote the formation of a well-ordered packing structure of the fluorinated moieties at the film surface. The ordering of the packing structure is to a certain extent more important than the content of the fluorinated moieties at the surface for improving the surface stability.

Protein-resistance performance enhanced by formation of highly-ordered perfluorinated alkyls on fluorinated polymer surfaces.

In this paper, the relationship between the surface structure of fluorinated polymers and their protein-resistant property was studied by preparing films of poly(n-alkyl methacrylate) end-capped with 2-perfluorooctylethyl methacrylate (FMA) (PFMA(y)-ec-PnAMA(x)-ec-PFMA(y)) with various ordered structures of perfluorinated alkyls. These fluorinated polymers were synthesized via a controlled/living atom-transfer radical polymerization (ATRP) method. Both the surface free energy and the CF(3)/CF(2) ratio obtained by X-ray photoelectron spectroscopy (XPS) were employed to scale the ordered structures of the perfluorinated alkyls. Protein adsorption studies using fibrinogen as a test molecule were undertaken on the various films by XPS. The results show that the adsorbed mass of fibrinogen decreased linearly with increasing CF(3)/CF(2) ratio on the fluorinated polymer surfaces. When the CF(3)/CF(2) ratio reaches 0.26, there was almost no fibrinogen adsorption. This work not only demonstrates the design of a fluorinated copolymer film on glass substrate with desirable protein-resistant performance, but also provides a fundamental understanding of how the orientation of perfluoroalkyl side chains affects protein-resistant behavior on fluorinated surfaces.

Alternative Splicing of the Amelogenin Gene in a Caudate Amphibian, Plethodon cinereus.

As the major enamel matrix protein contributing to tooth development, amelogenin has been demonstrated to play a crucial role in tooth enamel formation. Previous studies have revealed amelogenin alternative splicing as a mechanism for amelogenin heterogeneous expression in mammals. While amelogenin and its splicing forms in mammalian vertebrates have been characterized, splicing variants of amelogenin gene still remains largely unknown in non-mammalian species. Here, using PCR and sequence analysis we discovered two novel amelogenin transcript variants in tooth organ extracts from a caudate amphibian, the salamander Plethodoncinereus. The one was shorter -S- (416 nucleotides including untranslated regions, 5 exons) and the other larger -L- (851 nt, 7 exons) than the previously published "normal" gene in this species -M- (812 nucleotides, 6 exons). This is the first report demonstrating the amelogenin alternative splicing in amphibian, revealing a unique exon 2b and two novel amelogenin gene transcripts in Plethodoncinereus.

Identification of a novel splicing form of amelogenin gene in a reptile, Ctenosaura similis.

Amelogenin, the major enamel matrix protein in tooth development, has been demonstrated to play a significant role in tooth enamel formation. Previous studies have identified the alternative splicing of amelogenin in many mammalian vertebrates as one mechanism for amelogenin heterogeneous expression in teeth. While amelogenin and its splicing forms in mammalian vertebrates have been cloned and sequenced, the amelogenin gene, especially its splicing forms in non-mammalian species, remains largely unknown. To better understand the mechanism underlying amelogenin evolution, we previously cloned and characterized an amelogenin gene sequence from a squamate, the green iguana. In this study, we employed RT-PCR to amplify the amelogenin gene from the black spiny-tailed iguana Ctenosaura similis teeth, and discovered a novel splicing form of the amelogenin gene. The transcript of the newly identified iguana amelogenin gene (named C. Similis-T2L) is 873 nucleotides long encoding an expected polypeptide of 206 amino acids. The C. Similis-T2L contains a unique exon denominated exon X, which is located between exon 5 and exon 6. The C. Similis-T2L contains 7 exons including exon 1, 2, 3, 5, X, 6, and 7. Analysis of the secondary and tertiary structures of T2L amelogenin protein demonstrated that exon X has a dramatic effect on the amelogenin structures. This is the first report to provide definitive evidence for the amelogenin alternative splicing in non-mammalian vertebrates, revealing a unique exon X and the splicing form of the amelogenin gene transcript in Ctenosaura similis.

Enhanced surface segregation of poly(methyl methacrylate) end-capped with 2-perfluorooctylethyl methacrylate by introduction of a second block.

New fluorinated copolymers of poly(methyl methacrylate)-b-poly(butyl methacrylate) or poly(n-octadecyl methacrylate) end-capped with 2-perfluorooctylethyl methacrylate (PMMA(x)-b-PBMA(y)-ec-PFMA(z) or PMMA(x)-b-PODMA(y)-ec-PFMA(z)) were synthesized by living atom transfer radical polymerization. Thin films made of PMMA(230)-b-PODMA(y)-ec-PFMA(1) were characterized by differential scanning calorimetry, angle-resolved X-ray photoelectron spectroscopy and X-ray diffraction. These films were found to exhibit robust surface segregation of the end groups. Furthermore, the fluorine enrichment factor at the film surface was found to increase linearly with increasing degree of polymerization of poly(n-octadecyl methacrylate) and its increasing fusion enthalpy in the second block, which enhances the segregation of the fluorinated moieties.

Studies on the effects of the alkyl group on the surface segregation of poly(n-alkyl methacrylate) end-capped 2-perfluorooctylethyl methacrylate films.

