Polymer interfaces and biopharmaceuticals: Chemistry, designs and challenges

The complex interactions between biological components and polymer materials has an extensive technical history. Virtually every surface property has been invoked as being important to biological interfacial response: texture, roughness, topology, porosity, hydrophilic, hydrophobic, polar, apolar, (non)-wettable, non-fouling brushes, surface mobility, rigidity, flexibility, crystalline versus amorphous, aspect ratio. Few surface properties alone, however, provide consistent, global technical solutions to vexing biomedical technology problems, particularly with cell culture, blood, plasma, microbial milieu, and protein solutions. Bio-interface materials performance must therefore be tailored specifically to each application. Short-term contact use (minutes/hours) has different materials interface requirements than long-term (days) use; globular proteins have particularly difficult needs not readily satisfied by any materials solution. Viable biologics interfaces (i.e., fresh blood harvests, cell cultures) must also consider selective gas permeability, leachables, and sterilization issues. Film properties, lamination, cutting, chemical stability, sealing and handling issues are additional considerations for single-use materials. Lastly cost-of-goods and materials economics must be considered, especially for single use technologies. No one-size-fits-all surface solutions currently satisfy all bio-interface materials needs. This talk will review design principles, dogma and actual polymer chemistries to modulate, modify and manipulate polymer surfaces in contact with biological components. Several polymer surface properties will be discussed with regard to their physical chemistry in aqueous media. Traditional and recent developments in non-fouling interfaces and polymer approaches and their hypothesized influences on biophysical interactions with proteins and cells will be presented

The Escherichia coli RutR transcription factor binds at targets within genes as well as intergenic regions.

The Escherichia coli RutR protein is the master regulator of genes involved in pyrimidine catabolism. Here we have used chromatin immunoprecipitation in combination with DNA microarrays to measure the binding of RutR across the chromosome of exponentially growing E. coli cells. Twenty RutR-binding targets were identified and analysis of these targets generated a DNA consensus logo for RutR binding. Complementary in vitro binding assays showed high-affinity RutR binding to 16 of the 20 targets, with the four low-affinity RutR targets lacking predicted key binding determinants. Surprisingly, most of the DNA targets for RutR are located within coding segments of the genome and appear to have little or no effect on transcript levels in the conditions tested. This contrasts sharply with other E. coli transcription factors whose binding sites are primarily located in intergenic regions. We suggest that either RutR has yet undiscovered function or that evolution has been slow to eliminate non-functional DNA sites for RutR because they do not have an adverse effect on cell fitness

DNA Sampling: a method for probing protein binding at specific loci on bacterial chromosomes

We describe a protocol, DNA sampling, for the rapid isolation of specific segments of DNA, together with bound proteins, from Escherichia coli K-12. The DNA to be sampled is generated as a discrete fragment within cells by the yeast I-SceI meganuclease, and is purified using FLAG-tagged LacI repressor and beads carrying anti-FLAG antibody. We illustrate the method by investigating the proteins bound to the colicin K gene regulatory region, either before or after induction of the colicin K gene promoter

Autoregulation of the Escherichia coli melR promoter: repression involves four molecules of MelR

The Escherichia coli MelR protein is a transcription activator that autoregulates its own promoter by repressing transcription initiation. Optimal repression requires MelR binding to a site that overlaps the melR transcription start point and to upstream sites. In this work, we have investigated the different determinants needed for optimal repression and their spatial requirements. We show that repression requires a complex involving four DNA-bound MelR molecules, and that the global CRP regulator plays little or no role

The environmentally-regulated interplay between local three-dimensional chromatin organisation and transcription of <i>proVWX</i> in <i>E. coli</i>

Nucleoid associated proteins (NAPs) maintain the architecture of bacterial chromosomes and regulate gene expression. Thus, their role as transcription factors may involve three-dimensional chromosome re-organisation. While this model is supported by in vitro studies, direct in vivo evidence is lacking. Here, we use RT-qPCR and 3C-qPCR to study the transcriptional and architectural profiles of the H-NS (histone-like nucleoid structuring protein)-regulated, osmoresponsive proVWX operon of Escherichia coli at different osmolarities and provide in vivo evidence for transcription regulation by NAP-mediated chromosome re-modelling in bacteria. By consolidating our in vivo investigations with earlier in vitro and in silico studies that provide mechanistic details of how H-NS re-models DNA in response to osmolarity, we report that activation of proVWX in response to a hyperosmotic shock involves the destabilization of H-NS-mediated bridges anchored between the proVWX downstream and upstream regulatory elements (DRE and URE), and between the DRE and ygaY that lies immediately downstream of proVWX. The re-establishment of these bridges upon adaptation to hyperosmolarity represses the operon. Our results also reveal additional structural features associated with changes in proVWX transcript levels such as the decompaction of local chromatin upstream of the operon, highlighting that further complexity underlies the regulation of this model operon. H-NS and H-NS-like proteins are wide-spread amongst bacteria, suggesting that chromosome re-modelling may be a typical feature of transcriptional control in bacteria

