Mechanical Signaling on the Single Protein Level Studied Using Steered Molecular Dynamics

Efficient communication between the cell and its external environment is of the utmost importance to the function of multicellular organisms. While signaling events can be generally characterized as information exchange by means of controlled energy conversion, research efforts have hitherto mainly been concerned with mechanisms involving chemical and electrical energy transfer. Here, we review recent computational efforts addressing the function of mechanical force in signal transduction. Specifically, we focus on the role of steered molecular dynamics (SMD) simulations in providing details at the atomic level on a group of protein domains, which play a fundamental role in signal exchange by responding properly to mechanical strain. We start by giving a brief introduction to the SMD technique and general properties of mechanically stable protein folds, followed by specific examples illustrating three general regimes of signal transfer utilizing mechanical energy: purely mechanical, mechanical to chemical, and chemical to mechanical. Whenever possible the physiological importance of the example at hand is stressed to highlight the diversity of the processes in which mechanical signaling plays a key role. We also provide an overview of future challenges and perspectives for this rapidly developing field.     

Glucose biosensor based on electrodeposited platinum nanoparticles and three-dimensional porous chitosan membranes

The chitosan with three-dimensional porous structure greatly increased the effective electrode surface for loading of platinum nanoparticles and promoted efficient electron transfer. The resulting biosensor had a response time (within 5 s) and a linear response from 6 μM to 4.2 mM glucose with a detection limit of 2 μM (S/N = 3). Moreover, the methodology can be applied for the immobilization of other enzymes.     

A review of the chronostratigraphical ages of Middle Triassic to Late Jurassic dinoflagellate cyst biozones of the North West Shelf of Australia

The chronostratigraphical ages of the 20 dinoflagellate cyst zones and one dinoflagellate cyst assemblage for the Middle Triassic (Ladinian) to the Jurassic-Cretaceous transition of the North West Shelf of Australia are comprehensively reviewed. Evidence from macro- and micropalaeontology, palynology and strontium isotopes made available after the establishment of these biozones in the 1980s has been used to reassess the ages of this important zonal scheme and to calibrate it to the international stratigraphical stages. The Shublikodinium Superzone is renamed herein as the Rhaetogonyaulax Superzone, and based on conodont evidence is determined to span the Ladinian to Early Sinemurian. This is significantly shorter in duration than was originally envisaged (Late Anisian to Late Pliensbachian). The Luehndea Assemblage is a low diversity dinoflagellate cyst association which marks a eustatic rise; it is subdivided into two subzones. It is of latest Pliensbachian to Early Toarcian age, based largely on palynological evidence. The Bajocian to earliest Oxfordian Pareodinia ceratophora Superzone represents the inception of a continuous Mesozoic-Cenozoic dinoflagellate cyst record in Australia. It comprises seven zones, which are considered to be slightly older than originally interpreted. The overlying Pyxidiella Superzone is characterised by diverse dinoflagellate cyst associations. It is Early Oxfordian to Kimmeridgian in age, and comprises three zones. The bases of the Wanaea spectabilis and Wanaea clathrata zones are reinterpreted as being slightly older than originally proposed. The superjacent Fromea cylindrica Superzone is Tithonian to earliest Valanginian and modified ages are indicated for four of the nine zones. This unit is dominated by endemic dinoflagellate cysts, reflecting a global trend towards provincialism at this time due to a regressive eustatic regime.     

Causal relationships between frequency bands of extracellular signals in visual cortex revealed by an information theoretic analysis

Characterizing how different cortical rhythms interact and how their interaction changes with sensory stimulation is important to gather insights into how these rhythms are generated and what sensory function they may play. Concepts from information theory, such as Transfer Entropy (TE), offer principled ways to quantify the amount of causation between different frequency bands of the signal recorded from extracellular electrodes; yet these techniques are hard to apply to real data. To address the above issues, in this study we develop a method to compute fast and reliably the amount of TE from experimental time series of extracellular potentials. The method consisted in adapting efficiently the calculation of TE to analog signals and in providing appropriate sampling bias corrections. We then used this method to quantify the strength and significance of causal interaction between frequency bands of field potentials and spikes recorded from primary visual cortex of anaesthetized macaques, both during spontaneous activity and during binocular presentation of naturalistic color movies. Causal interactions between different frequency bands were prominent when considering the signals at a fine (ms) temporal resolution, and happened with a very short (ms-scale) delay. The interactions were much less prominent and significant at coarser temporal resolutions. At high temporal resolution, we found strong bidirectional causal interactions between gamma-band (40–100 Hz) and slower field potentials when considering signals recorded within a distance of 2 mm. The interactions involving gamma bands signals were stronger during movie presentation than in absence of stimuli, suggesting a strong role of the gamma cycle in processing naturalistic stimuli. Moreover, the phase of gamma oscillations was playing a stronger role than their amplitude in increasing causations with slower field potentials and spikes during stimulation. The dominant direction of causality was mainly found in the direction from MUA or gamma frequency band signals to lower frequency signals, suggesting that hierarchical correlations between lower and higher frequency cortical rhythms are originated by the faster rhythms.     

