Study of the mechanical properties of fresh and cryopreserved individual human oocytes Abstract In assisted reproduction technologies, the cryopreservation of oocytes is a common procedure used to circumvent female infertility. However, some morphological and functional alterations of oocytes have been observed depending on the protocol applied. In this work, the mechanical response of individual human oocytes before and after a freeze-thawing procedure was characterised. Oocytes, immediately after retrieval, were morphologically evaluated by bright-field optical microscopy and their elasticity measured by indentation measurements using atomic force microscopy. Oocytes were then frozen according to the open-vitrification protocol and stored in liquid nitrogen. Afterwards, the same oocytes were thawed and the indentation measurements repeated. Using this approach, we can follow the elasticity of a set of single oocytes from retrieval up to the freeze-thawing procedure. The analysis of the resulting data shows that the retrieved healthy oocytes, which preserve their healthy morphological features after cryopreservation, maintain unchanged also in stiffness values. In contrast, oocytes having dysmorphic characteristics, before and/or after freeze-thawing, show significant variations in their mechanical response. In addition, the dysmorphic oocytes are generally observed to be softer than the healthy oocytes. Our results indicate that stiffness of healthy oocytes is not considerably affected by the open-vitrification-thawing procedure, and that distinct elasticity ranges can be identified for healthy and dysmorphic oocytes. These findings indicate that the mechanical characterization of oocytes represents an opportunity to detect cellular defects, and assess the quality and bio-viability of processes such as cryopreservation.

Microenvironment of tryptophan residues in proteins of four structural classes: applications for fluorescence and circular dichroism spectroscopy Abstract In this study we examined microenvironment of Trp residues in “dry” sets of nonhomologous proteins that belong to four structural classes, as well as in a “wet” set. In silico experiments showed that residues of Trp demonstrate higher surface accessibility in proteins of “alpha/beta” class where they are rarely included in beta strands. However, this feature has not caused “red” shift in fluorescence spectra in “alpha/beta” proteins in vitro, since there are several factors that should be combined together to cause it: high surface accessibility and high hydrophilicity of the microenvironment, the presence of destabilizing contacts with Asp, Asn, Leu, and multiple Tyr residues, as well as the lack of stabilizing interactions with Arg, Thr, and Pro. The occurrence of Trp residues has the highest value in beta-structural proteins, while they are not involved in aromatic–aromatic interactions with each other as frequently, as they do in proteins of “alpha + beta” class in which Trp residues are overrepresented near each other in the primary sequence. That is why the deformation of circular dichroism spectra because of Trp–Trp interactions is expected to be more frequent in proteins of “alpha + beta” class. In all four classes of proteins Trp residues are involved in long-range interactions with some hydrophobic (Leu, Val, Ile) and aromatic residues (Trp, Phe, and Tyr) more frequently than it is expected. They are involved in long-range interactions with some hydrophilic residues (Asp, Glu, Ser, and Lys) rarely than it is expected. Short-range interactions between Arg and Trp are overrepresented just in alpha-helical proteins.

Network analysis of dynamically important residues in protein structures mediating ligand-binding conformational changes Abstract According to the generalized conformational selection model, ligand binding involves the co-existence of at least two conformers with different ligand-affinities in a dynamical equilibrium. Conformational transitions between them should be guaranteed by intramolecular vibrational dynamics associated to each conformation. These motions are, therefore, related to the biological function of a protein. Positions whose mutations are found to alter these vibrations the most can be defined as key positions, that is, dynamically important residues that mediate the ligand-binding conformational change. In a previous study, we have shown that these positions are evolutionarily conserved. They correspond to buried aliphatic residues mostly localized in regular structured regions of the protein like β-sheets and α-helices. In the present paper, we perform a network analysis of these key positions for a large dataset of paired protein structures in the ligand-free and ligand-bound form. We observe that networks of interactions between these key positions present larger and more integrated networks with faster transmission of the information. Besides, networks of residues result that are robust to conformational changes. Our results reveal that the conformational diversity of proteins seems to be guaranteed by a network of strongly interconnected key positions rather than individual residues.

