A new look at osteoarthritis: Threshold potentials and an analogy to hypocalcemia.

Cartilage is a tissue that consist of very few cells embedded in a highly negatively charged extracellular matrix (ECM). This tissue is dealing with several electrical potentials which have been shown to control the production of ECM. Cartilage is present at joints and is constantly prone to degradation. Failing to repair the damage will result in the occurrence of osteoarthritis (OA). This perspective aims to link biophysical insights with biomolecular research in order to provide an alternative view on the possible causes of OA. Firstly, we hypothesize the existence of a threshold potential, which should be reached in order to initiate repair but if not met, unrepaired damage will evolve to OA. Measurements of the magnitude of this threshold electrical potential would be a helpful diagnostic tool. Secondly, since electrical potential alterations can induce chondrocytes to synthesize ECM, a cellular sensor must be present. We here propose an analogy to the hypocalcemia 'unshielding' situation to comprehend electrical potential generation and explore possible sensing mechanisms translating the electrical message into cellular responses. A better understanding of the cellular voltage sensors and down-stream signalling mechanisms may lead to the development of novel treatments for cartilage regeneration.

A Bibliometric and Visualized Overview for the Evolution of Process Safety and Environmental Protection.

This paper presents a bibliometric overview of the publications in the principal international journal Process Safety and Environmental Protection (PSEP) from 1990 to 2020 retrieved in the Web of Science (WoS) database to explore the evolution in safety and environmental engineering design and practice, as well as experimental or theoretical innovative research. Therefore, based on the WoS database and the visualization of similarities (VOS) viewer software, the bibliometric analysis and scientometric mapping of the literature have been performed from the perspectives of document types, publication and citation distribution over time, leading authors, countries (regions), institutions, the corresponding collaboration networks, most cited publications and references, focused research fields and topics, research trend evolution over time, etc. The paper provides a comprehensive and quantitative overview and significant picture representation for the journal's leading and evolutionary trends by employing specific aforementioned bibliometric analysis factors. In addition, by reviewing the evolutionary trends of the journal and the proposed investigated factors, such as the influential works, main research topics, and the research frontiers, this paper reveals the scientific literature production's main research objectives and directions that could be addressed and explored in future studies.

Time-Varying Risk Measurement for Ship Collision Prevention.

We propose an innovative time-varying collision risk (TCR) measurement for ship collision prevention in this article. The proposed measurement considers the level of danger of the approaching ships and the capability of a ship to prevent collisions. We define the TCR as the probability of the overlap of ships' positions in the future, given the uncertainty of maneuvers. Two sets are identified: (1) the velocity obstacle set as the maneuvers of the own ship that lead to collisions with target ships, and (2) the reachable velocity set as the maneuvers that the own ship can reach regarding its maneuverability. We then measure the TCR as the time-dependent percentage of overlap between these two sets. Several scenarios are presented to illustrate how the proposed measurement identifies the time-varying risk levels, and how the approach can be used as an intuitively understandable tool for collision avoidance.

[Improving the biocompatibility of silicone implants using spider silk coatings: immunohistochemical analysis of capsule formation]. / Verbesserung der Biokompatibilität von Silikonimplantaten durch Spinnenseidenbeschichtung: Immunhistochemische Untersuchungen zum Einfluss auf die Kapselbildung.

INTRODUCTION: Optimisation of the biocompatibility of silicone implants and reduction of capsule formation around the surface of such implants are in the focus of plastic surgical biomaterial research. In addition to its extraordinary physical and biochemical properties, spider silk shows high biocompatibility. Therefore, the coating of silicone implant surfaces with recombinant spider silk was analysed regarding foreign body reactions. MATERIALS AND METHODS: In the context of a preclinical study, miniaturised silicone implants were implanted in the back of 60 Sprague-Dawley rats. The animals were randomised; 30 animals received a texturised implant coated with the recombinant spider silk protein eADF4(C16) and 30 animals received uncoated implants. 3, 6 and 12 months after implantation, implants together with the surrounding capsules were removed and submitted to histological and immunohistochemical assessment. RESULTS: Coating of silicone implants with the recombinant spider silk protein eADF4(C16) resulted in a delayed and significantly decreased foreign body reaction and a reduced capsule manifestation. CONCLUSION: eADF4(C16) seems to be a promising candidate for the reduction of foreign body-associated capsule formation. Moreover, coating of other medical implants with this recombinant spider silk protein may improve their biocompatibility with little additional effort.

