Fold Surface Research Articles

Mantle margin morphogenesis in Nodipecten nodosus (Mollusca: Bivalvia): new insights into the development and the roles of bivalve pallial folds.

BackgroundDespite extensive knowledge on bivalve anatomy and development, the formation and differentiation of the mantle margin and its associated organs remain largely unclear. Bivalves from the family Pectinidae (scallops) are particularly promising to cast some light on these issues, because they exhibit a complex mantle margin and their developmental stages are easily obtained from scallop farms. We investigated the mantle margin of the scallop Nodipecten nodosus (L. 1758) during larval and postmetamorphic development.MethodsA thorough analysis of the mantle margin development in Nodipecten nodosus, from veliger larvae to mature adults, was conducted by means of integrative microscopy techniques, i.e., light, electron, and confocal microscopy.ResultsInitially unfolded, the pallial margin is divided into distal and proximal regions by the periostracum-forming zone. The emergence of the pallial musculature and its neural innervation are crucial steps during bivalve larval development. By the late pediveliger stage, the margin becomes folded, resulting in a bilobed condition (i.e., outer and inner folds), a periostracal groove, and the development of different types of cilia. After metamorphosis, a second outgrowth process is responsible for emergence of the middle mantle fold from the outer surface of the inner fold. Once the three-folded condition is established, the general adult features are rapidly formed.ConclusionsOur data show that the middle mantle fold forms from the outer surface of the inner fold after metamorphosis and that the initial unfolded mantle margin may represent a common condition among bivalves. The first outgrowth process, which gives rise to the outer and inner folds, and the emergence of the pallial musculature and innervation occur during larval stages, highlighting the importance of the larval period for mantle margin morphogenesis in Bivalvia.

The family 6 Carbohydrate Binding Module (CtCBM6) of glucuronoxylanase (CtXynGH30) of Clostridium thermocellum binds decorated and undecorated xylans through cleft A

CtCBM6 of glucuronoxylan-xylanohydrolase (CtXynGH30) from Clostridium thermocellum was cloned, expressed and purified as a soluble ∼14kDa protein. Quantitative binding analysis with soluble polysaccharides by affinity electrophoresis and ITC revealed that CtCBM6 displays similar affinity towards decorated and undecorated xylans by binding wheat- and rye-arabinoxylans, beechwood-, birchwood- and oatspelt-xylan. Protein melting studies confirmed thermostable nature of CtCBM6 and that Ca2+ ions did not affect its structure stability and binding affinity significantly. The CtCBM6 structure was modeled and refined and CD spectrum displayed 44% β-strands supporting the predicted structure. CtCBM6 displays a jelly roll β-sandwich fold presenting two potential carbohydrate binding clefts, A and B. The cleft A, is located between two loops connecting β4–β5 and β8–β9 strands. Tyr28 and Phe84 present on these loops make a planar hydrophobic binding surface to accommodate sugar ring of ligand. The cleft B, is located on concave surface of β-sandwich fold. Tyr34 and Tyr104 make a planar hydrophobic platform, which may be inaccessible to ligand due to hindrance by Pro68. Site-directed mutagenesis revealed Tyr28 and Phe84 in cleft A, playing a major role in ligand binding. The results suggest that CtCBM6 interacts with carbohydrates through cleft A, which recognizes equally well both decorated and un-decorated xylans.

Engineering tandem single-chain Fv as cell surface reporters with enhanced properties of fluorescence detection.

A recently described fluorescence biosensor platform utilizes single-chain Fv (scFvs) that selectively bind and activate fluorogen molecules. In this report we investigated the display of tandem scFv biosensors at the surface of mammalian cells with the aim of advancing current fluorescence detection strategies. We initially screened different peptide linkers to separate each scFv unit, and discovered that tandem proteins joined by either flexible or α-helical linkers properly fold and display at the surface of mammalian cells. Accordingly, we performed a combinatorial scFv-dimer study and identified that fluorescence activation correlated with the cellular location (membrane distal versus proximal) and selections of the different scFvs. Furthermore, in vitro measurements showed that the stability of each scFv monomer unit influenced the folding and cell surface activities of tandem scFvs. Additionally, we investigated the absence or poor signals from some scFv-dimer combinations and discovered that intramolecular and intermolecular scFv chain mispairings led to protein misfolding and/or secretory-pathway-mediated degradation. Furthermore, when tandem scFvs were utilized as fluorescence reporter tags with surface receptors, the biosensor unit and target protein showed independent activities. Thus, the live cell application of tandem scFvs permitted advanced detection of target proteins via fluorescence signal amplification, Förster resonance energy transfer resulting in the increase of Stokes shift and multi-color vesicular traffic of surface receptors.