Solution structure and backbone dynamics for S1 domain of ribosomal protein S1 from Mycobacterium tuberculosis Abstract The pro-drug pyrazinamide is hydrolyzed to pyrazinoic acid (POA) in its use for the treatment of tuberculosis. As a molecule with bactericidal activity, POA binds to the C-terminal S1 domain of ribosomal protein S1 from Mycobacterium tuberculosis(MtRpsACTD_S1) to inhibit trans-translation. Trans-translation is a critical component of protein synthesis quality control, and is mediated by transfer-messenger RNA. Here, we have determined the solution structure of MtRpsACTD_S1(280–368), and analyzed its structural dynamics by NMR spectroscopy. The solution structure of MtRpsACTD_S1(280–368) mainly consists of five anti-parallel β strands, two α helices, and two 310 helices. Backbone dynamics reveals that the overall structure of MtRpsACTD_S1(280–368) is rigid, but segment L326–V333 undergoes large amplitude fluctuations on picosecond to nanosecond time scales. In addition, residues V321, H322, V331 and D335 with large Rex values exhibit significant chemical or conformational exchange on microsecond to millisecond time scale. Titration of the truncated MtRpsACTD_S1(280–368) with POA shows similar characteristics to titration of MtRpsACTD_S1(280–438) with POA. In addition, diverse length fragments of MtRpsACTD_S1 show various HN resonance signals, and we find that the interaction of MtRpsA(369–481) with MtRpsACTD_S1(280–368) [Kd = (4.25 ± 0.15) mM] is responsible for the structural difference between MtRpsACTD_S1(280–368) and MtRpsACTD_S1. This work may shed light on the underlying molecular mechanism of MtRpsACTD recognizing and binding POA or mRNA, as well as the detailed mechanism of interactions between MtRpsACTD_S1(280–368) and the additional C-terminal MtRpsA(369–481).

U-turn trajectories of magnetotactic cocci allow the study of the correlation between their magnetic moment, volume and velocity Abstract Magnetotactic bacteria are microorganisms that present intracellular chains of magnetic nanoparticles, the magnetosome chain. A challenge in the study of magnetotactic bacteria is the measurement of the magnetic moment associated with the magnetosome chain. Several techniques have been used to estimate the average magnetic moment of a population of magnetotactic bacteria, and others permit the measurement of the magnetic moment of individual bacteria. The U-turn technique allows the measurement of the individual magnetic moment and other parameters associated with the movement and magnetotaxis, such as the velocity and the orientation angle of the trajectory relative to the applied magnetic field. The aim of the present paper is to use the U-turn technique in a population of uncultured magnetotactic cocci to measure the magnetic moment, the volume, orientation angle and velocity for the same individuals. Our results showed that the magnetic moment is distributed in a log-normal distribution, with a mean value of 8.2 × 10–15 Am2 and median of 5.4 × 10–15 Am2. An estimate of the average magnetic moment using the average value of the orientation cosine produces a value similar to the median of the distribution and to the average magnetic moment obtained using transmission electron microscopy. A strong positive correlation is observed between the magnetic moment and the volume. There is no correlation between the magnetic moment and the orientation cosine and between the magnetic moment and the velocity. Those null correlations can be explained by our current understanding of magnetotaxis.

Growth of a gas bubble in a perfused tissue in an unsteady pressure field with source or sink Abstract In the context of decompression sickness, this paper presents analytical formulae and explanations for growth of a gas bubble in blood and other tissues in an unsteady diffusion field with a source or a sink. The formulae are valid for variable (through decompression) and constant (concerning diving stops/at sea level) ambient pressure. Under a linear decompression regime for ambient pressure, the gas bubble growth is proportional to ascent rate, tissue diffusivity and initial tissue tension and inversely proportional to surface tension, initial ambient pressure and the strength of the source/sink parameter \(k\) which gives the conditions for bubble growth. We find that the growth process is noticeably affected by changing k-values within a specified range, with no significant effect on the value of the bubble radius when k is outside this range. We discuss the effect of the presence of multiple bubbles, and of repetitive diving. Of the three available models for bubble growth, the predicted time to completion is longest in the model by Srinivasan et al. (J Appl Physiol 86:732–741, 1999), where the bubble grows in a steady diffusion field, but shortest in the model we describe for k-values closest to the boundaries of the interval \([0. 9 5 8 7,\;\;1.0]\) . This is because our model considers the effect of the presence of a source, increasing the bubble growth rate and not taken into account in our previous (2010) model predicting an intermediate timeframe for bubble growth. We believe our new model provides a more accurate and widely applicable description of bubble growth in decompression sickness than previous versions.