Statistical flaws in design and analysis of fertility treatment -studies on cryopreservation raise doubts on the conclusions.

Decisions about pharmacotherapy are being taken by medical doctors and authorities based on comparative studies on the use of medications. In studies on fertility treatments in particular, the methodological quality is of utmost -importance in the application of evidence-based medicine and systematic reviews. Nevertheless, flaws and omissions appear quite regularly in these types of studies. Current study aims to present an overview of some of the typical statistical flaws, illustrated by a number of example studies which have been published in peer reviewed journals. Based on an investigation of eleven studies at random selected on fertility treatments with cryopreservation, it appeared that the methodological quality of these studies often did not fulfil the -required statistical criteria. The following statistical flaws were identified: flaws in study design, patient selection, and units of analysis or in the definition of the primary endpoints. Other errors could be found in p-value and power calculations or in critical p-value definitions. Proper -interpretation of the results and/or use of these study results in a meta analysis should therefore be conducted with care.

Fault tree analysis for urban flooding.

Traditional methods to evaluate flood risk generally focus on heavy storm events as the principal cause of flooding. Conversely, fault tree analysis is a technique that aims at modelling all potential causes of flooding. It quantifies both overall flood probability and relative contributions of individual causes of flooding. This paper presents a fault model for urban flooding and an application to the case of Haarlem, a city of 147,000 inhabitants. Data from a complaint register, rainfall gauges and hydrodynamic model calculations are used to quantify probabilities of basic events in the fault tree. This results in a flood probability of 0.78/week for Haarlem. It is shown that gully pot blockages contribute to 79% of flood incidents, whereas storm events contribute only 5%. This implies that for this case more efficient gully pot cleaning is a more effective strategy to reduce flood probability than enlarging drainage system capacity. Whether this is also the most cost-effective strategy can only be decided after risk assessment has been complemented with a quantification of consequences of both types of events. To do this will be the next step in this study.

Improved methods for modelling drinking water treatment in quantitative microbial risk assessment; a case study of Campylobacter reduction by filtration and ozonation.

Quantitative microbial risk assessment (QMRA) is increasingly applied to estimate drinking water safety. In QMRA the risk of infection is calculated from pathogen concentrations in drinking water, water consumption and dose response relations. Pathogen concentrations in drinking water are generally low and monitoring provides little information for QMRA. Therefore pathogen concentrations are monitored in the raw water and reduction of pathogens by treatment is modelled stochastically with Monte Carlo simulations. The method was tested in a case study with Campylobacter monitoring data of rapid sand filtration and ozonation processes. This study showed that the currently applied method did not predict the monitoring data used for validation. Consequently the risk of infection was over estimated by one order of magnitude. An improved method for model validation was developed. It combines non-parametric bootstrapping with statistical extrapolation to rare events. Evaluation of the treatment model was improved by presenting monitoring data and modelling results in CCDF graphs, which focus on the occurrence of rare events. Apart from calculating the yearly average risk of infection, the model results were presented in FN curves. This allowed for evaluation of both the distribution of risk and the uncertainty associated with the assessment.

Bovine viral diarrhoea virus antigen in foetal calf serum batches and consequences of such contamination for vaccine production.

A protocol to test foetal calf serum (FCS) for contamination with bovine viral diarrhoea virus (BVDV) is described. Following this protocol, which combines cell culture methods and detection of pestivirus RNA, seven batches of FCS were tested. Infectious BVDV was detected in four of those batches. One of the remaining batches contained a relatively high number of non-infectious BVDV particles. A sample of this batch was formulated with aluminium hydroxide and aluminium phosphate as adjuvant into an experimental vaccine preparation. This product was injected twice into BVDV seronegative cattle with a 4 week interval. Blood samples taken 4 weeks after the second application were negative for BVDV specific antibodies. Our data stress that detection of BVDV RNA is not sufficient for a complete risk assessment on FCS. Discrimination between infectious and non-infectious BVDV is essential. This can only be achieved by cell culture methods.

