Enhanced exploration of the mode of action of a five-layer foam dressing: critical properties to support wound healing.

OBJECTIVE: The aim of this in vitro experimental series was to explore the mode of action of a hydrocellular polyurethane foam dressing (HPFD) and how its advanced features support beneficial interactions with the wound bed to address common barriers to wound healing, thus supporting improved clinical outcomes. METHOD: Multiple in vitro microbiological tests were performed, assessing prevention of bacterial ingress, surface removal of bacteria, bacterial sequestration and retention into the dressing in a clinically relevant environment. Odour molecule concentrations were measured using gas chromatography and further assays explored matrix metalloproteinase (MMP)-9 retention in the dressing using enzyme linked immunosorbent assay. RESULTS: The HPFD demonstrated marked reductions in bioburden levels across multiple tests. These included prevention of bacterial ingress for seven days, removal of surface bacteria and absorption into the dressing. Further tests identified that most bacteria were sequestered into the hyperabsorbent layer (90.5% for Pseudomonas aeruginosa and 89.6% for meticillin-resistant Staphylococcus aureus). Moreover, the majority of bacteria (99.99% for both test organisms) were retained within the dressing, even upon compression. Additional tests demonstrated a marked reduction of odour molecules following incubation with HPFD and total retention of protease MMP-9 within the dressing. CONCLUSIONS: Proactive management of the wound environment with an appropriate advanced wound dressing, such as the HPFD examined in these in vitro investigations, can not only help to minimise the barriers to healing, as observed across this test series by direct interaction with the wound bed, but may, as a result, provide an ideal environment for wound progression with minimal disturbance.

An integrated design and fabrication strategy for entirely soft, autonomous robots.

Soft robots possess many attributes that are difficult, if not impossible, to achieve with conventional robots composed of rigid materials. Yet, despite recent advances, soft robots must still be tethered to hard robotic control systems and power sources. New strategies for creating completely soft robots, including soft analogues of these crucial components, are needed to realize their full potential. Here we report the untethered operation of a robot composed solely of soft materials. The robot is controlled with microfluidic logic that autonomously regulates fluid flow and, hence, catalytic decomposition of an on-board monopropellant fuel supply. Gas generated from the fuel decomposition inflates fluidic networks downstream of the reaction sites, resulting in actuation. The body and microfluidic logic of the robot are fabricated using moulding and soft lithography, respectively, and the pneumatic actuator networks, on-board fuel reservoirs and catalytic reaction chambers needed for movement are patterned within the body via a multi-material, embedded 3D printing technique. The fluidic and elastomeric architectures required for function span several orders of magnitude from the microscale to the macroscale. Our integrated design and rapid fabrication approach enables the programmable assembly of multiple materials within this architecture, laying the foundation for completely soft, autonomous robots.

Cadexomer iodine provides superior efficacy against bacterial wound biofilms in vitro and in vivo.

Examination of clinical samples indicates bacterial biofilms are present in the majority of chronic wounds, and substantial evidence suggests biofilms contribute significantly to delayed healing. Bacteria in biofilms are highly tolerant of antimicrobials, and little data exist to guide the choice of anti-biofilm wound therapy. Cadexomer iodine (CI) was recently reported to have superior efficacy compared to diverse wound dressings against Pseudomonas aeruginosa biofilms in an ex vivo model. In the current study, the strong performance of CI vs. P. aeruginosa biofilm was confirmed using colony and colony drip-flow in vitro wound biofilm models. Similar in vitro efficacy of CI was also demonstrated against mature Staphylococcus aureus biofilms using the same models. Additionally, the rapid kill of mature S. aureus and P. aeruginosa colony biofilms was visualized by confocal microscopy using Live/Dead fluorescent stains. Superior in vitro efficacy of CI vs. staphylococcal biofilms was further demonstrated against methicillin-resistant S. aureus (MRSA) using multiple biofilm models with log reduction, Live/Dead, and metabolic endpoints. Comparator antimicrobial dressings, including silver-based dressings used throughout and other active agents used in individual models, elucidated only limited effects against the mature biofilms. Given the promising in vitro activity, CI was tested in an established mouse model of MRSA wound biofilm. CI had significantly greater impact on MRSA biofilm in mouse wounds than silver dressings or mupirocin based on Gram-stained histology sections and quantitative microbiology from biopsy samples (>4 log reduction in CFU/g vs. 0.7-1.6, p < 0.0001). The superior efficacy for CI in these in vitro and in vivo models suggests CI topical products may represent a better choice to address established bacterial biofilm in chronic wounds.

