Mucolytic activity of bacterial and human chitinases

Several pulmonary pathologies, like cystic fibrosis (CF), are characterized by hypersecretion and stasis of tenacious mucus. Bacterial glycosidases are known to degrade mucins but their use as mucolytic agents is questionable. The observation that bacterial chitinases degrade mucins and the recent discovery of human chitinases, which have been proposed to be involved in the genesis of asthma, prompted us to evaluate the mucolytic properties of human derived chitinases. The effect of these human chitinases, and bacterial chitinases (positive control), on the viscoelasticity of CF sputa and on the electrophoretic mobility of human mucins was tested. Commercial bacterial chitinase drastically degraded CF sputum, while human derived chitinases did not. Accordingly, the commercial bacterial chitinase was found to degrade mucins, whereas recombinant human chitinases did not. A thorough analysis of the commercial chitinase elucidated that contaminating proteases and also nucleases assisted in the mucolytic effect. Indeed, recombinant bacterial chitinases very slightly reduced the viscoelasticity of CF sputum, but they caused a significant degradation of the CF sputum when they were combined with proteases. In conclusion, this work shows that recombinant human and recombinant bacterial chitinases have no or very low mucolytic activities, respectively. The observed mucolytic properties of commercial bacterial chitinase are due to a synergistic effect between chitinolytic and proteolytic enzymes at one hand and at the other hand also due to the presence of contaminating nucleases.     

Proteomic analysis of bovine skeletal muscle hypertrophy

Myostatin plays a major role in muscle growth and development and animals with disruption of this gene display marked increases in muscle mass. Little is known about muscle physiological adaptations in relation to this muscle hypertrophy. To provide a more comprehensive view, we analyzed bovine muscles from control, heterozygote and homozygote young Belgian blue bulls for myostatin deletion, which results in a normal level of inactive myostatin. Heterozygote and homozygote animals were characterized by a higher proportion of fast-twitch glycolytic fibers in Semitendinosus muscle. Differential proteomic analysis of this muscle was performed using two-dimensional gel electrophoresis followed by mass spectrometry. Thirteen proteins, corresponding to 28 protein spots, were significantly altered in response to the myostatin deletion. The observed changes in protein expression are consistent with an increased fast muscle phenotype, suggesting that myostatin negatively controls mainly fast-twitch glycolytic fiber number. Finally, we demonstrated that differential mRNA splicing of fast troponin T is altered by the loss of myostatin function. The structure of mutually exclusive exon 16 appears predominantly expressed in muscles from heterozygote and homozygote animals. This suggests a role for exon 16 of fast troponin T in the physiological adaptation of the fast muscle phenotype.     

The impact of nanoparticle-driven lysosomal alkalinization on cellular functionality

Background

The biomedical use of nanosized materials is rapidly gaining interest, which drives the quest to elucidate the behavior of nanoparticles (NPs) in a biological environment. Apart from causing direct cell death, NPs can affect cellular wellbeing through a wide range of more subtle processes that are often overlooked. Here, we aimed to study the effect of two biomedically interesting NP types on cellular wellbeing.

Results

In the present work, gold and SiO₂ NPs of similar size and surface charge are used and their interactions with cultured cells is studied. Initial screening shows that at subcytotoxic conditions gold NPs induces cytoskeletal aberrations while SiO₂ NPs do not. However, these transformations are only transient. In-depth investigation reveals that Au NPs reduce lysosomal activity by alkalinization of the lysosomal lumen. This leads to an accumulation of autophagosomes, resulting in a reduced cellular degradative capacity and less efficient clearance of damaged mitochondria. The autophagosome accumulation induces Rac and Cdc42 activity, and at a later stage activates RhoA. These transient cellular changes also affect cell functionality, where Au NP-labelled cells display significantly impeded cell migration and invasion.

Conclusions

These data highlight the importance of in-depth understanding of bio-nano interactions to elucidate how one biological parameter (impact on cellular degradation) can induce a cascade of different effects that may have significant implications on the further use of labeled cells.

     

Improved Optics in Monolithic Perovskite/Silicon Tandem Solar Cells with a Nanocrystalline Silicon Recombination Junction

Perovskite/silicon tandem solar cells are increasingly recognized as promi­sing candidates for next‐generation photovoltaics with performance beyond the single‐junction limit at potentially low production costs. Current designs for monolithic tandems rely on transparent conductive oxides as an intermediate recombination layer, which lead to optical losses and reduced shunt resistance. An improved recombination junction based on nanocrystalline silicon layers to mitigate these losses is demonstrated. When employed in monolithic perovskite/silicon heterojunction tandem cells with a planar front side, this junction is found to increase the bottom cell photocurrent by more than 1 mA cm^?2. In combination with a cesium‐based perovskite top cell, this leads to tandem cell power‐conversion efficiencies of up to 22.7% obtained from J–V measurements and steady‐state efficiencies of up to 22.0% during maximum power point tracking. Thanks to its low lateral conductivity, the nanocrystalline silicon recombination junction enables upscaling of monolithic perovskite/silicon heterojunction tandem cells, resulting in a 12.96 cm² monolithic tandem cell with a steady‐state efficiency of 18%.     