The effects of the alkyl group on the surface segregation of poly(n-alkyl methacrylate) end-capped with various numbers of units of 2-perfluorooctylethyl methacrylate (FMA) (PnAMA-ec-PFMA) were investigated by differential scanning calorimetry, angle-resolved XPS analysis, contact angle measurements, and X-ray diffraction (XRD). The results show that with similar numbers of FMA units at the polymer chain end the extent of fluorine segregation (Q) increased with increasing the number of carbon atoms in the side n-alkyl chains of poly(n-alkyl methacrylate). The surface fluorine content within 5 nm deep of the film of poly(n-octadecyl methacrylate) end-capped with one FMA unit (PODMA(160)-ec-PFMA(1.0)) was 208-fold higher than that of the bulk level. These observed differences in Q values were found due to the aggregate structure of the end-capped polymers in the solution, the flexibility, and the crystallinity of the n-alkyl side chains. When the nonfluorinated block was completely amorphous, the molecular aggregate structure of the end-capped polymers in the solution played an important role in the surface segregation of the fluorinated moieties on the resulting film. However, when the nonfluorinated block was crystalline, crystallinity would enhance greatly the segregation of the fluorinated moieties.

Investigation on surface molecular conformations and pervaporation performance of the poly(vinyl alcohol) (PVA) membrane.

A simple method of changing pre-treatment temperature in the course of film formation was used to tune the surface structures of PVA membranes. Surface structure and property of the resulting membranes were characterized by X-ray photoelectron spectroscopy (XPS), sum frequency generation (SFG) vibrational spectroscopy, and contact angle measurements. The results show that PVA have different molecular conformations at the membrane surface while those membranes were prepared at different pre-treatment temperature. At higher pre-treatment temperatures, polar acetoxyl residues and hydroxyl groups of the PVA chains oriented in a more orderly fashion, as induced by the faster evaporation of water. When the membranes were in air, CH(3) groups adjacent to the acetoxyl groups covered the surface in order to minimize the surface free energy, while backbones of the PVA were rarely observed. These surfaces exhibited a hydrophilic nature upon contact with water due to rapid surface reconstruction. Conversely, at lower pre-treatment temperatures, the backbone CH(2) groups dominated the surface, forming a less hydrophilic surface. When the PVA membranes were employed to separate ethanol/water mixtures, it was found that the PVA membranes with more hydrophilic surface exhibited higher water selectivity. Our investigation indicates that molecular conformations on the membrane surface have considerable influence on pervaporation performance.

Amelogenin evolution and tetrapod enamel structure.

Amelogenins are the major proteins involved in tooth enamel formation. In the present study, we have cloned and sequenced four novel amelogenins from three amphibian species in order to analyze similarities and differences between mammalian and non-mammalian amelogenins. The newly sequenced amphibian amelogenin sequences were from a red-eyed tree frog (Litoria chloris) and a Mexican axolotl (Ambystoma mexicanum). We identified two amelogenin isoforms in the Eastern red-backed salamander (Plethodon cinereus). Sequence comparisons confirmed that non-mammalian amelogenins are overall shorter than their mammalian counterparts, contain less proline and less glutamine, and feature shorter polyproline tripeptide repeat stretches than mammalian amelogenins. We propose that unique sequence parameters of mammalian amelogenins might be a pre-requisite for complex mammalian enamel prism architecture.

Identification and characterization of a squamate reptilian amelogenin gene: Iguana iguana.

As the principal components of the developing tooth enamel matrix, amelogenins play a significant role in tooth enamel formation and organization. In order to elucidate the structure and function of amelogenins in the evolution of enamel, we have selected the Iguana iguana as a squamate model organism. Here we report the first complete squamate amelogenin sequence available as of yet and document unique features of Iguana amelogenins and enamel. Transmission electron microscopy documented randomly oriented Iguana enamel crystals during the elongation phase compared with organized enamel crystal patterns at comparable stages in mammals. Sequencing of PCR amplified products revealed a full-length I. iguana amelogenin cDNA containing 877 nucleotides with a 564 nucleotide coding sequence encoding 187 amino acids. The homologies of the newly discovered I. iguana amelogenin amino acid sequence with the published mouse, caiman (Palaeosuchus), and snake (Elaphe) amelogenin were 41.3%, 53.5%, and 55.5%, respectively. On Western blots one major protein with a molecular weight of 24 kDa, and two minor proteins with molecular weights of 28 and 13.5 kDa, respectively, were detected based on the cross-reactivity of antisera against recombinant Rana pipiens amelogenin proteins. Sequence analysis revealed a moderate sequence homology between mammalian and reptilian amelogenin genes. A significant alteration was the deletion of the hydrophilic GSP sequence from exon 3 in the mouse sequence resulting in a conversion to a hydrophobic region in Iguana. Together, these findings identified a novel amelogenin cDNA sequence in the squamate reptilian I. iguana and functional implications for the evolution of amelogenins and enamel in squamates.

Amelogenin sequence and enamel biomineralization in Rana pipiens.

The amelogenin gene contributes the majority of tooth enamel proteins and plays a significant role in enamel biomineralization. While several mammalian and reptilian amelogenins have been cloned and sequenced, basal vertebrate amelogenin evolution remains to be understood. In order to start elucidating the structure and function of amelogenins in the evolution of enamel, the leopard frog (Rana pipiens) was used as a model. Tissues from Rana pipiens teeth were analyzed for enamel structure and RNA extracts were processed for sequence analysis. Electron microscopy revealed that Rana pipiens enamel contains long and parallel crystals similar to mammalian enamel, while immunoreactions confirmed the site-specific localization of cross-reactive amelogenins in Rana pipiens enamel. Sequencing of amelogenin PCR products revealed a 782bp cDNA with a 546-nucleotide coding sequence encoding 181 amino acids. The homology of the newly discovered Rana pipiens amelogenin nucleotide and amino acid sequence with the published mouse amelogenin was 38.6% and 45%, respectively. These findings report the first complete amelogenin cDNA sequence in amphibians and indicate a close homology between mammalian enamel formation and Rana pipiens enamel biomineralization.