Pluripotency of embryonic stem cells lacking clathrin-mediated endocytosis cannot be rescued by restoring cellular stiffness

Mouse embryonic stem cells (mESCs) display unique mechanical properties, including low cellular stiffness in contrast to differentiated cells, which are stiffer. We have previously shown that mESCs lacking the clathrin heavy chain (Cltc), an essential component for clathrin-mediated endocytosis (CME), display a loss of pluripotency and an enhanced expression of differentiation markers. However, it is not known whether physical properties such as cellular stiffness also change upon loss of Cltc, similar to what is seen in differentiated cells, and if so, how these altered properties specifically impact pluripotency. Using atomic force microscopy (AFM), we demonstrate that mESCs lacking Cltc display higher Young''s modulus, indicative of greater cellular stiffness, compared with WT mESCs. The increase in stiffness was accompanied by the presence of actin stress fibers and accumulation of the inactive, phosphorylated, actin-binding protein cofilin. Treatment of Cltc knockdown mESCs with actin polymerization inhibitors resulted in a decrease in the Young''s modulus to values similar to those obtained with WT mESCs. However, a rescue in the expression profile of pluripotency factors was not obtained. Additionally, whereas WT mouse embryonic fibroblasts could be reprogrammed to a state of pluripotency, this was inhibited in the absence of Cltc. This indicates that the presence of active CME is essential for the pluripotency of embryonic stem cells. Additionally, whereas physical properties may serve as a simple readout of the cellular state, they may not always faithfully recapitulate the underlying molecular fate.     

The surface structure of a complex substrate revealed by enzyme kinetics and Freundlich constants for α-amylase interaction with the surface of starch

Background

Starch is a main source of carbohydrate in human diets, but differences are observed in postprandial glycaemia following ingestion of different foods containing identical starch contents. Such differences reflect variations in rates at which different starches are digested in the intestine. In seeking explanations for these differences, we have studied the interaction of α-amylase with starch granules. Understanding this key step in digestion should help with a molecular understanding for observed differences in starch digestion rates.

Methods

For enzymes acting upon solid substrates, a Freundlich equation relates reaction rate to enzyme adsorption at the surface. The Freundlich exponent (n) equals 2/3 for a liquid-smooth surface interface, 1/3 for adsorption to exposed edges of ordered structures and 1.0 for solution–solution interfaces. The topography of a number of different starch granules, revealed by Freundlich exponents, was compared with structural data obtained by differential scanning calorimetry and Fourier transform infrared spectroscopy with attenuated total internal reflectance (FTIR-ATR).

Results

Enzyme binding rate and FTIR-ATR peak ratio were directly proportional to n and Δ_gelH was inversely related to n. Amylase binds fastest to solubilised starch and to granules possessing smooth surfaces at the solid–liquid interface and slowest to granules possessing ordered crystalline surfaces.

Conclusions

Freundlich exponents provide information about surface blocklet structures of starch that supplements knowledge obtained from physical methods.

General Significance

Nanoscale structures at the surface of starch granules influence hydrolysis by α-amylase. This can be important in understanding how dietary starch is digested with relevance to diabetes, cardiovascular health and cancer.     

Attachment strength of an adhesive nuisance mussel,limnoperna fortunei,against water flow

The attachment strength of the freshwater mussel Limnoperna fortunei against water flow was studied. Newton's expression successfully described the hydrodynamic drag force acting on the mussel with a drag coefficient value of 1.03. The drag‐resistant force (defined as hydrodynamic drag force at mussel detachment) was smaller than the detachment force measured using a tensile load test. A fairly good correlation was obtained between the drag‐resistant force and the number of secreted threads. The drag‐resistant force divided by the number of threads increased with shell size, suggesting that byssal thread strength increased with mussel growth. For the mussel specimens obtained from a water transmission pipe, thread width increased with shell size. However, thread width was not dependent on current velocity. There was no correlation between the number of secreted threads and shell length, which indicated that the number of secreted threads did not change with mussel size. Therefore, the water velocity needed to detach mussels increases with shell size of the mussel when the number of secreted threads is constant. The increases in the water velocity to detach mussels with larger shells suggests that the mussel becomes more resistant to water flow as it grows. It is estimated that a flow velocity of around lms^‐1 is critical for attachment/detachment of a juvenile mussel with a shell length of a few millimeters and one hundred byssal threads.     