Structure and mechanism of the chromatin remodelling factor ISW1a.

Site-specific recognition of DNA in eukaryotic organisms depends on the arrangement of nucleosomes in chromatin. In the yeast Saccharomyces cerevisiae, ISW1a and related chromatin remodelling factors are implicated in establishing the nucleosome repeat during replication and altering nucleosome position to affect gene activity. Here we have solved the crystal structures of S. cerevisiae ISW1a lacking its ATPase domain both alone and with DNA bound at resolutions of 3.25 Å and 3.60 Å, respectively, and we have visualized two different nucleosome-containing remodelling complexes using cryo-electron microscopy. The composite X-ray and electron microscopy structures combined with site-directed photocrosslinking analyses of these complexes suggest that ISW1a uses a dinucleosome substrate for chromatin remodelling. Results from a remodelling assay corroborate the dinucleosome model. We show how a chromatin remodelling factor could set the spacing between two adjacent nucleosomes acting as a 'protein ruler'.

Report from AmSECT's International Consortium for Evidence- Based Perfusion Consensus Statement: Minimal Criteria for Reporting Cardiopulmonary Bypass-Related Contributions to Red Blood Cell Transfusions Associated With Adult Cardiac Surgery.

Gaps remain in our understanding of the contribution of bypass-related practices associated with red blood cell (RBC) transfusions after cardiac surgery. Variability exists in the reporting of bypass-related practices in the peer-reviewed literature. In an effort to create uniformity in reporting, a draft statement outlining proposed minimal criteria for reporting cardiopulmonary bypass (CPB)- related contributions (i.e., RBC data collection/documentation, clinical considerations for transfusions, equipment details, and clinical endpoints) was presented in conjunction with the American Society of ExtraCorporeal Technology's (AmSECT's) 2014 Quality and Outcomes Meeting (Baltimore, MD). Based on presentations and feedback from the conference, coauthors (n = 14) developed and subsequently voted on each proposed data element. Data elements receiving a total of 4 votes were dropped from further consideration, 5-9 votes were considered as "Recommended," and elements receiving ≥10 votes were considered as "Mandatory." A total of 52 elements were classified as mandatory, 16 recommended, and 14 dropped. There are 8 mandatory data elements for RBC data collection/documentation, 24 for clinical considerations for transfusions, 13 for equipment details, and 7 for clinical endpoints. We present 52 mandatory data elements reflecting CPB-related contributions to RBC transfusions. Consistency of such reporting would offer our community an increased opportunity to shed light on the relationship between intra-operative practices and RBC transfusions.

Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway.

The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.

Kinetic mechanism for single-stranded DNA binding and translocation by Saccharomyces cerevisiae Isw2.