Modeling the biomechanics of fetal movements

Fetal movements in the uterus are a natural part of development and are known to play an important role in normal musculoskeletal development. However, very little is known about the biomechanical stimuli that arise during movements in utero, despite these stimuli being crucial to normal bone and joint formation. Therefore, the objective of this study was to create a series of computational steps by which the forces generated during a kick in utero could be predicted from clinically observed fetal movements using novel cine-MRI data of three fetuses, aged 20–22 weeks. A custom tracking software was designed to characterize the movements of joints in utero, and average uterus deflection of \(6.95 \pm 0.41\) mm due to kicking was calculated. These observed displacements provided boundary conditions for a finite element model of the uterine environment, predicting an average reaction force of \(0.52 \pm 0.15\) N generated by a kick against the uterine wall. Finally, these data were applied as inputs for a musculoskeletal model of a fetal kick, resulting in predicted maximum forces in the muscles surrounding the hip joint of approximately 8 N, while higher maximum forces of approximately 21 N were predicted for the muscles surrounding the knee joint. This study provides a novel insight into the closed mechanical environment of the uterus, with an innovative method allowing elucidation of the biomechanical interaction of the developing fetus with its surroundings.     

Chemically Induced Polyploidization in Spathiphyllum wallisii Regel through Somatic Embryogenesis

Polyploid Spathiphyllum wallisii Regel 'Speedy' plants were obtained from somatic embryos induced on anther filaments exposed to mitosis inhibitors. Primary embryos yielded less polyploid plantlets than secondary embryos. Colchicine could be efficiently replaced by oryzalin or trifluralin (10 μM), resulting in an average yield of 5% polyploids. The mitosis inhibitors were directly added to the induction medium. Morphological differences between diploid and tetraploid S. wallisii hybrids were observed. Throughout our experiments flow cytometry was applied as an unambiguous screening tool. Ploidy breeding within the genus Spathiphyllum holds promises towards the development of tetraploid hybrids with an altered morphology, or triploids with a reduced fertility.     

Composition of organic matter in sandy relict and cultivated heathlands as examined by pyrolysis-field ionization MS

Unusually high SOC levels have been reported for sandy cropland soils in North-Western Europe. A potential link with their general heathland land-use history was investigated by comparing two soil pairs of relict heathland and cultivated former heathland in the Belgian sandy region. A sequential chemical fractionation yielded similar sizes in corresponding SOM fractions between the heathland and cropland soils (i.e. NaOCl resistant: 12.3–15.0 g C kg⁻¹ and NaOCl + HF resistant: 2.6–5.3 g C kg⁻¹). Higher amounts of clay sized N in the cropland plots can be attributed to N additions from mineral fertilizers and animal manure. Temperature resolved Pyrolysis Field Ionization Mass Spectroscopy analysis showed that the composition of both relict heathland and cultivated soils was surprisingly similar, in spite of over 60 years of intense cropland management. The mass spectra of SOM in both heathland-cropland soil pairs investigated was dominated by signals from lipids, alkylaromatics and sterols. The accumulation of this SOM rich in aliphatics was logically linked to the high input of lipids, long-chain aliphatics and sterols from heathland vegetation and the low soil pH and microbial activity. Based on the relatively high OC surface loadings of HF-extractable OM (13–44 mg C m⁻² Fe and 1.2–2.3 mg C m⁻² clay), direct organo-mineral bonds between OM and Fe-oxides or clay minerals seem to be only partly involved as a stabilization mechanism in these soils. The distinct bimodal shape of the thermograms indicates that OM-crosslinking could furthermore contribute substantially to SOM stabilization in these soils. This study therefore corroborates the previously proposed view that lipids may be bound in networks of alkylaromatics, the structural building blocks of OM macromolecules. We hypothesize that such binding is able to explain the measured retention of these OM components, even under several decades of cropland management.     

On the role of calcium as second messenger in liver for the hormonally induced activation of glycogen phosphorylase

We have studied the mode of action of three hormones (angiotensin, vasopressin and phenylephrine, an α-adrenergic agent) which promote liver glycogenolysis in a cyclic AMP-independent way, in comparison with that of glucagon, which is known to act essentially via cyclic AMP. The following observations were made using isolated rat hepatocytes: (a) In the normal Krebs-Henseleit bicarbonate medium, the hormones activated glycogen phosphorylase (EC 2.4.1.1) to about the same degree. In contrast to glucagon, the cyclic AMP-independent hormones did not activate either protein kinase (EC 2.7.1.37) or phosphorylase $\text{b}$ kinase (EC 2.7.1.38). (b) The absence of Ca²⁺ from the incubation medium prevented the activation of glycogen phosphorylase by the cyclic AMP-independent agents and slowed down that induced by glucagon. (c) The ionophore A 23187 produced the same degree of activation of glycogen phosphorylase, provided that Ca²⁺ was present in the incubation medium (d) Glucagon, cyclic AMP and three cyclic AMP-independent hormones caused an enhanced uptake of ⁴⁵Ca; it was verified that concentrations of angiotensin and of vasopressin known to occur in haemorrhagic conditions were able to produce phosphorylase activation and stimulate ⁴⁵Ca uptake. (e) Appropriate antagonists (i.e. phentolamine against phenylephrine and an angiotensin analogue against angiotensin) prevented both the enhanced ⁴⁵Ca uptake and the phosphorylase activation.We interpret our data in favour of a role of calcium (1) as the second messenger in liver for the three cyclic AMP-independent glycogenolytic hormones and (2) as an additional messenger for glucagon which, via cyclic AMP, will make calcium available to the cytoplasm either from extracellular or from intracellular pools. The target enzyme for Ca²⁺ is most probably phosphorylase $\text{b}$ kinase.