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The aim of this study was to identify the post–percutaneous coronary intervention (PCI) target value of instantaneous wave-free ratio (iFR) that would best discriminate clinical events at 1 year in the DEFINE PCI (Physiologic Assessment of Coronary Stenosis Following PCI) study. The impact of residual ischemia detected by iFR post-PCI on clinical and symptom-related outcomes is unknown. Blinded iFR pull back was performed after successful stent implantation in 500 patients. The primary endpoint was the rate of residual ischemia, defined as iFR ≤0.89, after operator-assessed angiographically successful PCI. Secondary endpoints included clinical events at 1 year and change in Seattle Angina Questionnaire angina frequency (SAQ-AF) score during follow-up. As reported, 24.0% of patients had residual ischemia (iFR ≤0.89) after successful PCI, with 81.6% of cases attributable to angiographically inapparent focal lesions. Post-PCI iFR ≥0.95 (present in 182 cases [39%]) was associated with a significant reduction in the composite of cardiac death, spontaneous myocardial infarction, or clinically driven target vessel revascularization compared with post-PCI iFR <0.95 (1.8% vs 5.7%; P = 0.04). Baseline SAQ-AF score was 73.3 ± 22.8. For highly symptomatic patients (baseline SAQ-AF score ≤60), SAQ-AF score increased by ≥10 points more frequently in patients with versus without post-PCI iFR ≥0.95 (100.0% vs 88.5%; P = 0.01). In DEFINE PCI, despite angiographically successful PCI, highly symptomatic patients at baseline without residual ischemia by post-PCI iFR had greater reductions in anginal symptoms at 1 year compared with patients with residual ischemia. Achieving post-PCI iFR ≥0.95 was also associated with improved 1-year event-free survival. (Physiologic Assessment of Coronary Stenosis Following PCI [DEFINE PCI]; NCT03084367) [Display omitted] [ABSTRACT FROM AUTHOR]

For over 20 years, peptide materials in their hydrogel or soluble fibril form have been used for biomedical applications such as drug delivery, cell culture, vaccines, and tissue regeneration. To facilitate the translation of these materials, key areas of research still need to be addressed. Their structural characterization lags compared to amyloid proteins. Many of the structural features designed to guide materials formation are primarily being characterized by their observation in atomic resolution structures of amyloid assemblies. Herein, these motifs are examined in relation to peptide designs identifying common interactions that drive assembly and provide structural specificity. Current efforts to design complex structures, as reviewed here, highlight the need to extend the structural revolution of amyloid proteins to peptide assemblies to validate design principles. With respect to clinical applications, the fundamental interactions and responses of proteins, cells, and the immune system to peptide materials are still not well understood. Only a few trends are just now emerging for peptide materials interactions with biological systems. Understanding how peptide material properties influence these interactions will enable the translation of materials towards current and emerging applications. [ABSTRACT FROM AUTHOR]

Objectives The aim of this study was to prospectively study and confirm the safety and efficacy of the Tryton Side Branch Stent in the treatment of coronary artery bifurcations involving large side branches (SBs). Background The TRYTON Pivotal randomized controlled trial (RCT) was designed to compare the Tryton stent with standard provisional SB stenting in large vessels. The trial inadvertently enrolled patients with too small SBs (<2.25 mm). The overall trial did not meet its primary endpoint, because of an increased rate of periprocedural myocardial infarction in the Tryton stent arm. A post hoc analysis restricted to the intended population showed that the trial would have met its endpoint if only patients with SBs ≥2.25 mm in diameter (by core laboratory quantitative coronary angiography) had been enrolled. Methods The Tryton Confirmatory Study was a prospective, single-arm extension of the TRYTON Pivotal RCT that enrolled an additional 133 patients treated with the Tryton Side Branch Stent. It was designed to confirm the results of the post hoc analysis and emphasized the inclusion of appropriately sized SBs. The primary endpoint was noninferiority with regard to periprocedural myocardial infarction (creatine kinase myocardial band 3 times the upper limit of normal) compared with a performance goal based on the TRYTON Pivotal RCT. Results Among the 133 enrolled patients, 132 (99.2%) had SBs ≥2.25 mm. Baseline clinical and angiographic parameters were similar in this study and the RCT. Periprocedural myocardial infarction occurred in 10.5% of patients, which was numerically lower than the provisional group in the TRYTON Pivotal RCT (11.9%). The 95% confidence bounds did not extend beyond the pre-defined performance goal of 17.9%, meeting the noninferiority primary endpoint. Conclusions The Tryton Confirmatory Study, in conjunction with the post hoc analysis of the intended population in the TRYTON Pivotal RCT, supports the safety and efficacy of the Tryton Side Branch Stent for treatment of bifurcation lesions involving large SBs. [ABSTRACT FROM AUTHOR]