The chromatin remodeling complex Isw2 from Saccharomyces cerevisiae (yIsw2) mobilizes nucleosomes through an ATP-dependent reaction that is coupled to the translocation of the helicase domain of the enzyme along intranucleosomal DNA. In this study, we demonstrate that yIsw2 is capable of translocating along single-stranded DNA in a reaction that is coupled to ATP hydrolysis. We propose that single-stranded DNA translocation by yIsw2 occurs through a series of repeating uniform steps with an overall macroscopic processivity (P) of 0.90 +/- 0.02, corresponding to an average translocation distance of 20 +/- 2 nucleotides before dissociation. This processivity corresponds well to the processivity of nucleosome sliding by yIsw2, thus arguing that single-stranded DNA translocation or tracking may be fundamental to the double-stranded DNA translocation required for effective nucleosome mobilization. Furthermore, we find evidence that a slow initiation process, following DNA binding, may be required to make yIsw2 competent for DNA translocation. We also provide evidence that this slow initiation process may correspond to the second step of a two-step DNA binding mechanism by yIsw2 and a quantitative description of the kinetics of this DNA binding mechanism.

Neuronal calcium sensor proteins are unable to modulate NFAT activation in mammalian cells.

Calcium activated gene transcription through Nuclear Factor of Activated T-cells, (NFAT) proteins, is emerging as a ubiquitous mechanism for the control of important physiological processes. Of the five mammalian NFAT isoforms, transcriptional activities of NFATs 1-4 are stimulated by a calcium driven association between the ubiquitous phosphatase calcineurin and the calcium-sensing protein calmodulin. Published in vitro evidence has suggested that other members of the calmodulin super-family, in particular the neuronal calcium sensor (NCS) proteins, can similarly modulate calcineurin activity. In this study we have assessed the ability of NCS proteins to interact directly with calcineurin in vitro and report a specific if weak association between various NCS proteins and the phosphatase. In an extension to these analyses we have also examined the effects of over-expression of NCS-1 or NCS-1 mutants on calcineurin signalling in HeLa cells in experiments examining the dephosphorylation of an NFAT-GFP reporter construct as a readout of calcineurin activity. Results from these experiments indicate that NCS-1 was not able to detectably modulate calcineurin/NFAT signalling in a live mammalian cell system, findings that are consistent with the idea that calmodulin and not NCS-1 or other NCS family proteins is the physiologically relevant modulator of calcineurin activity.

Multiprotein expression strategy for structural biology of eukaryotic complexes.

The concept of the cell as a collection of multisubunit protein machines is emerging as a cornerstone of modern biology, and molecular-level study of these machines in most cases will require recombinant production. Here, we present and validate a strategy to rapidly produce, permutate, and posttranslationally modify large, eukaryotic multiprotein complexes by using DNA recombination in a process that is fully automatable. Parallel production of 12 protein complex variants within a period of weeks resulted in specimens of sufficient quantity and homogeneity for structural biology applications.

MultiBac: Baculovirus-Mediated Multigene DNA Cargo Delivery in Insect and Mammalian Cells.

The baculovirus/insect cell system (BICS) is widely used in academia and industry to produce eukaryotic proteins for many applications, ranging from structure analysis to drug screening and the provision of protein biologics and therapeutics. Multi-protein complexes have emerged as vital catalysts of cellular function. In order to unlock the structure and mechanism of these essential molecular machines and decipher their function, we developed MultiBac, a BICS particularly tailored for heterologous multigene transfer and multi-protein complex production. Baculovirus is unique among common viral vectors in its capacity to accommodate very large quantities of heterologous DNA and to faithfully deliver this cargo to a host cell of choice. We exploited this beneficial feature to outfit insect cells with synthetic DNA circuitry conferring new functionality during heterologous protein expression, and developing customized MultiBac baculovirus variants in the process. By altering its tropism, recombinant baculovirions can be used for the highly efficient delivery of a customized DNA cargo in mammalian cells and tissues. Current advances in synthetic biology greatly facilitate the construction or recombinant baculoviral genomes for gene editing and genome engineering, mediated by a MultiBac baculovirus tailored to this purpose. Here, recent developments and exploits of the MultiBac system are presented and discussed.

High-Throughput Production of Influenza Virus-Like Particle (VLP) Array by Using VLP-factory™, a MultiBac Baculoviral Genome Customized for Enveloped VLP Expression.