The role of matrix metalloproteinases 2 and 9 during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. The purpose of this study was to characterize the role of MMPs as depicted by the expression of MMP-2 and MMP-9 during 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis. Male Wistar rats were distributed into three groups of 10 animals each and treated with 4-nitroquinoline 1-oxide solution at 50 ppm through their drinking water for 4, 12, and 20 weeks. Ten animals were used as control group. No histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure; however, immunoexpression of MMP-2 was noticed. The same picture occurred to MMP-9, in which positive expression was detected for this immunomarker. MMP-2 and MMP-9 showed positive expression either in pre-neoplastic lesions at 12 weeks following carcinogen exposure or in well-differentiated squamous cell carcinoma induced after 20 weeks of treatment with 4NQO. Taken together, our results support the belief that MMP-2 and MMP-9 play important role during malignant transformation and conversion of oral mucosa as assessed by immunohistochemistry.     

Optimisation of the purification process of a tumour-targeting antibody produced in <Emphasis Type="Italic">N. benthamiana</Emphasis> using vacuum-agroinfiltration

It was previously demonstrated that the tumour-targeting antibody mAb H10 can be transiently expressed and purified at high levels in Nicotiana benthamiana by using a vacuum-agroinfiltration system boosted by the use of a virus silencing suppressor protein. Scope of this work was to analyse different steps of protein extraction from agroinfiltrated leaves to optimise the purification process of the secretory mAb H10 providing new insights in the field of large-scale plant production. Two different extraction procedures (mechanical shearing/homogenisation and recovery of intercellular fluids -IFs-) were evaluated and compared in terms of purified antibody yields, antibody degradation and total phenolic compounds content. Mechanical grinding from fresh leaf tissues gave the highest purification yield (75 mg/kg Fresh Weight -75% intact tetrameric IgG-) and total phenolics concentration in the range of 420 μg/g FW. The second extraction procedure, based on the recovery of IFs, gave purification yields of 15–20 mg/kg FW (corresponding to 27% of total soluble protein) in which about 40% of purified protein is constituted by fully assembled IgG with a total phenolic compounds content reduced by one order of magnitude (21 μg/g FW). Despite a higher antibody degradation, purification from intercellular fluids demonstrated to be very promising since extraction procedures resulted extremely fast and amenable to scaling-up. Overall data highlight that different extraction procedures can dramatically affect the proteolytic degradation and quality of antibody purified from agroinfiltrated N. benthamiana leaves. Based on these results, we optimised a pilot-scale purification protocol using a two-step purification procedure from batches of fresh agroinfiltrated leaves (250 g) allowing purification of milligram quantities (average yield 40 mg/kg FW) of fully assembled and functional IgG with a 99.4% purity, free of phenolic and alkaloid compounds with low endotoxin levels (<1 EU/ml).     

Proteomic Analysis of Interactions Between the Generalist Herbivore <Emphasis Type="Italic">Spodoptera exigua</Emphasis> (Lepidoptera: Noctuidae) and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In this study, comparative proteomics was used to investigate the interaction of Spodoptera exigua and Arabidopsis thaliana. By using 2-D electrophoresis of differentially expressed proteins, combined with high-throughput matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF/TOF MS, the changes in the abundance of proteins induced by insect feeding were studied in A. thaliana. More than 1,100 protein spots were reproducibly detected on each gel. The intensities of 30 protein spots in particular changed significantly, showing differences in volume of at least twofold. Among these, 17 protein spots were upregulated, and 13 were downregulated following an 8-h insect feeding period. Nineteen insect-feeding-responsive proteins were identified, all of which were involved in metabolic regulation, binding functions or cofactor requirement of protein, cell rescue, and defense and virulence, as assessed by Munich Information Center for Protein Sequences function category. About 50% of these were involved in metabolism, including transketolase, S-adenosylmethionine synthase 3, 2,3-biphosphoglycerate-independent phosphoglycerate mutase, beta-ureidopropionase, GDP-d-mannose 3′,5′-epimerase, and fatty acid synthase. The identification of insect-feeding-responsive proteins on Arabidopsis provides not only new insights into insect stress but also a good start for further investigation of their functions. Understanding how the plant responses to insects in the proteomic level will provide tools for a better management of insect pest in the field.     

Structural and cellular features in metaphyseal and diaphyseal periosteum of osteoporotic rats

Despite the important physiological role of periosteum in the pathogenesis and treatment of osteoporosis, little is known about the structural and cellular characteristics of periosteum in osteoporosis. To study the structural and cellular differences in both diaphyseal and metaphyseal periosteum of osteoporotic rats, samples from the right femur of osteoporotic and normal female Lewis rats were collected and tissue sections were stained with hematoxylin and eosin, antibodies or staining kit against tartrate resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF), von Willebrand (vWF), tyrosine hydroxylase (TH) and calcitonin gene-related peptide (CGRP). The results showed that the osteoporotic rats had much thicker and more cellular cambial layer of metaphyseal periosteum compared with other periosteal areas and normal rats (P < 0.001). The number of TRAP⁺ osteoclasts in bone resorption pits, VEGF⁺ cells and the degree of vascularization were found to be greater in the cambial layer of metaphyseal periosteum of osteoporotic rats (P < 0.05), while no significant difference was detected in the number of ALP⁺ cells between the two groups. Sympathetic nerve fibers identified by TH staining were predominantly located in the cambial layer of metaphyseal periosteum of osteoporotic rats. No obvious difference in the expression of CGRP between the two groups was found. In conclusion, periosteum may play an important role in the cortical bone resorption in osteoporotic rats and this pathological process may be regulated by the sympathetic nervous system.