An overview of quantitative risk measures for loss of life and economic damage.

A comprehensive overview of methods to quantify and limit risks arising from different sources is still missing in literature. Therefore, a study of risk literature was carried out by the authors. This article summarises about 25 quantitative risk measures. A risk measure is defined as a mathematical function of the probability of an event and the consequences of that event. The article focuses mainly on risk measures for loss of life (individual and societal risk) and economic risk, concentrating on risk measurement experiences in The Netherlands. Other types of consequences and some international practices are also considered. For every risk measure the most important characteristics are given: the mathematical formulation, the field of application and the standard set in this field. Some of the measures have been used in a case study to calculate the flood risks for an area in The Netherlands.

Use of linearity of the Sokoloff model to improve performance of non-linear search.

The three-parameter Sokoloff equation is used to measure the rates of glucose consumption in the brain in vivo. This equation depends linearly on one of its parameters, k1, which is responsible for the glucose transport from plasma to tissue. By equating to zero the first derivative of the minimization function with respect to k1, it is possible to express this parameter as a function of the other two and reduce the non-linear search from three to two dimensions. This approach was examined by the Nelder-Mead simplex method and the Levenberg-Marquardt algorithm. In both cases the process of convergence was more robust and required fewer iterations to achieve a given accuracy than the direct three-parameter non-linear search.

Serum-free produced Bovine Herpesvirus type 1 and Bovine Parainfluenza type 3 virus vaccines are efficacious and safe.

The studies described in this report were performed to determine, whether it is possible to produce live virus vaccines without serum or fractions thereof used during any cell or virus passage, thus completely serum-free. Two viruses were included in the experiments: Bovine Herpesvirus 1 (BHV-1) and Bovine Parainfluenza type 3 virus (PI3). Both viruses were found to grow to satisfactory titers, and to be stable after freeze-drying and subsequent storage at temperatures of +4 degrees C and -20 degrees C for at least one year. Moreover, a vaccine containing serum free produced BHV-1 was tested in a vaccination-challenge experiment. For comparison, a vaccine batch with BHV-1 grown in serum-containing cell culture medium was included in the study. Both vaccine preparations performed equally well and both met the strict requirements as laid down in the European Phamacopeia. Moreover, in two separate experiments the safety of serum-free produced BHV-1 and PI3 after overdose and repeated administration even in very young calves and even after four administrations has been demonstrated. This report is the first, which to our knowledge demonstrates the safety and efficacy of serum-free produced live vaccines in the target animal as well as the stability of these products.

Sucrose transport through maltoporin mutants of Escherichia coli.

Maltoporin (LamB) and sucrose porin (ScrY) reside in the bacterial outer membrane and facilitate the passive diffusion of maltodextrins and sucrose, respectively. To gain further insight into the determinants of solute specificity, LamB mutants were designed to allow translocation of sucrose, which hardly translocates through wild-type LamB. Three LamB mutants were studied. (a) Based on sequence and structure alignment of LamB with ScrY, two LamB triple mutants were generated (R109D, Y118D,D121F; R109N,Y118D,D121F) to mimic the ScrY constriction. The crystal structure of the first of these mutants was determined to be 3.2 A and showed an increased ScrY-like cross-section except for D109 that protrudes into the channel. (b) Based on this crystal structure a double mutant was generated by truncation of the two residues that obstruct the channel most in LamB (R109A,Y118A). Analysis of liposome swelling and in vivo sugar uptake demonstrated substantial sucrose permeation through all mutants with the double alanine mutant performing best. The triple mutants did not show a well-defined binding site as indicated by sugar-induced ion current noise analysis, which can be explained by remaining steric interference as deduced from the crystal structure. Binding, however, was observed for the double mutant that had the obstructing residues truncated to alanines.