Particle size and traffic of phagocytes between the turbot peritoneal cavity and lymphoid organs

New adjuvants based on microparticles are being developed for use in fish vaccines. The size of the microparticles may affect the immune response generated, as the adjuvant can either be retained at the site of injection or transported to lymphoid organs. The objectives of this study were to evaluate the maximum size of particles that can be exported out of the cavity, to determine the phagocytosis kinetics and to establish the routes whereby particle-containing cells move from the peritoneal cavity after injection. Fish were injected intraperitoneally with fluorescent cyclodextrins or with fluorescent particles of different size (0.1–10 μm). Phagocytes containing beads of size 4 μm or larger did not reach lymphoid organs, although some were able to cross the peritoneal mesothelium. The number of free peritoneal neutrophils and macrophage-like cells containing beads peaked at 6 and 24 h respectively, and the numbers then decreased quickly, indicating migration of cells to the peritoneum or other body areas. Migration of cells containing beads mainly occurs through the visceral peritoneum. These cells were found on the latero-ventral surfaces of the peritoneal folds that connect the visceral organs. Except for some vascularised areas, the surfaces of liver, stomach and intestine were devoid of particle-containing cells. Some cells containing beads were also found attached to the parietal peritoneum, although in lower numbers than in the visceral peritoneum. Such cells were also found in high numbers in the spleen and kidney 6 h post injection. Because cells containing phagocytosed material quickly become attached to the peritoneum or migrate to lymphoid organs, the immune response generated by a vaccine or by an inflammatory stimulus should probably be evaluated in attached cells as well as in free peritoneal cells.

Specificity and competitive cation association to Phosphatidylinositol‐4,5‐bisphosphate model membranes

Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is an active signaling lipid implicated, among other functions, in the regulation of cell growth by activating the tumor suppressor PTEN. Using synchrotron surface‐sensitive x‐ray diffraction and fluorescence techniques we determined the preferential cation binding to PIP2 monolayers. The natural, highly unsaturated PIP2 was spread as a Langmuir monolayer on a physiological buffer containing 100 mM KCl at pH 7.2 to which divalent cations (Ca2+ and Mg2+) were added. X‐ray fluorescence of the PIP2 monolayer on the buffer shows an eight fold surface enhancement of monovalent K+ compared to its bulk concentration. When physiological levels of calcium are added (1‐100 µM), the Ca2+ gradually replaces bound K+ ions, leading to a significant change in the organization of the PIP2 model membrane. At higher concentrations (100‐1000 µM), which might be achieved during calcium signaling, we observe a 1000 fold surface enhancement of Ca2+. Similar experiments with Mg2+ ions also show strong ion binding to PIP2 at physiological levels (1 mM) with a lesser structural effect on the monolayer compared to that induced by Ca2+. For mixed solutions of Mg2+ and Ca2+ we find that Ca2+ occupies the majority of binding sites, and at mM concentrations completely removes the Mg2+ ions from the interface. Surprisingly, with both 1 mM Mg2+ and 1 mM Ca2+ in the subphase there is still a fourfold surface enrichment of K+ ions at the headgroup region.

Classification of laryngeal disorders based on shape and vascular defects of vocal folds