Objectives The purpose of this study was to assess the diagnostic accuracy of the instantaneous wave-free ratio (iFR) to characterize, outside of a pre-specified range of values, stenosis severity, as defined by fractional flow reserve (FFR) ≤0.80, in a prospective, independent, controlled, core laboratory–based environment. Background Studies with methodological heterogeneity have reported some discrepancies in the classification agreement between iFR and FFR. The ADVISE II (ADenosine Vasodilator Independent Stenosis Evaluation II) study was designed to overcome limitations of previous iFR versus FFR comparisons. Methods A total of 919 intermediate coronary stenoses were investigated during baseline and hyperemia. From these, 690 pressure recordings (n = 598 patients) met core laboratory physiology criteria and are included in this report. Results The pre-specified iFR cut-off of 0.89 was optimal for the study and correctly classified 82.5% of the stenoses, with a sensitivity of 73.0% and specificity of 87.8% (C statistic: 0.90 [95% confidence interval (CI): 0.88 to 0.92, p < 0.001]). The proportion of stenoses properly classified by iFR outside of the pre-specified treatment (≤0.85) and deferral (≥0.94) values was 91.6% (95% CI: 88.8% to 93.9%). When combined with FFR use within these cut-offs, the percent of stenoses properly classified by such a pre-specified hybrid iFR-FFR approach was 94.2% (95% CI: 92.2% to 95.8%). The hybrid iFR-FFR approach obviated vasodilators from 65.1% (95% CI: 61.1% to 68.9%) of patients and 69.1% (95% CI: 65.5% to 72.6%) of stenoses. Conclusions The ADVISE II study supports, on the basis rigorous methodology, the diagnostic value of iFR in establishing the functional significance of coronary stenoses, and highlights its complementariness with FFR when used in a hybrid iFR-FFR approach. (ADenosine Vasodilator Independent Stenosis Evaluation II–ADVISE II; NCT01740895 ) [ABSTRACT FROM AUTHOR]

Abstract: A growing number of medications must be administered through parenteral delivery, i.e., intravenous, intramuscular, or subcutaneous injection, to ensure effectiveness of the therapeutic. For some therapeutics, the use of delivery vehicles in conjunction with this delivery mechanism can improve drug efficacy and patient compliance. Macromolecular self-assembly has been exploited recently to engineer materials for the encapsulation and controlled delivery of therapeutics. Self-assembled materials offer the advantages of conventional crosslinked materials normally used for release, but also provide the ability to tailor specific bulk material properties, such as release profiles, at the molecular level via monomer design. As a result, the design of materials from the “bottom up” approach has generated a variety of supramolecular devices for biomedical applications. This review provides an overview of self-assembling molecules, their resultant structures, and their use in therapeutic delivery. It highlights the current progress in the design of polymer- and peptide-based self-assembled materials. [Copyright &y& Elsevier]

Photolithography enables the precise construction of nanodevices in two-dimensional formats. However, self-assembly of designed molecules serves as an alternative for the construction of three-dimensional nanoscale systems and is particularly appealing in that material properties can potentially be engineered at the molecular level. Peptides and proteins hold promise as building blocks for self-assembled systems because of their exquisite three-dimensional structures and evolutionarily fine-tuned functions. [Copyright &y& Elsevier]

Aim: A ‘Silver Standard’ for HLA data collection and reporting has been described at ImmPort (immport.niaid.nih.gov, “Proposal for HLA Data Validation”) to address ambiguity resolution in the recording and reporting of HLA typing results. While standards are critical for HLA data interoperability, they are not meaningful until useful tools are developed and made available for community use. We are developing distributable tools that implement this silver standard. Here we describe the development a web service to create, update, and retrieve HLA typing data in standardized formats without the need for NMDP allele codes and the corresponding inherent introduction of new ambiguities. Methods: ReST web services with HTTP negotiation are being developed employing a Java library that manages HLA typing data using standardized formats. These formats include the XML based Histoimmunogenetics Markup Language (HML) and a simple character-delimited string format (GL String) able to encode ambiguity within HLA typing. Resources are identified with a simple Uniform Resource Identifier (URI). Results: The services build on a foundation of an open access database schema for IMGT/HLA reference sequence data (updated quarterly), and objects such as alleles, lists of alleles, haplotypes, genotypes, lists of genotypes and multi-locus unphased genotypes. Public services include creating, updating, and retrieving these objects. Content negotiation allows data retrieval in a variety of formats including GL String, HML, HTML, JSON, and QR Code. Conclusions: The tools being developed here provide the HLA researcher, clinician and lab technician a common resource for managing HLA data in a standardized way. We envision these tools to augment workflows through creating new instances of HLA typing objects when needed, and retrieval of those objects and their associated metadata when called upon. [Copyright &y& Elsevier]

