Mechanical Strain Stabilizes Reconstituted Collagen Fibrils against Enzymatic Degradation by Mammalian Collagenase Matrix Metalloproteinase 8 (MMP-8)

Background

Collagen, a triple-helical, self-organizing protein, is the predominant structural protein in mammals. It is found in bone, ligament, tendon, cartilage, intervertebral disc, skin, blood vessel, and cornea. We have recently postulated that fibrillar collagens (and their complementary enzymes) comprise the basis of a smart structural system which appears to support the retention of molecules in fibrils which are under tensile mechanical strain. The theory suggests that the mechanisms which drive the preferential accumulation of collagen in loaded tissue operate at the molecular level and are not solely cell-driven. The concept reduces control of matrix morphology to an interaction between molecules and the most relevant, physical, and persistent signal: mechanical strain.

Methodology/Principal Findings

The investigation was carried out in an environmentally-controlled microbioreactor in which reconstituted type I collagen micronetworks were gently strained between micropipettes. The strained micronetworks were exposed to active matrix metalloproteinase 8 (MMP-8) and relative degradation rates for loaded and unloaded fibrils were tracked simultaneously using label-free differential interference contrast (DIC) imaging. It was found that applied tensile mechanical strain significantly increased degradation time of loaded fibrils compared to unloaded, paired controls. In many cases, strained fibrils were detectable long after unstrained fibrils were degraded.

Conclusions/Significance

In this investigation we demonstrate for the first time that applied mechanical strain preferentially preserves collagen fibrils in the presence of a physiologically-important mammalian enzyme: MMP-8. These results have the potential to contribute to our understanding of many collagen matrix phenomena including development, adaptation, remodeling and disease. Additionally, tissue engineering could benefit from the ability to sculpt desired structures from physiologically compatible and mutable collagen.     

Effect of muscle length on cross-bridge kinetics in intact cardiac trabeculae at body temperature

Dynamic force generation in cardiac muscle, which determines cardiac pumping activity, depends on both the number of sarcomeric cross-bridges and on their cycling kinetics. The Frank–Starling mechanism dictates that cardiac force development increases with increasing cardiac muscle length (corresponding to increased ventricular volume). It is, however, unclear to what extent this increase in cardiac muscle length affects the rate of cross-bridge cycling. Previous studies using permeabilized cardiac preparations, sub-physiological temperatures, or both have obtained conflicting results. Here, we developed a protocol that allowed us to reliably and reproducibly measure the rate of tension redevelopment (k_tr; which depends on the rate of cross-bridge cycling) in intact trabeculae at body temperature. Using K⁺ contractures to induce a tonic level of force, we showed the k_tr was slower in rabbit muscle (which contains predominantly β myosin) than in rat muscle (which contains predominantly α myosin). Analyses of k_tr in rat muscle at optimal length (L_opt) and 90% of optimal length (L₉₀) revealed that k_tr was significantly slower at L_opt (27.7 ± 3.3 and 27.8 ± 3.0 s⁻¹ in duplicate analyses) than at L₉₀ (45.1 ± 7.6 and 47.5 ± 9.2 s⁻¹). We therefore show that k_tr can be measured in intact rat and rabbit cardiac trabeculae, and that the k_tr decreases when muscles are stretched to their optimal length under near-physiological conditions, indicating that the Frank–Starling mechanism not only increases force but also affects cross-bridge cycling kinetics.     

Lentiviral Expression of Rabies Virus Glycoprotein in the Rat Hippocampus Strengthens Synaptic Plasticity

Rabies virus (RABV) is a neurotropic virus exclusively infecting neurons in the central nervous system. RABV encodes five proteins. Among them, the viral glycoprotein (RVG) plays a key role in viral entry into neurons and rabies pathogenesis. It was shown that the nature of the C-terminus of the RABV G protein, which possesses a PDZ-binding motif (PBM), modulates the virulence of the RABV strain. The neuronal protein partners recruited by this PBM may alter host cell function. This study was conducted to investigate the effect of RVG on synaptic function in the hippocampal dentate gyrus (DG) of rat. Two μl (10⁸ T.U./ml) of the lentiviral vector containing RVG gene was injected into the DG of rat hippocampus. After 2 weeks, the rat’s brain was cross-sectioned and RVG-expressing cells were detected by fluorescent microscopy. Hippocampal synaptic activity of the infected rats was then examined by recording the local field potentials from DG after stimulation of the perforant pathway. Short-term synaptic plasticity was also assessed by double pulse stimulation. Expression of RVG in DG increased long-term potentiation population spikes (LTP-PS), whereas no facilitation of LTP-PS was found in neurons expressing δRVG (deleted PBM). Furthermore, RVG and δRVG strengthened paired-pulse facilitation. Heterosynaptic long-term depression (LTD) in the DG was significantly blocked in RVG-expressing group compared to the control group. This blockade was dependent to PBM motif as rats expressing δRVG in the DG-expressed LTD comparable to the RVG group. Our data demonstrate that RVG expression facilitates both short- and long-term synaptic plasticity in the DG indicating that it may involve both pre- and postsynaptic mechanisms to alter synaptic function. Further studies are needed to elucidate the underlying mechanisms.