Baculovirus-based expression of proteins in insect cell cultures has emerged as a powerful technology to produce complex protein biologics for many applications ranging from multiprotein complex structural biology to manufacturing of therapeutic proteins including virus-like particles (VLPs). VLPs are protein assemblies that mimic live viruses but typically do not contain any genetic material, and therefore are safe and attractive alternatives to life attenuated or inactivated viruses for vaccination purposes. MultiBac is an advanced baculovirus expression vector system (BEVS) which consists of an engineered viral genome that can be customized for tailored applications. Here we describe the creation of a MultiBac-based VLP-factory™, based on the M1 capsid protein from influenza, and its application to produce in a parallelized fashion an array of influenza-derived VLPs containing functional mutations in influenza hemagglutinin (HA) thought to modulate the immune response elicited by the VLP.

Baculovirus expression system for heterologous multiprotein complexes.

The discovery of large multiprotein complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant multiprotein production and multigene transfer applications.

Structure-function analysis of the vanillin molecule and its antifungal properties.

The aim of the present study was to evaluate which structural elements of the vanillin molecule are responsible for its observed antifungal activity. MICs of vanillin, its six direct structural analogues, and several other related compounds were determined in yeast extract peptone dextrose broth against a total of 18 different food spoilage molds and yeasts. Using total mean MICs after 4 days of incubation at 25 degrees C, the antifungal activity order was 3-anisaldehyde (1.97 mM) > benzaldehyde (3.30 mM) > vanillin (5.71 mM) > anisole (6.59 mM) > 4-hydroxybenzaldehyde (9.09 mM) > phenol (10.59 mM) > guaiacol (11.66 mM). No correlation was observed between the relative antifungal activity of the test compounds and log P(o/w). Furthermore, phenol (10.6 mM) was found to exhibit a greater activity than cyclohexanol (25.3 mM), whereas cyclohexanecarboxaldehyde (2.13 mM) was more active than benzaldehyde (3.30 mM). Finally, the antifungal order of isomers of hydroxybenzaldehyde and anisaldehyde was found to be 2- > 3- > 4- and 3- > 2- > 4-, respectively. In conclusion, the aldehyde moeity of vanillin plays a key role in its antifungal activity, but side-group position on the benzene ring also influences this activity. Understanding how the structure of natural compounds relates to their antimicrobial function is fundamentally important and may help facilitate their application as novel food preservatives.

Analysis of the inhibition of food spoilage yeasts by vanillin.

The antimicrobial potential of vanillin, the major component of vanilla flavour, was examined against the growth of three yeasts associated with food spoilage, Saccharomyces cerevisiae, Zygosaccharomyces bailii and Zygosaccharomyces rouxii. Minimum inhibitory concentration (MIC) values of 21, 20 and 13 mM vanillin were determined for the three yeast strains, respectively. The observed inhibition was found to be biostatic. During fermentation, the bioconversion of sub-MIC levels of vanillin in the culture medium was demonstrated. The major bioconversion product was identified as vanillyl alcohol, however low levels of vanillic acid were also detected. Neither the vanillyl alcohol nor the vanillic acid was found to be antagonistic to yeast cell growth. The results indicate the importance of the aldehyde moiety in the vanillin structure regarding its antimicrobial activity and that the bioconversion of vanillin could be advantageous for the yeasts, but only at levels below MIC. These bioconversion activities, presumably catalysed by non-specific dehydrogenases, were shown to be expressed constitutively. It was observed that increased vanillin concentrations inhibited its own bioconversion suggesting that the activity required intact cells with metabolic capacity.

The potential application of vanillin in preventing yeast spoilage of soft drinks and fruit juices.