Unrelated stem cell registries have been collecting HLA typing of volunteer bone marrow donors for over 25 years. Donor selection for hematopoietic stem cell transplantation is based primarily on matching the alleles of donors and patients at five polymorphic HLA loci. As HLA typing technologies have continually advanced since the beginnings of stem cell transplantation, registries have accrued typings of varied HLA typing ambiguity. We present a new typing resolution score (TRS), based on the likelihood of self-match, that allows the systematic comparison of HLA typings across different methods, data sets and populations. We apply the TRS to chart improvement in HLA typing within the Be The Match Registry of the United States from the initiation of DNA-based HLA typing to the current state of high-resolution typing using next-generation sequencing technologies. In addition, we present a publicly available online tool for evaluation of any given HLA typing. This TRS objectively evaluates HLA typing methods and can help define standards for acceptable recruitment HLA typing. [ABSTRACT FROM AUTHOR]

Hydrogels formed from self-assembling peptides are finding use in tissue engineering and drug delivery applications. Given the notorious difficulties associated with producing self-assembling peptides by recombinant expression, most are typically prepared by chemical synthesis. Herein, we report the design of a family of self-assembling β-hairpin peptides amenable to efficient production using an optimized bacterial expression system. Ex pressing peptides, EX1, EX2 and EX3 contain identical eight-residue amphiphilic β-strands connected by varying turn sequences that are responsible for ensuring chain reversal and the proper intramolecular folding and consequent self-assembly of the peptide into a hydrogel network under physiological conditions. EX1 was initially used to establish and optimize the bacterial expression system by which all the peptides could be eventually individually expressed. Expression clones were designed to allow exploration of possible fusion partners and investigate both enzymatic and chemical cleavage as means to liberate the target peptide. A systematic analysis of possible expression systems followed by fermentation optimization lead to a system in which all three peptides could be expressed as fusions with BAD-BH3, the BH3 domain of the proapoptotic BAD (Bcl-2 Associated Death) Protein. CNBr cleavage followed by purification afforded 50, 31, and 15 mg/L yields of pure EX1, EX2 and EX3, respectively. CD spectroscopy, TEM, and rheological analysis indicate that these peptides fold and assembled into well-defined fibrils that constitute hydrogels having shear-thin/recovery properties. [ABSTRACT FROM AUTHOR]

Abstract: Controlled biodegradation specific to matrix metalloproteinase-13 was incorporated into the design of self-assembling β-hairpin peptide hydrogels. Degrading Peptides (DP peptides) are a series of five peptides that have varying proteolytic susceptibilities toward MMP-13. These peptides undergo environmentally triggered folding and self-assembly under physiologically relevant conditions (150 mm NaCl, pH 7.6) to form self-supporting hydrogels. In the presence of enzyme, gels prepared from distinct peptides are degraded at rates that differ according to the primary sequence of the single peptide comprising the gel. Material degradation was monitored by oscillatory shear rheology over the course of 14 days, where overall degradation of the gels vary from 5% to 70%. Degradation products were analyzed by HPLC and identified by electrospray-ionization mass spectrometry. This data shows that proteolysis of the parent peptides constituting each gel occurs at the intended sequence location. DP hydrogels show specificity to MMP-13 and are only minimally cleaved by matrix metalloproteinase-3 (MMP-3), another common enzyme present during tissue injury. In vitro migration assays performed with SW1353 cells show that migration rates through each gel differs according to peptide sequence, which is consistent with the proteolysis studies using exogenous MMP-13. [Copyright &y& Elsevier]