Vocal fold disorders such as laryngitis, vocal nodules, and vocal polyps may cause hoarseness, breathing and swallowing difficulties due to vocal fold malfunction. Despite the fact that state of the art medical imaging techniques help physicians to obtain more detailed information, difficulty in differentiating minor anomalies of vocal folds encourages physicians to research new strategies and technologies to aid the diagnostic process. Recent studies on vocal fold disorders note the potential role of the vascular structure of vocal folds in differential diagnosis of anomalies. However, standards of clinical usage of the blood vessels have not been well established yet due to the lack of objective and comprehensive evaluation of the vascular structure.In this paper, we present a novel approach that categorizes vocal folds into healthy, nodule, polyp, sulcus vocalis, and laryngitis classes exploiting visible blood vessels on the superior surface of vocal folds and shapes of vocal fold edges by using image processing techniques and machine learning methods. We first detected the vocal folds on videolaryngostroboscopy images by using Histogram of Oriented Gradients (HOG) descriptors. Then we examined the shape of vocal fold edges in order to provide features such as size and splay portion of mass lesions. We developed a new vessel centerline extraction procedure that is specialized to the vascular structure of vocal folds. Extracted vessel centerlines were evaluated in order to get vascular features of vocal folds, such as the amount of vessels in the longitudinal and transverse form. During the last step, categorization of vocal folds was performed by a novel binary decision tree architecture, which evaluates features of the vocal fold edge shape and vascular structure.The performance of the proposed system was evaluated by using laryngeal images of 70 patients. Sensitivity of 86%, 94%, 80%, 73%, and 76% were obtained for healthy, polyp, nodule, laryngitis, and sulcus vocalis classes, respectively. These results indicate that visible vessels of vocal folds can act as a prognostic marker for vocal fold pathologies, as well as the vocal fold shape features, and may play a critical role in more effective diagnosis.

Effect of Structural Changes in Proteins Derived from GATA4 Nonsynonymous Single Nucleotide Polymorphisms in Congenital Heart Disease.

Congenital heart disease is the most common type of birth defect. The single nucleotide polymorphism in GATA4 is associated with various congenital heart disease phenotypes. In the present study, we analysed the nonsynonymous single nucleotide polymorphism of GATA4, which are involved in congenital heart disease by predicting the changes in protein structures. Total of 49 nonsynonymous single nucleotide polymorphisms of GATA4 was screened from congenital heart disease patients of Mysore and also globally reported nonsynonymous single nucleotide polymorphisms. To understand the role of nonsynonymous single nucleotide polymorphisms, we mutated the sequence and translated into amino acids. Further the mutated protein secondary structure is predicted and tertiary structure is predicted using homology modeling. The quantitative evaluation of protein structure quality was verified with Volume Area Dihedral Angle Reporter server. Results revealed the secondary, tertiary structural changes along with changes in free energy of folding, volume and accessible surface area. Thus, the structural changes in the mutated proteins impaired the normal function of GATA4.

5 Analysis of Fault-Related Folding in South of Birjand

Folds have a significant development in the Cretaceous-Tertiary rock units of the northern part of Bagheran Mountains in southwest Birjand between Lut and Sistan structural zones. The general trends of fold axis and axial surface are E-W and the folds are less exposed by distance from mountain and plain boundary. Geometric and kinematic status investigation of folds (such as f2 fold in the middle part) and faults shows that faulting process has created some of the folds leading to their development. Such structures are described as fault-related folds. Also, analysis of geometry and mechanism of faults indicate that back thrusts have the largest influence on generation and development of folds in this region.

Specificity and Competitive Cation Association to Phosphatidylinositol-4,5-Bisphosphate Model Membranes

Phosphatidylinositol-4,5-bisphosphate (PIP2) is an active signaling lipid implicated, among other functions, in the regulation of cell growth by activating the tumor suppressor PTEN. Using synchrotron surface-sensitive x-ray diffraction and fluorescence techniques we determined the preferential cation binding to PIP2 monolayers. The natural, highly unsaturated PIP2 was spread as a Langmuir monolayer on a physiological buffer containing 100 mM KCl at pH 7.2 to which divalent cations (Ca2+ and Mg2+) were added. X-ray fluorescence of the PIP2 monolayer on the buffer shows an eight fold surface enhancement (within the x-ray penetration depth below the critical angle, ∼5 nm) of monovalent K+ compared to its bulk concentration. When physiological levels of calcium are added (1-100 µM), the Ca2+ gradually replaces bound K+ ions, leading to a significant change in the organization of the PIP2 model membrane. At higher concentrations (100-1000 µM), which might be achieved during calcium signaling, we observe a 1000 fold surface enhancement of Ca2+. Similar experiments with Mg2+ ions also show strong ion binding to PIP2 at physiological levels (1 mM) with a lesser structural effect on the monolayer compared to that induced by Ca2+. For mixed solutions of Mg2+ and Ca2+ we find that Ca2+ occupies the majority of binding sites, and at mM concentrations completely removes the Mg2+ ions from the interface. Surprisingly, with both 1 mM Mg2+ and 1 mM Ca2+ in the subphase there is still a fourfold surface enrichment of K+ ions at the headgroup region.