The preservative effect of vanillin, the major constituent of vanilla beans, was studied in an apple juice and peach-flavored soft drink. Vanillin activity was tested against Saccharomyces cerevisiae and Candida parapsilosis at 8 and 25 degrees C over an 8-week storage period. Initial results in laboratory media indicated minimum inhibitory concentration values of 17 and 9 mM vanillin for the two yeast strains. Concentrations of 20 and 10 mM vanillin, respectively, were required to achieve complete inhibition of both yeast strains inoculated at a level of approximately 10(4) CFU/ml in the apple juice and peach-flavored soft drink over the 8-week storage at 25 degrees C. These effective levels were reduced to 5 and 1 mM, when the storage temperature was reduced to 8 degrees C. A biocidal effect against both yeasts was observed within 96 h to 8 weeks, with vanillin concentrations of 5 to 40 mM depending on the beverage and the storage temperatures used. The increased activity of vanillin in the peach-flavored soft drink (pH 3.1) in comparison to the apple juice (pH 3.5) is probably a result of the lower intrinsic pH of the former; however, variation in vitamin and mineral levels or the presence of other phenolic compounds between the two drinks might also have contributed to the observed differences. Furthermore, the increased activity at the lower temperature could be linked to the combination of the increased membrane fluidity and the membrane-perturbing action of vanillin. We conclude that vanillin has the potential to preserve fruit juices and soft drinks that are low in both lipid and protein content against S. cerevisiae and C. parapsilosis.

Gene gymnastics: Synthetic biology for baculovirus expression vector system engineering.

Most essential activities in eukaryotic cells are catalyzed by large multiprotein assemblies containing up to ten or more interlocking subunits. The vast majority of these protein complexes are not easily accessible for high resolution studies aimed at unlocking their mechanisms, due to their low cellular abundance and high heterogeneity. Recombinant overproduction can resolve this bottleneck and baculovirus expression vector systems (BEVS) have emerged as particularly powerful tools for the provision of eukaryotic multiprotein complexes in high quality and quantity. Recently, synthetic biology approaches have begun to make their mark in improving existing BEVS reagents by de novo design of streamlined transfer plasmids and by engineering the baculovirus genome. Here we present OmniBac, comprising new custom designed reagents that further facilitate the integration of heterologous genes into the baculovirus genome for multiprotein expression. Based on comparative genome analysis and data mining, we herein present a blueprint to custom design and engineer the entire baculovirus genome for optimized production properties using a bottom-up synthetic biology approach.

2011 update to the Society of Thoracic Surgeons and the Society of Cardiovascular Anesthesiologists blood conservation clinical practice guidelines.

BACKGROUND: Practice guidelines reflect published literature. Because of the ever changing literature base, it is necessary to update and revise guideline recommendations from time to time. The Society of Thoracic Surgeons recommends review and possible update of previously published guidelines at least every three years. This summary is an update of the blood conservation guideline published in 2007. METHODS: The search methods used in the current version differ compared to the previously published guideline. Literature searches were conducted using standardized MeSH terms from the National Library of Medicine PUBMED database list of search terms. The following terms comprised the standard baseline search terms for all topics and were connected with the logical 'OR' connector--Extracorporeal circulation (MeSH number E04.292), cardiovascular surgical procedures (MeSH number E04.100), and vascular diseases (MeSH number C14.907). Use of these broad search terms allowed specific topics to be added to the search with the logical 'AND' connector. RESULTS: In this 2011 guideline update, areas of major revision include: 1) management of dual anti-platelet therapy before operation, 2) use of drugs that augment red blood cell volume or limit blood loss, 3) use of blood derivatives including fresh frozen plasma, Factor XIII, leukoreduced red blood cells, platelet plasmapheresis, recombinant Factor VII, antithrombin III, and Factor IX concentrates, 4) changes in management of blood salvage, 5) use of minimally invasive procedures to limit perioperative bleeding and blood transfusion, 6) recommendations for blood conservation related to extracorporeal membrane oxygenation and cardiopulmonary perfusion, 7) use of topical hemostatic agents, and 8) new insights into the value of team interventions in blood management. CONCLUSIONS: Much has changed since the previously published 2007 STS blood management guidelines and this document contains new and revised recommendations.