As the prevalence of cancer and transmittable disease persists, the development of new and more advanced therapies remains a priority in medical research. An emerging platform for the treatment of these illnesses is the use of materials formed via peptide assembly where the bulk material itself acts as the therapeutic. Higher ordered peptide structures with defined chemistry are capable of cellular targeting, recognition, and internalization. Recent design efforts are being made to exploit the nanoscale definition of the materials formed by assembling peptides to target cancer and microbial cells and to function as vaccines. This review focuses on assembled peptide materials that actively participate in the biological processes important to cancer and transmittable diseases to exert an anticipated functional outcome. [Copyright &y& Elsevier]

Abstract: A family of penta- and hexadentate metal ligating α-amino acids, suitably protected for Fmoc solid-phase chemistry, has been prepared. These residues incorporate the mono-amides of ethanolaminetriacetic acid, ethylenediaminetriacetic acid, and ethylenediaminetetraacetic acid as side chains. Side chains are tethered varying distances (n) from the Cα-carbon to allow metal binding events to occur at distinct distances from the peptide backbone. These residues are designed to allow the facile installation of metal chelates along a peptide backbone. [Copyright &y& Elsevier]

Allogeneic Hematopoietic Cell Transplantation (HCT) is a curative therapy for hematologic disorders and often requires human leukocyte antigen (HLA)-matched donors. Donor registries have recruited donors utilizing evolving technologies of HLA genotyping methods. This necessitates in-silico ambiguity resolution and statistical imputation based on haplotype frequencies estimated from donor data stratified by self-identified race and ethnicity (SIRE). However, SIRE has limited genetic validity and presents a challenge for individuals with unknown or mixed SIRE. We present MR-GRIMM "Multi-Race Graph IMputation and Matching" that simultaneously imputes the race/ethnic category and HLA genotype using a SIRE based prior. Additionally, we propose a novel method to impute HLA typing inconsistent with current haplotype frequencies. The performance of MR-GRIMM was validated using a dataset of 170,000 donor-recipient pairs. MR-GRIMM has an average 20 % lower matching error (1-AUC) than single-race imputation. The recall metric (sensitivity) of the race/ethnic category imputation from HLA was measured by comparing the imputed donor race with the donor-provided SIRE. Accuracies of 0.74 and 0.55 were obtained for the prediction of 5 broad and 21 detailed US population groups respectively. The operational implementation of this algorithm in a registry search could help improve match predictions and access to HLA-matched donors. [ABSTRACT FROM AUTHOR]

Several gene transfer clinical trials are currently ongoing with the common aim of delivering a shortened version of dystrophin, termed a microdystrophin, for the treatment of Duchenne muscular dystrophy (DMD). However, one of the main differences between these trials is the microdystrophin protein produced following treatment. Each gene transfer product is based on different selections of dystrophin domain combinations to assemble microdystrophin transgenes that maintain functional dystrophin domains and fit within the packaging limits of an adeno-associated virus (AAV) vector. While domains involved in mechanical function, such as the actin-binding domain and β-dystroglycan binding domain, have been identified for many years and included in microdystrophin constructs, more recently the neuronal nitric oxide synthase (nNOS) domain has also been identified due to its role in enhancing nNOS membrane localization. As nNOS membrane localization has been established as an important requirement for prevention of functional ischemia in skeletal muscle, inclusion of the nNOS domain into a microdystrophin construct represents an important consideration. The aim of this mini review is to highlight what is currently known about the nNOS domain of dystrophin and to describe potential implications of this domain in a microdystrophin gene transfer clinical trial. [ABSTRACT FROM AUTHOR]

Abstract Mechanical feedback from the tumor microenvironment regulates an array of processes underlying cancer biology. For example, increased stiffness of mammary extracellular matrix (ECM) drives malignancy and alters the phenotypes of breast cancer cells. Despite this link, the role of substrate stiffness in chemotherapeutic response in breast cancer remains unclear. This is complicated by routine culture and adaptation of cancer cell lines to unnaturally rigid plastic or glass substrates, leading to profound changes in their growth, metastatic potential and, as we show here, chemotherapeutic response. We demonstrate that primary breast cancer cells undergo dramatic phenotypic changes when removed from the host microenvironment and cultured on rigid surfaces, and that drug responses are profoundly altered by the mechanical feedback cells receive from the culture substrate. Conversely, primary breast cancer cells cultured on substrates mimicking the mechanics of their host tumor ECM have a similar genetic profile to the in situ cells with respect to drug activity and resistance pathways. These results suggest substrate stiffness plays a significant role in susceptibility of breast cancer to clinically-approved chemotherapeutics, and presents an opportunity to improve drug discovery efforts by integrating mechanical rigidity as a parameter in screening campaigns. [ABSTRACT FROM AUTHOR]