Stochastic and deterministic approaches to modelling calcium release in cardiac myocytes at different spatial arrangements of ryanodine receptors Abstract Calcium release sites (CRSs) play a key role in excitation–contraction coupling of cardiac myocytes. Recent studies based on electron tomography and super-resolution imaging revealed that CRSs are not completely filled with ryanodine receptors (RyRs) and that the spatial arrangement of RyRs is neither uniform nor static. In this work, we studied the effect of spatial arrangement of RyRs on RyR activation using simulations based on Monte Carlo (MC) and mean-field (MF) approaches. Both approaches showed that activation of RyRs is sensitive to the arrangement of RyRs in the CRS. However, the MF simulations did not reproduce results of MC simulations for non-compact CRSs, suggesting that the approximations used in the MF approach are not suitable for simulation studies of RyRs arrangements observed experimentally. MC simulations revealed the importance of realistic spatial arrangement of RyRs for adequate modelling of calcium release in cardiac myocytes.

Liposome production and concurrent loading of drug simulants by microfluidic hydrodynamic focusing Abstract Liposomes are spherical vesicles enclosed by phospholipid bilayers. Nanoscale liposomes are widely employed for drug delivery in the pharmaceutical industry. In this study, nanoscale liposomes are fabricated using the microfluidic hydrodynamic focusing (MHF) approach, and the effects of flow rate ratio (FRR) on liposome size and drug loading efficiency are studied. Fluorescein isothiocyanate modified dextran is used as a hydrophilic drug simulant and Nile red is used as a hydrophobic drug simulant. The experiment results show that hydrophilic drug simulant loading efficiency increases as FRR increases and eventually plateaues at around 90% loading efficiency. The hydrophobic drug simulant loading efficiency and FRR have a positive linear correlation when FRR varies from 10 to 50. Concurrent loading of both hydrophilic and hydrophobic drug simulants maintains the same loading efficiencies as those of loading each drug simulant alone. A negative correlation between liposome size and FRR is also confirmed. Unloaded liposomes and hydrophilic drug-loaded liposomes are of the same sizes, and are smaller than the ones loaded with the hydrophobic drug simulants alone or combined. The results suggest tunable liposome size and drug loading efficiency with the MHF technique. This provides evidence to encourage further studies of microfluidic liposome fabrication in the pharmaceutical industry.

An estimate to the first approximation of microtubule rupture force Abstract Microtubule mechanical properties are essential for understanding basic cellular processes, including cell motility and division, but the forces that result in microtubule rupture or breakage have not yet been measured directly. These forces are essential to understand the mechanical properties of the cytoskeleton and responses by cells to both normal conditions and stress caused by injury or disease. Here we estimate the force required to rupture a microtubule by analyzing kinesin-14 Ncd motor-induced microtubule breakage in ensemble motility assays. We model the breakage events as caused by Ncd motors pulling or pushing on single microtubules that are clamped at one end by other motors attached to the glass surface. The number of pulling or pushing Ncd motors is approximated from the length of the microtubule bound to the surface and the forces produced by the pulling or pushing motors are estimated from forces produced by the Ncd motor in laser-trap assays, reported by others. Our analysis provides an estimate, to the first approximation, of ~ 500 pN for the minimal force required to rupture a 13-pf microtubule. The value we report is close to the forces estimated from microtubule stretching/fragmentation experiments and overlaps with the forces applied by AFM in microtubule indentation assays that destabilize microtubules and break microtubule protofilaments. It is also consistent with the forces required to disrupt protein noncovalent bonds in force spectroscopy experiments. These findings are relevant to microtubule deformation and breakage caused by cellular tension in vivo.

Inhibition of influenza virus activity by the bovine seminal plasma protein PDC-109 Abstract A number of viruses causing sexually transmissible diseases are transmitted via mammalian seminal plasma. Several components of seminal plasma have been shown to influence those viruses and their physiological impact. To unravel whether components of seminal plasma could affect viruses transmitted via other pathways, it was investigated here whether the bovine seminal plasma protein PDC-109, belonging to the Fn-type 2 protein family, influences the activity of influenza A viruses, used as a model for enveloped viruses. We found that PDC-109 inhibits the fusion of influenza virus with human erythrocyte membranes and leads to a decreased viral infection in MDCK cells. In the presence of the head group of the phospholipid phosphatidylcholine, phosphorylcholine, the inhibitory effect of PDC-109 was attenuated. This indicates that the impact of the protein is mainly caused by its binding to viral and to erythrocyte membranes thereby interfering with virus-cell binding. Our study underlines that Fn-type 2 proteins have to be considered as new antiviral components present in mammalian seminal plasma.