An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells.

Transferred DNA (T-DNA) transfer from Agrobacterium tumefaciens into eukaryotic cells is the only known example of interkingdom DNA transfer. T-DNA is a single-stranded segment of Agrobacterium's tumor-inducing plasmid that enters the plant cell as a complex with the bacterial virulence proteins VirD2 and VirE2. The VirE2 protein is highly induced on contact of A. tumefaciens with a plant host and has been reported to act in late steps of transfer. One of its previously demonstrated functions is binding to the single-stranded (ss) T-DNA and protecting it from degradation. Recent experiments suggest other functions of the protein. A combination of planar lipid bilayer experiments, vesicle swelling assays, and DNA transport experiments demonstrated that VirE2 can insert itself into artificial membranes and form channels. These channels are voltage gated, anion selective, and single-stranded DNA-specific and can facilitate the efficient transport of single-stranded DNA through membranes. These experiments demonstrate a VirE2 function as a transmembrane DNA transporter, which could have applications in gene delivery systems.

Understanding the function of bacterial outer membrane channels by reconstitution into black lipid membranes

Structural and functional information is obtained by the reconstitution of membrane channel forming proteins into black lipid membranes. Due to this outstanding sensitivity only little material is needed and single molecule detection can be easily achieved. An overview on different types of detection will be given.

Structure and function of bacterial outer membrane proteins: barrels in a nutshell.

The outer membrane protects Gram-negative bacteria against a harsh environment. At the same time, the embedded proteins fulfil a number of tasks that are crucial to the bacterial cell, such as solute and protein translocation, as well as signal transduction. Unlike membrane proteins from all other sources, integral outer membrane proteins do not consist of transmembrane alpha-helices, but instead fold into antiparallel beta-barrels. Over recent years, the atomic structures of several outer membrane proteins, belonging to six families, have been determined. They include the OmpA membrane domain, the OmpX protein, phospholipase A, general porins (OmpF, PhoE), substrate-specific porins (LamB, ScrY) and the TonB-dependent iron siderophore transporters FhuA and FepA. These crystallographic studies have yielded invaluable insight into and decisively advanced the understanding of the functions of these intriguing proteins. Our review is aimed at discussing their common principles and peculiarities as well as open questions associated with them.

Extended sugar slide function for the periplasmic coiled coil domain of ScrY.

Several bacterial outer membrane proteins have a periplasmic extension whose structure and function remain elusive. Here, the structure/function relationship of the N-terminal periplasmic domain of the sucrose-specific outer membrane channel ScrY was investigated. Circular dichroism and analytical centrifugation demonstrated that the N-terminal domain formed a parallel, three-stranded coiled coil. When this domain was fused to the maltose-specific channel LamB, permeation of maltooligosaccharides in liposomes increased with increasing sugar chain length whereas wild-type LamB showed the opposite effect. Current fluctuation analysis demonstrated increased off-rates for sugar transport through the fusion protein. Moreover, equilibrium dialysis showed an affinity of sucrose for the isolated N-terminal peptide. Together these results demonstrate a novel function for coiled coil domains, operating as an extended sugar slide.

Role of the lipase-specific foldase of Burkholderia glumae as a steric chaperone.

Most lipases of Gram-negative bacteria require a lipase-specific foldase (Lif) in order to fold in the periplasm into their active, protease-resistant conformation prior to their secretion. The periplasmic domain of the Lif (amino acids 44-353) of Burkholderia glumae was purified as a His-tagged protein, and its function in the folding of lipase was studied in vitro. Refolding of the denatured lipase into its active conformation was dependent on the presence of the Lif. Circular dichroism revealed that the lipase refolded in the absence of Lif into a form with a native-like conformation, which was more stable against heat-induced denaturation than the native form, but was enzymatically inactive. This form of the protein could be activated by adding Lif after several hours, which demonstrates that the function of this chaperone is to help lipase to overcome an energetic barrier in the productive folding pathway rather than to prevent it from entering a non-productive pathway. The Lif was shown to interact with the native lipase in protease-protection experiments as well as by affinity chromatography, consistent with a role of the Lif late in the folding process. These results demonstrate that the Lif functions in a way analogous to the propeptides of many bacterial proteases and indicate that the amino acid sequence of the lipase does not contain all the information required for the protein to adopt its three-dimensional structure.