Assessment of a Siloxane Poly(Urethane‐Urea) Elastomer Designed for Implantable Heart Valve Leaflets

Synthetic polymer leaflets in prosthetic cardiac valves hold the potential to reduce calcification and thrombus, while improving blood flow, durability, and device economics. A recently developed siloxane poly(urethane‐urea) (LifePolymer™, LP) exhibits properties essential for heart valve leaflets, including low dynamic modulus, high tensile strength, minimal creep, and excellent biostability. LP properties result from carefully designed “linked co‐macrodiol” chemistry that maximizes silicone content and virtual crosslinks between soft and hard phases. Characterization of multiple commercial batches demonstrates a robust synthesis process with minimal variation. Extensive ISO 10993‐based biocompatibility testing resulted in no observable toxicity or other adverse reactions. An ex vivo AV shunt thrombogenicity investigation revealed nearly undetectable levels of platelet attachment and thrombus formation on LP surfaces. Chronic ovine implantation of prototype heart valves with LP leaflets showed no differences in thrombogenicity or systemic tissue response when compared to a clinically standard tissue‐based valve. Toxicological risk assessment, based on extractables and leachables analysis of LP‐based heart valves, confirmed minimal toxicological risk. Lastly, 24‐week, strain‐accelerated in vivo LP biostability testing confirmed previous favorable in vitro biostability findings. These studies demonstrate that this newly developed elastomer exhibits ideal biomaterial properties for the flexible leaflets of a totally synthetic heart valve replacement

Transcription factor distribution in Escherichia coli: studies with FNR protein

Using chromatin immunoprecipitation (ChIP) and high-density microarrays, we have measured the distribution of the global transcription regulator protein, FNR, across the entire Escherichia coli chromosome in exponentially growing cells. Sixty-three binding targets, each located at the 5′ end of a gene, were identified. Some targets are adjacent to poorly transcribed genes where FNR has little impact on transcription. In stationary phase, the distribution of FNR was largely unchanged. Control experiments showed that, like FNR, the distribution of the nucleoid-associated protein, IHF, is little altered when cells enter stationary phase, whilst RNA polymerase undergoes a complete redistribution

Accepting higher morbidity in exchange for sacrificing fewer animals in studies developing novel infection-control strategies.

Preventing bacterial infections from becoming the leading cause of death by the year 2050 requires the development of novel, infection-control strategies, building heavily on biomaterials science, including nanotechnology. Pre-clinical (animal) studies are indispensable for this development. Often, animal infection outcomes bear little relation to human clinical outcome. Here, we review conclusions from pathogen-inoculum dose-finding pilot studies for evaluation of novel infection-control strategies in murine models. Pathogen-inoculum doses are generally preferred that produce the largest differences in quantitative infection outcome parameters between a control and an experimental group, without death or termination of animals due to having reached an inhumane end-point during the study. However, animal death may represent a better end-point for evaluation than large differences in outcome parameters or number of days over which infection persists. The clinical relevance of lower pre-clinical outcomes, such as bioluminescence, colony forming units (CFUs) retrieved or more rapid clearance of infection is unknown, as most animals cure infection without intervention, depending on pathogen-species and pathogen-inoculum dose administered. In human clinical practice, patients suffering from infection present to hospital emergency wards, frequently in life-threatening conditions. Animal infection-models should therefore use prevention of death and recurrence of infection as primary efficacy targets to be addressed by novel strategies. To compensate for increased animal morbidity and mortality, animal experiments should solely be conducted for pre-clinical proof of principle and safety. With the advent of sophisticated in vitro models, we advocate limiting use of animal models when exploring pathogenesis or infection mechanisms

Clinical translation of the assets of biomedical engineering - a retrospective analysis with looks to the future

Introduction: Biomedical-engineering (BME) plays a major role in modern medicine. Many BME-based assets have been brought to clinical translation in the twentieth century, but translation currently stagnates. Here, we compare the impact of past and present scientific, economic and societal climates on the translation of BME-based assets, in order to provide the BME-community with incentives to address current stagnation. Areas covered: In the twentieth century, W.J. Kolff brought kidney dialysis, the total artificial heart, artificial vision and limbs to clinical application. This success raises the question whether Kolff and other past giants of clinical translation had special mind-sets, or whether their problem selection, their training, or governmental and regulatory control played roles. Retrospective analysis divides the impact of BME-based assets to clinical application into three periods: 1900-1970: rapid translation from bench-to-bedside, 1970-1990: new diseases and increased governmental control, and the current translational crisis from 1990 onward. Expert opinion: Academic and societal changes can be discerned that are concurrent with BME's translational success: mono-disciplinary versus multi-disciplinary training, academic reward systems based on individual achievements versus team achievements with strong leadership, increased governmental and regulatory control, and industrial involvement. From this, recommendations can be derived for accelerating clinical translation of BME-assets