Estimation of inferior-superior vocal fold kinematics from high-speed stereo endoscopic data in vivo.

Despite being an indispensable tool for both researchers and clinicians, traditional endoscopic imaging of the human vocal folds is limited in that it cannot capture their inferior-superior motion. A three-dimensional reconstruction technique using high-speed video imaging of the vocal folds in stereo is explored in an effort to estimate the inferior-superior motion of the medial-most edge of the vocal folds under normal muscle activation in vivo. Traditional stereo-matching algorithms from the field of computer vision are considered and modified to suit the specific challenges of the in vivo application. Inferior-superior motion of the medial vocal fold surface of three healthy speakers is reconstructed over one glottal cycle. The inferior-superior amplitude of the mucosal wave is found to be approximately 13 mm for normal modal voice, reducing to approximately 3 mm for strained falsetto voice, with uncertainty estimated at σ ≈ 2 mm and σ ≈ 1 mm, respectively. Sources of error, and their relative effects on the estimation of the inferior-superior motion, are considered and recommendations are made to improve the technique.

Pulse-chase analysis for studying protein synthesis and maturation.

Pulse-chase analysis is a well-established and highly adaptable tool for studying the life cycle of endogenous proteins, including their synthesis, folding, subunit assembly, intracellular transport, post-translational processing, and degradation. This unit describes the performance and analysis of a radiolabel pulse-chase experiment for following the folding and cell surface trafficking of a trimeric murine MHC class I glycoprotein. In particular, the unit focuses on the precise timing of pulse-chase experiments to evaluate early/short-time events in protein maturation in both suspended and strictly adherent cell lines. The advantages and limitations of radiolabel pulse-chase experiments are discussed, and a comprehensive section for troubleshooting is provided. Further, ways to quantitatively represent pulse-chase results are described, and feasible interpretations on protein maturation are suggested. The protocols can be adapted to investigate a variety of proteins that may mature in very different ways.

A hybrid approach to the computational aeroacoustics of human voice production.

The aeroacoustic mechanisms in human voice production are complex coupled processes that are still not fully understood. In this article, a hybrid numerical approach to analyzing sound generation in human voice production is presented. First, the fluid flow problem is solved using a parallel finite-volume computational fluid dynamics (CFD) solver on a fine computational mesh covering the larynx. The CFD simulations are run for four geometrical configurations: both with and without false vocal folds, and with fixed convergent or convergent-divergent motion of the medial vocal fold surface. Then the aeroacoustic sources and propagation of sound waves are calculated using Lighthill's analogy or acoustic perturbation equations on a coarse mesh covering the larynx, vocal tract, and radiation region near the mouth. Aeroacoustic sound sources are investigated in the time and frequency domains to determine their precise origin and correlation with the flow field. The problem of acoustic wave propagation from the larynx and vocal tract into the free field is solved using the finite-element method. Two different vocal-tract shapes are considered and modeled according to MRI vocal-tract data of the vowels /i/ and /u/. The spectra of the radiated sound evaluated from acoustic simulations show good agreement with formant frequencies known from human subjects.

Morphology and crystallization behavior of poly(l‐lactide)/poly(butylene oxalate) blends

ABSTRACTThe isothermal crystallization of poly(l‐lactide) (PLLA) in blends with poly(butylene oxalate) (PBOX) is investigated by time‐resolved small‐angle X‐ray scattering, differential scanning calorimetry, and optical microscopy. We focus on the temperatures at which only PLLA crystallizes while PBOX is amorphous. It is obtained that the addition of PBOX causes a reduction of the melting temperature of PLLA. The lamellar thickness of PLLA crystals decreases whereas the amorphous layer thickness increases with blend composition, suggesting the occurrence of the interlamellar incorporation upon the addition of PBOX. The crystal growth rate and morphology of PLLA/PBOX blends are analyzed by polarized optical microscopy. The spherulite growth rate of PLLA is found to increase with the addition of PBOX. Analysis of the isothermal crystallization in terms of the Lauritzen and Hoffman equation give the reduction of the fold surface free energy upon the addition of PBOX in PLLA, indicating that the mobility of the PLLA chains is significantly improved due to the presence of PBOX. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015, 53, 192–202