We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines ( miring.immunogenomics.org ). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS. [ABSTRACT FROM AUTHOR]

Aim As clinical matching definitions for transplantation evolve there is a need to quantify the degree of uncertainty in a typing result relative to a particular resolution target. We aimed to develop such a measure and then applied it to quantifying the improvement in typing resolution of the Be The Match® Registry since 1993. Methods We describe and apply a new typing ambiguity score, based on the likelihood of self-match which allows for comparison of HLA typings across different methods, data sets and populations. In order to compute the typing ambiguity score, we perform HLA genotype imputation on 14 million donors using high-resolution haplotype frequencies generated from unrelated donors from the National Marrow Donor Program database for 5 population categories. For this experiment the resolution target was 5-locus ARS exons with equivalent amino acid sequence. Results The Registry has seen an increasing trend in the score over time for all populations and all HLA loci, with the overall scores for recruitment typing rising from 0.31 in 1993 to 0.96 in 2015. Many discontinuities in scores coincide with changes in recruitment typing policy, such as the transition of HLA-A and B typing from serology to DNA, the start of sequence based typing, the inclusion of HLA-C and DQB1 at recruitment, etc. We find evidence that oligo-based kits were tuned to reduce typing ambiguity primarily for majority race/ethnic groups as European American donors generally had the most rapid increase in scores, while African American donors have the lowest scores and a slower increase in scores. Finally, we show that new recruitment HLA typing performed for the US registry today has very little ambiguity under the current standard of matching at HLA-A, C, B, DRB1, and DQB1, using the current laboratory methods that employ next-generation sequencing or sequence-based typing with panels of group-specific sequencing primers. Conclusion Our typing ambiguity score objectively measured the improvement in HLA typing within the US registry from 1993 to the current state of high-resolution typing. We next aim to assess ambiguity among global registries in BMDW. This method is general and can be applied to other loci (e.g. DPB1, DPA1, DQA1) other systems (KIR) and other definitions of allele (all-exons or full-gene). [ABSTRACT FROM AUTHOR]

DNA that encodes tumor-specific antigens represents potential immunostimulatory agents. However, rapid enzymatic degradation and fragmentation of DNA during administration can result in limited vector expression and, consequently, poor efficacy. These challenges have necessitated the use of novel strategies for DNA delivery. Herein, we study the ability of cationic self-assembling peptide hydrogels to encapsulate plasmid DNA, and enhance its immunostimulatory potential in vivo . The effect of network charge on the gel's ability to retain the DNA was assessed employing three gel-forming peptides that vary systematically in formal charge. The peptide HLT2, having a formal charge of +5 at neutral pH, was optimal in encapsulating microgram quantities of DNA with little effect on its rheological properties, allowing its effective syringe delivery in vivo . The plasmid, DNA(TA), encapsulated within these gels encodes for a melanoma-specific gp100 antigen fused to the alarmin protein adjuvant HMGN1. Implantation of DNA(TA)-loaded HLT2 gels into mice resulted in an acute inflammatory response with the presence of polymorphonuclear cells, which was followed by infiltrating macrophages. These cellular infiltrates aid in the processing of encapsulated DNA, promoting increased lymphoproliferation and producing an enhanced immune response mediated by CD4+/IFNγ+ expressing Th1 cells, and complemented by the formation of gp100-specific antibodies. [ABSTRACT FROM AUTHOR]

Aim: We previously showed that Shannon’s entropy could be successfully used to quantify the typing resolution of HLA data in stem cell registries. Here, we present an improvement of this measure, termed typing ambiguity score, which enables direct and systematic comparison across methods, data sets and populations. Methods: Typing ambiguity score is calculated from normalized Shannon’s entropy. It ranges between 0 and 1, where 1 is given to a typing with no ambiguity and 0 to a typing with maximum ambiguity. We differentiate between ambiguity in (phased) HLA genotypes and ambiguity in un-phased genotypes, which has a more practical application for donor and patient matching. Furthermore, we implement a web-based typing ambiguity assessment tool, integrated with an existing application called HaploStats that facilitates access to HLA haplotype frequencies. Results: Maximum ambiguity is obtained for a typing that results in equally probable set of outcomes, since for such cases we have the least information on what the true outcome is. For example, outcome with probabilities p = (0.5, 0.5) has a score equal to 1, while outcome with probabilities p = (0.95, 0.05) has a score of 0.28. To demonstrate how this score can be used to evaluate typings in a stem cell registry, we compute overall and per-locus ambiguity scores for HLA typings in the BeTheMatch® (US) registry, and explore how it varies with data resolution, typing method, time, and population. Conclusions: We present a method to objectively quantify and compare ambiguity in HLA typing data obtained via different methods. An analysis of HLA typing data in the US registry demonstrates the utility of this method for stem cell registries and HLA typing labs. We show that the typing ambiguity score can compare typing methodologies, determine which methods are best suited for which populations, and help define acceptable HLA typing for donor recruitment. [ABSTRACT FROM AUTHOR]