Impact of cranberry juice on initial adhesion of the EPS producing bacterium Burkholderia cepacia

The impact of cranberry juice was investigated with respect to the initial adhesion of three isogenic strains of the bacterium Burkholderia cepacia with different extracellular polymeric substance (EPS) producing capacities, viz. a wild-type cepacian EPS producer PC184 and its mutant strains PC184rml with reduced EPS production and PC184bceK with a deficiency in EPS production. Adhesion experiments conducted in a parallel-plate flow chamber demonstrated that, in the absence of cranberry juice, strain PC184 had a significantly higher adhesive capacity compared to the mutant strains. In the presence of cranberry juice, the adhesive capacity of the EPS-producing strain PC184 was largely reduced, while cranberry juice had little impact on the adhesion behavior of either mutant strain. Thermodynamic modeling supported the results from adhesion experiments. Surface force apparatus (SFA) and scanning electron microscope (SEM) studies demonstrated a strong association between cranberry juice components and bacterial EPS. It was concluded that cranberry juice components could impact bacterial initial adhesion by adhering to the EPS and impairing the adhesive capacity of the cells, which provides an insight into the development of novel treatment strategies to block the biofilm formation associated with bacterial infection.     

Mechanical properties of a mature biofilm from a wastewater system: from microscale to macroscale level

A fundamental understanding of biofilm mechanical stability is critical in order to describe detachment and develop biofouling control strategies. It is thus important to characterise the elastic deformation and flow behaviour of the biofilm under different modes of applied force. In this study, the mechanical properties of a mature wastewater biofilm were investigated with methods including macroscale compression and microscale indentation using atomic force microscopy (AFM). The mature biofilm was found to be mechanically isotropic at the macroscale level as its mechanical properties did not depend on the scales and modes of loading. However, the biofilm showed a tendency for mechanical inhomogeneity at the microscale level as indentation progressed deeper into the matrix. Moreover, it was observed that the adhesion force had a significant influence on the elastic properties of the biofilm at the surface, subjected to microscale tensile loading. These results are expected to inform a damage-based model for biofilm detachment.     

Review – Interactions between diatoms and stainless steel: focus on biofouling and biocorrosion

There is a considerable body of information regarding bacterially enhanced corrosion, however, this review focuses on diatoms (unicellular algae) whose contribution to biocorrosion is less well studied. The reasons why diatoms have been neglected in studies of biocorrosion in natural waters are discussed and the question whether diatoms should be considered as inert with respect of electrochemical processes is considered. A particular focus is given to the case of stainless steels (SS), which are widely used in variety of applications in natural waters. Basic information on the cell biology of diatoms is included in the review, particularly with respect to their ability to ‘sense’ and adhere to surfaces. Investigations at the nanoscale are reviewed as these studies provide information about the behavior of cells at interfaces. Recent advances include the use of atomic force microscopy (AFM), although only a few studies have been applied to diatoms. Regarding the electrochemical behavior of SS, the mechanisms by which diatoms influence the potential ennoblement process is discussed. Such studies reveal the association of diatoms, in addition to bacteria, with biocorrosion processes.     

Microfluidic detachment assay to probe the adhesion strength of diatoms

Fouling release (FR) coatings are increasingly applied as an environmentally benign alternative for controlling marine biofouling. As the technology relies on removing fouling by water currents created by the motion of ships, weakening of adhesion of adherent organisms is the key design goal for improved coatings. In this paper, a microfluidic shear force assay is used to quantify how easily diatoms can be removed from surfaces. The experimental setup and the optimization of the experimental parameters to study the adhesion of the diatom Navicula perminuta are described. As examples of how varying the physico-chemical surface properties affects the ability of diatoms to bind to surfaces, a range of hydrophilic and hydrophobic self-assembled monolayers was compared. While the number of cells that attached (adhered) was barely affected by the coatings, the critical shear stress required for their removal from the surface varied significantly.     

Controlling the secondary-structure-forming tendencies of proteins by a backbone torsion-energy term

We examined a new backbone torsion-energy term proposed by us in the force field for protein systems. This torsion-energy term is represented by a double Fourier series in two variables, namely the backbone dihedral angles φ and ψ. It gives a natural representation of the torsion energy in the Ramachandran space in the sense that any two-dimensional energy surface periodic in both φ and ψ can be expanded by the double Fourier series. We can then easily control secondary-structure-forming tendencies by modifying the torsion-energy surface. For instance, we can increase or decrease the α-helix-forming-tendencies by lowering or raising the torsion-energy surface in the α-helix region and likewise increase or decrease the β-sheet-forming tendencies by lowering or raising the surface in the β-sheet region in the Ramachandran space. We applied this torsion-energy modification method to six force fields, AMBER parm94, AMBER parm96, AMBER parm99, CHARMM27, OPLS-AA and OPLS-AA/L, and demonstrated that our modifications of the torsion-energy terms resulted in the expected changes of secondary-structure-forming tendencies by performing folding simulations of α-helical and β-hairpin peptides.