Porcine oocyte maturation in vitro : role of cAMP and oocyte-secreted factors: a practical approach

Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling

Effects of grain shape on packing and dilatancy of sheared granular materials

Granular material exposed to shear shows a variety of unique phenomena: Reynolds dilatancy, positional order and orientational order effects may compete in the shear zone. We study granular packings consisting of macroscopic prolate, oblate and spherical grains and compare their behaviour. X-ray tomography is used to determine the particle positions and orientations in a cylindrical split bottom shear cell. Packing densities and the arrangements of individual particles in the shear zone are evaluated. For anisometric particles, we observe the competition of two opposite effects. One the one hand, the sheared granulate is dilated, but on the other hand the particles reorient and align with respect to the streamlines. Even though aligned cylinders in principle may achieve higher packing densities, this alignment compensates for the effect of dilatancy only partially. The complex rearrangements lead to a depression of the surface above the well oriented region while neigbouring parts still show the effect of dilation in the form of heaps. For grains with isotropic shapes, the surface remains rather flat. Perfect monodisperse spheres crystallize in the shear zone, whereby positional order partially overcompensates dilatancy effects. However, already slight deviations from the ideal monodisperse sphere shape inhibit crystallization.Comment: 12 pages, 13 figures, accepted in Soft Matte

Heaping, Secondary Flows and Broken Symmetry in Flows of Elongated Granular Particles

In this paper we report experiments where we shear granular rods in split-bottom geometries, and find that a significant heap of height of least 40% of the filling height can form at the particle surface. We show that heaping is caused by a significant secondary flow, absent for spherical particles. Flow reversal transiently reverses the secondary flow, leading to a quick collapse and slower regeneration of the heap. We present a symmetry argument and experimental data that show that the generation of the secondary flow is driven by a misalignment of the mean particle orientation with the streamlines of the flow. This general mechanism is expected to be important in all flows of sufficiently anisometric grains.Comment: Accepted for Soft Matte

Jeffery Orbits with Noise Revisited

The behavior of non-spherical particles in a shear-flow is of significant practical and theoretical interest. These systems have been the object of numerous investigations since the pioneering work of Jeffery a century ago. His eponymous orbits describe the deterministic motion of an isolated, rod-like particle in a shear flow. Subsequently, the effect of adding noise was investigated. The theory has been applied to colloidal particles, macromolecules, anisometric granular particles and most recently to microswimmers, for example bacteria. We study the Jeffery orbits of elongated particles subject to noise using Langevin simulations and a Fokker-Planck equation. We extend the analytical solution for infinitely thin needles ($\beta=1$) obtained by Doi and Edwards to particles with arbitrary shape factor ($0\le \beta\le 1$) and validate the theory by comparing it with simulations. We examine the rotation of the particle around the vorticity axis and study the orientational order matrix. We use the latter to obtain scalar order parameters $s$ and $r$ describing nematic ordering and biaxiality from the orientational distribution function. The value of $s$ (nematic ordering) increases monotonically with increasing P\'eclet number, while $r$ (measure of biaxiality) displays a maximum value. From perturbation theory we obtain simple expressions that provide accurate descriptions at low noise (or large P\'eclet numbers). We also examine the orientational distribution in the v-grad v plane and in the perpendicular direction. Finally we present the solution of the Fokker-Planck equation for a strictly two-dimensional (2D) system. For the same noise amplitude the average rotation speed of the particle in 3D is larger than in 2D

Drug and dye binding induced folding of the intrinsically disordered antimicrobial peptide CM15

The rapid increase of antimicrobial resistance against conventional antibiotics has resulted in a significant focus on the use of peptides as antimicrobial agents. Understanding the structure and function relationships of these compounds is thus highly important, however, their in vivo actions are a complex issue, including interactions with small molecule agents. Here we report the folding inducing capability of some pharmaceutical substances and synthetic dyes on the intrinsically disordered (ID) cationic antimicrobial peptide CM15 (KWKLFKKIGAVLKVL). By employing circular dichroism (CD) spectroscopy, it is shown that some therapeutic drugs (suramin, pamoic acid, cromolyn) and polysulfonated dyes (Congo red, trypan blue) trigger the disorder-to-order conformational transition of CM15. The cooperative binding of 2-4 acidic molecules per peptide chain provokes its folding in a concentration dependent manner. Secondary structure analysis indicated the sharp and moderate rise of the [small alpha]-helical and [small beta]-sheet content, respectively. According to semi-empirical quantum chemical calculations, these organic molecules may induce folding by forming multiple salt-bridges with lysine residues from both N- and C-terminals as well as from the middle of the CM15 sequence. Due to the mutual neutralization of the positive and negative charges, the water solubility of the resulting complexes decreases which favours their aggregation as detected by dynamic light scattering measurements. Our findings suggest that small molecules can dramatically affect the structure of antimicrobial peptides, which may potentially alter, either enhancing or attenuating, their efficiency. It is proposed that CM15 or similar ID peptides could be useful for preliminary screening of folding inducer effect of anionic drugs and biomolecules. The data presented herein may stimulate further studies on the structural and functional impacts of related compounds on ID peptides