A study on the effect of intraglottal vortical structures on vocal fold vibration

Recent investigations suggested possible formation of the vortical structures in the intraglottal region during the closing phase of the phonation cycle. Vortical regions in the flow field are locations of negative pressure, and it has been hypothesized that this negative pressure might facilitate the glottal closure and thus affects the vibration pattern and voice production for high subglottal pressures. However, it is unclear whether the vortex-induced negative pressure is large enough, compared with vocal fold inertia and elastic recoil, to have a noticeable effect on glottal closure. In addition, the intraglottal vortical structures generally exist only for a small fraction of the closing phase when the glottis becomes divergent enough to induce flow separation. In the current work, oscillation of the vocal folds and the flow field are modeled using a non-linear finite element solver and a reduced order flow solver, respectively. The effect of vortical structures is modeled as a sinusoidal negative pressure wave applied to vocal fold surface between the flow separation point and the superior edge of the vocal folds. The effects of this vortex-induced negative pressure are quantified at different conditions of vocal fold stiffness and subglottal pressures. [Work supported by NIH.]

Poly(styrene‐co‐maleic anhydride) ionomers as nucleating agent on the crystallization behavior of poly(ethylene terephthalate)

ABSTRACTPoly(styrene‐co‐maleic anhydride) (SMA) ionomers were synthesized and designed as a new kind of nucleation agent according to the crystallization theory for improving the crystallization of poly(ethylene terephthalate) (PET). The crystallization behavior of PET with the addition of nucleation agents was investigated by differential scanning calorimetry, polarized‐light microscope, and X‐ray diffraction (XRD). Avrami equation and Hoffman–Lauritzen theory are adopted for analyzing isothermal and non‐isothermal crystallization kinetics, respectively. The results show that the addition of 1 wt % SMA ionomers effectively accelerates the crystallization rate and reduces the fold surface free energy of PET at high temperature regions. PLM results also indicated that the crystals impinge on each other, thus decreasing the spherulite size for PET/SMA ionomers samples compared with PET. XRD measurement revealed that the introduction of SMA ionomers does not change the crystal structure but indeed accelerates the crystallinity of PET. The results clearly demonstrate that our synthesized SMA ionomers are an efficient nucleating agent for PET. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 41240.

The role of glottal surface adhesion on vocal folds biomechanics.

The airway surface liquid (ASL) is a very thin mucus layer and covers the vocal fold (VF) surface. Adhesion mediated by the ASL occurs during phonation as the VFs separate after collision. Such adhesion is hypothesized to determine voice quality and health. However, biomechanical insights into the adhesive processes during VF oscillation are lacking. Here, a computational study is reported on self-sustained VF vibration involving contact and adhesion. The VF structural model and the glottal airflow are considered fully three-dimensional. The mechanical behavior of the ASL is described through a constitutive traction-separation law where mucosal cohesive strength, cohesive energy, and rupture length enter. Cohesive energy values considered are bound below by the cohesive energy of water at standard temperature and pressure. Cohesive strength values considered are bound above by prior reported data on the adhesive strength of mucosal surface of rat small intestine. This model introduces a mechanical length scale into the analysis. The sensitivity of various aspects of VF dynamics such as flow-declination rate, VF separation under adhesive condition, and formation of multiple local fluid bridges is determined in relation to specific ASL adhesive properties. It is found that for the ASL considered here, the characteristics of the VF separation process are of debond type. Instabilities lead to the breakup of the bond area into several smaller bond patches. Such finding is consistent with in vivo observations.

Correlating polymer crystals via self-induced nucleation.