Sugar transport through maltoporin of Escherichia coli. Role of polar tracks.

The three-dimensional structure of the maltooligosaccharide specific outer membrane channel LamB of Escherichia coli complexed with sugar molecules revealed a hypothetical transport pathway. Sugars are supposed to slide over a stretch of aromatic residues facilitated by continuous making/breaking of hydrogen bonds between the hydroxyl groups of the sugars and charged amino acids, the "polar tracks." The effect of nine single and three multiple mutations in the polar track residues was investigated by current fluctuations, liposome swelling assays, and in vivo uptake of radiolabeled substrates. Additionally, sugar transport through wild-type LamB was investigated by current fluctuation analysis in water and deuterium. This way the effects on k(on) and k(off) could be investigated separately. Analyses of the various mutants revealed a strong effect on the k(on) values. Because steering to the binding site requires only a few interactions, consequently the loss of even one bond will have a strong effect. Deuterium experiments, which changed the characteristic of all hydrogen bonds, showed a strong effect on k(off) rates, because at this stage the sugar has numerous interactions with the channel. Furthermore, all the mutations induces a strong decrease of in vivo uptake of sugars. These results clearly demonstrate the importance of the polar track residues on both on and off rates in sugar transport and reveal a strong cooperative effect of hydrogen bond formation.

Oriented channels reveal asymmetric energy barriers for sugar translocation through maltoporin of Escherichia coli.

Sugar transport through maltoporin of Escherichia coli was investigated. This protein facilitates maltooligosaccharide translocation via a binding site in the channel. Because incorporation of the protein into the bilayer results in randomly orientated channels, we re-examined the postulated symmetric translocation model by reconstitution of maltoporin under an externally applied field. Upon binding of bacteriophage lambda, which exploit surface-exposed loops of maltoporin as the receptor, sugar permeation, but not the ion current, was blocked. Thus using the phage-to-probe orientation we were able to show that the channels were approximately 80% directionally inserted into the bilayer. Moreover, asymmetry of the channel was revealed because sugar entrance through the 'open' periplasmic side of maltoporin was similarly reduced. Here a new asymmetrical two-barrier model is presented. Based on liposome-swelling assays and current-fluctuation analysis we conclude that the periplasmic side of the porin shows a two- to threefold higher energy barrier than the extracellular loop-side of the channels.

The C-terminal domain of the Pseudomonas secretin XcpQ forms oligomeric rings with pore activity.

The Pseudomonas secretin XcpQ forms an oligomeric complex, which is involved in the translocation of proteins across the outer membrane via the type II secretion pathway. Pseudomonas aeruginosa produces only small amounts of this complex, 50 to 100 copies per bacterium, and overexpression is lethal to these cells. However, overexpression of Pseudomonas alcaligenes XcpQ could be achieved in the P. alcaligenes mutant strain 537. Protease protection experiments with P. alcaligenes XcpQ showed that the C-terminal domain of XcpQ, which is conserved in all the different members of the secretin family, is largely resistant to proteinase K. This protease-resistant fragment is embedded in the membrane and remains a stable complex, indicating that this domain is involved in complex formation. Both the intact and the protease-protected XcpQ complex showed a tendency to form two-dimensional crystal-like structures. Electron microscopic analysis of these structures showed that the overall oligomeric rings of the intact and of the protease-resistant complex are highly similar. The central cavity of the intact XcpQ complex contains structured mass. Both the intact and the protease-protected XcpQ complex showed pore-forming activity in planar lipid bilayers, consistent with their role as a translocation channel. However, the single-channel conductances observed were not uniform. Together, these results demonstrate that the C-terminal secretin homology domain of XcpQ is the structural domain that forms the channel through which macromolecules are being transported.