Disorder-to-helix conformational conversion of the human immunomodulatory peptide LL-37 induced by antiinflammatory drugs, food dyes and some metabolites

The human antimicrobial and immunomodulatory peptide LL-37 is ubiquitously expressed and secreted by epithelial cells of mucosal surfaces including the gastrointestinal tract, the primary absorption site of orally administered drugs and food components. Besides antimicrobial properties, LL-37 also contributes to the pathophysiology of various diseases such as ulcerative colitis, Crohn's disease and cancer. The non-covalent association of antiinflammatory drugs, porphyrin pigments, bile salts and food dyes to the peptide was uncovered and evaluated by circular dichroism (CD) spectroscopy. These agents induce the disorder-to-order conformational transition of the natively unstructured LL-37 leading to its helical folding. Even in the presence of chloride, where LL-37 is partially folded, these small molecules were able to rise the α-helix content. CD titration data indicated positive cooperativity between the ligand molecules accommodated to the peptide chain resulting in multimeric complexes with apparent dissociation constants ranged from 2 to 500 μM. Computational docking suggested the prominent role of the Lys8-Arg19 segment of LL-37 in the accommodation of ligand molecules, governed principally by salt bridges and H-bonding. Since pleiotropic biological functions of LL-37 are strongly conformation-dependent it could be anticipated that folding inducer compounds may modulate its in vivo actions and of related cationic peptides

Comparative Study of Molecular Mechanics Force Fields for β-Peptidic Foldamers: Folding and Self-Association

Computer-assisted study and design of non-natural peptidomimetics is increasingly important in the development of novel constructs with widespread applicability. Among these methods, molecular dynamics can accurately describe monomeric as well as oligomeric states of these compounds. We studied seven different sequences composed of cyclic and acyclic β-amino acids, the closest homologues of natural peptides, and compared the performance on them of three force field families in which specific modifications were made to improve reproduction of β-peptide structures. Altogether 17 systems were simulated, each for 500 ns, testing multiple starting conformations and in three cases also oligomer formation and stability from eight β-peptide monomers. The results indicated that our recently developed CHARMM force field extension, based on torsional energy path matching of the β-peptide backbone against quantum-chemical calculations, performs best overall, reproducing the experimental structures accurately in all monomeric simulations and correctly describing all the oligomeric examples. The Amber and GROMOS force fields could only treat some of the seven peptides (four in each case) without further parametrization. Amber was able to reproduce the experimental secondary structure of those β-peptides which contained cyclic β-amino acids, while the GROMOS force field had the lowest performance in this sense. From the latter two, Amber was able to hold together already formed associates in the prepared state but was not able to yield spontaneous oligomer formation in the simulations

Comparative Study of Molecular Mechanics Force Fields for β-Peptidic Foldamers: Folding and Self-Association

Computer-assisted study and design of non-natural peptidomimetics is increasingly important in the development of novel constructs with widespread applicability. Among these methods, molecular dynamics can accurately describe monomeric as well as oligomeric states of these compounds. We studied seven different sequences composed of cyclic and acyclic β-amino acids, the closest homologues of natural peptides, and compared the performance on them of three force field families in which specific modifications were made to improve reproduction of β-peptide structures. Altogether 17 systems were simulated, each for 500 ns, testing multiple starting conformations and in three cases also oligomer formation and stability from eight β-peptide monomers. The results indicated that our recently developed CHARMM force field extension, based on torsional energy path matching of the β-peptide backbone against quantum-chemical calculations, performs best overall, reproducing the experimental structures accurately in all monomeric simulations and correctly describing all the oligomeric examples. The Amber and GROMOS force fields could only treat some of the seven peptides (four in each case) without further parametrization. Amber was able to reproduce the experimental secondary structure of those β-peptides which contained cyclic β-amino acids, while the GROMOS force field had the lowest performance in this sense. From the latter two, Amber was able to hold together already formed associates in the prepared state but was not able to yield spontaneous oligomer formation in the simulations