Crystallizable polymers often form multiple stacks of uniquely oriented lamellae, which have good registry despite being separated by amorphous fold surfaces. These correlations require multiple synchronized, yet unidentified, nucleation events. Here, we demonstrate that in thin films of isotactic polystyrene, the probability of generating correlated lamellae is controlled by the branched morphology of a single primary lamella. The nucleation density n(s) of secondary lamellae is found to be dependent on the width w of the branches of the primary lamella such that n(s) ∼ w(-2). This relation is independent of molecular weight, crystallization temperature, and film thickness. We propose a nucleation mechanism based on the insertion of polymers into a branched primary lamellar crystal.

Isothermal Cold Crystallization and Melting Behaviors of Poly(lactic acid)/Epoxy Vinyl Polyhedral Oligomeric Silsesquioxanes Nanocomposites

Poly(l-lactic acid) (PLA)/epoxy vinyl polyhedral oligomeric silsesquioxane (EP-POSS) nanocomposites were prepared via melt blending. The isothermal cold crystallization and melting behavors of pure PLA and its nanocomposites were investigated using differential scanning calorimetry (DSC). The crystal growth model of PLA was a mixture of two-dimensional discotic growth and three-dimensional spherulite growth, as evidenced by the values of the Avrami exponent ranging from 2.15 to 2.66. The crystallization rate displayed a great dependence on the EP-POSS loading level and crystallization temperature, and a transition from regime II to regime III was observed around 115°C. The values of fold surface free energy were calculated from the nucleation parameter which was determined by the Hoffman-Lauritzen equation. Polarizing microscope images (POM) indicated that EP-POSS promoted the nucleation of PLA.

Dynamic interactions between poly(3-hexylthiophene) and single-walled carbon nanotubes in marginal solvent.

Interfacial interactions between conjugated polymers and carbon nanotubes are pivotal in determining the device performance of nanotube-based polymer electronic devices. Here, we report on interfacial structures and crystallization kinetics of poly(3-hexylthiophene) (P3HT) in the presence of single-walled carbon nanotubes (SWNTs) in anisole by means of transmission electron microscope (TEM) and ultraviolet-visible (UV-vis) absorption spectroscopy. Confined on SWNT surfaces, the P3HT forms nanofibril crystals perpendicular to the long axis of SWNTs. The equilibrium dissolution temperature of the P3HT crystals in anisole is determined to be 381 ± 10 K according to the Hoffman-Weeks extrapolation approach. Upon cooling, the polymer solution spontaneously undergoes a time-dependent chromism. Various kinetics factors such as crystallization temperature, concentration, and SWNT loading have been investigated. It is found that the growth rate (G) of the crystals scales with concentration (C) as G ∝ C(1.70±0.16). The Avrami model is utilized to analyze the nucleation mechanism and the Avrami exponents vary between 1.0 and 1.3. The Lauritzen-Hoffman theory is applied to study the chain-folding process. The fold surface free energy is calculated to be (5.28-11.9) × 10(-2) J m(-2). It is evident that the addition of 0.30 wt % SWNTs reduces the fold surface free energy by 55.6%.

Novel poly(3-hydroxybutyrate) nanocomposites containing WS2 inorganic nanotubes with improved thermal, mechanical and tribological properties

Poly(3-hydroxybutyrate) (PHB) nanocomposites containing environmentally-friendly tungsten disulphide inorganic nanotubes (INT–WS2) have been successfully prepared by a simple solution blending method. The dynamic and isothermal crystallization studies by differential scanning calorimetry (DSC) demonstrated that the INT–WS2 exhibits much more prominent nucleation activity on the crystallization of PHB than specific nucleating agents or other nanoscale fillers. Both crystallization rate and crystallinity significantly increase in the nanocomposites compared to neat PHB. These changes occur without modifying the crystalline structure of PHB in the nanocomposites, as shown by wide-angle X-ray diffraction (WAXS) and infrared/Raman spectroscopy. Other parameters such as the Avrami exponent, the equilibrium melting temperature, global rate constant and the fold surface free energy of PHB chains in the nanocomposites were obtained from the calorimetric data in order to determine the influence of the INT–WS2 filler. The addition of INT–WS2 remarkably influences the energetics and kinetics of nucleation and growth of PHB, reducing the fold surface free energy by up to 20%. Furthermore, these nanocomposites also show an improvement in both tribological and mechanical (hardness and modulus) properties with respect to pure PHB evidenced by friction and nanoindentation tests, which is of important potential interest for industrial and medical applications.