     

Tissue composition effect on dose distribution in neutron brachytherapy/neutron capture therapy

Aim

The aim of this study is to assess the effect of the compositions of various soft tissues and tissue-equivalent materials on dose distribution in neutron brachytherapy/neutron capture therapy.

Background

Neutron brachytherapy and neutron capture therapy are two common radiotherapy modalities.

Materials and methods

Dose distributions were calculated around a low dose rate ²⁵²Cf source located in a spherical phantom with radius of 20.0 cm using the MCNPX code for seven soft tissues and three tissue-equivalent materials. Relative total dose rate, relative neutron dose rate, total dose rate, and neutron dose rate were calculated for each material. These values were determined at various radial distances ranging from 0.3 to 15.0 cm from the source.

Results

Among the soft tissues and tissue-equivalent materials studied, adipose tissue and plexiglass demonstrated the greatest differences for total dose rate compared to 9-component soft tissue. The difference in dose rate with respect to 9-component soft tissue varied with compositions of the materials and the radial distance from the source. Furthermore, the total dose rate in water was different from that in 9-component soft tissue.

Conclusion

Taking the same composition for various soft tissues and tissue-equivalent media can lead to error in treatment planning in neutron brachytherapy/neutron capture therapy. Since the International Commission on Radiation Units and Measurements (ICRU) recommends that the total dosimetric uncertainty in dose delivery in radiotherapy should be within ±5%, the compositions of various soft tissues and tissue-equivalent materials should be considered in dose calculation and treatment planning in neutron brachytherapy/neutron capture therapy.     

Virus-induced silencing of genes encoding LEA protein in Tibetan hulless barley (Hordeum vulgare ssp. vulgare) and their relationship to drought tolerance

Expression of the late embryogenesis abundant (LEA) gene is usually associated with plant response to dehydration. In this study, a drought-tolerant genotype was screened from 48 accessions of Tibetan hulless barley (Hordeum vulgare ssp. vulgare). By using virus-induced gene silencing, the influence of two LEA genes (HVA1 and Dhn6) on drought tolerance of Tibetan hulless barley was investigated. Results of quantitative real-time PCR indicated that the relative expression levels of HVA1 and Dhn6 in silenced plants were significantly reduced compared with control plants. Both HVA1-silenced and Dhn6-silenced plants showed a consequently lower survival rate than control plants under drought stress. However, only HVA1-silenced plants exhibited a significantly higher water loss rate (WLR). These results suggested that HVA1 and Dhn6 might participate in adaptive responses to water deficit in different ways. Vegetative growth of HVA1-silenced plants was significantly retarded even under optimal growth conditions, and their biomass accumulation was also much lower than that of the controls. These results indicate that HVA1 might play a role in vegetative growth of Tibetan hulless barley.     

Protective effects of Helicobacter pylori against gastroesophageal reflux disease may be due to a neuroimmunological anti-inflammatory mechanism

There is some evidence that Helicobacter pylori infection has a protective effect against gastroesophageal reflux disease (GORD) and its complications such as Barrett's oesophagus and oesophageal adenocarcinoma. In this paper, we propose that a neuroimmunological mechanism is responsible for the protective effect of H. pylori on GORD. H. pylori infection of the gastric mucosa induces a T helper1-like immune response and production of pro-inflammatory cytokines. These cytokines can inhibit local sympathetic tone, whereas they increase systemic sympathetic tone. Increased sympathetic tone can induce an anti-inflammatory milieu, which in turn can inhibit inflammation in the oesophagus and lower oesophageal sphincter (LOS). Furthermore, H. pylori infection may stimulate the cholinergic anti-inflammatory pathway. It has been suggested that reflux-induced oesophageal inflammation plays an important role in the pathogenesis of reflux oesophagitis. Reduction of oesophageal inflammation by increased systemic sympathetic tone and vagal activity may lead to a decrease in reflux-induced oesophageal injury and LOS dysfunction in GORD.