Aim: Using Shannon’s entropy, we objectively measured the HLA typing ambiguity of various commercial Sequence-Specific Oligonucleotide (SSO) protocols (groups of SSO kits used in tandem). We previously compared the HLA typing ambiguity obtained by serology, allele family level DNA-based typing, SSO, and single-pass sequence based typing (SBT). However, commercial companies have made significant advances in SSO technology in recent years, and those results only evaluated early generic versions of SSO kits. Methods: For several US populations, and for 5 HLA loci relevant to transplantation (HLA-A, -B, -C, -DRB1, -DQB1), we identify a set of commonly used SSO protocols. We use population-level haplotype frequency data to generate a cohort of simulated subjects, followed by 5-locus genotype imputation to generate a list of genotypes that would result from SSO typing each simulated subject. The imputation step computes the relative likelihood of each genotype, which is then used for entropy calculation. Results: Distribution of entropies across populations and for each locus is generally similar, while the magnitudes differ. For example, entropy for locus HLA-A in African American population reaches 0.19, while the maximum entropy in Caucasians is 0.055. We generally observed that protocols comprised of newer kits had lower ambiguity across all populations. In certain populations, some newer kits had higher entropy than older ones. This may indicate those kits were designed to detect alleles uncommonly found in those populations, but resolving ambiguity in other populations. Conclusions: Ambiguity in SSO typing kits has reduced substantially since their introduction in the mid-1990s. Results also indicate a European focus toward selection of additional probes, which is suboptimal for typing diverse populations. We hope to use Shannon’s entropy next to evaluate group-specific sequence primer strategies with SBT. These results will guide selection of HLA typing methods by researchers and laboratories. [Copyright &y& Elsevier]

Abstract: Hydrogel materials that display inherent activity against bacteria can be used to directly treat accessible wounds to prevent or kill existing infection. Hydrogels composed of self-assembling β-hairpin peptides, having a high content of arginine, were found to be extremely effective at killing both gram-positive and gram-negative bacteria, including multi-drug resistant Pseudomonas aeruginosa. No added antibacterial agents are necessary to realize activity. Using self-assembling peptides for material construction allows facile structure–activity relationships to be determined since changes in peptide sequence at the monomer level are directly transposed to the bulk material''s antibacterial properties. SAR studies show that arginine content largely influences the hydrogel''s antibacterial activity, and influences their bulk rheological properties. These studies culminated in an optimized gel, composed of the peptide PEP6R (VKVRVRVRVDPPTRVRVRVKV). PEP6R gels prepared at 1.5 wt % or higher concentration, demonstrate high potency against bacteria, but are cytocompatible toward human erythrocytes as well as mammalian mesenchymal stem cells. Rheological studies indicate that the gel is moderately stiff and displays shear-thin recovery behavior, allowing its delivery via simple syringe. [Copyright &y& Elsevier]

Abstract: Iterative peptide design was used to generate two peptide-based hydrogels to study the effect of network electrostatics on primary chondrocyte behavior. MAX8 and HLT2 peptides have formal charge states of +7 and +5 per monomer, respectively. These peptides undergo triggered folding and self-assembly to afford hydrogel networks having similar rheological behavior and local network morphologies, yet different electrostatic character. Each gel can be used to directly encapsulate and syringe-deliver cells. The influence of network electrostatics on cell viability after encapsulation and delivery, extracellular matrix deposition, gene expression, and the bulk mechanical properties of the gel-cell constructs as a function of culture time was assessed. The less electropositive HLT2 gel provides a microenvironment more conducive to chondrocyte encapsulation, delivery, and phenotype maintenance. Cell viability was higher for this gel and although a moderate number of cells dedifferentiated to a fibroblast-like phenotype, many retained their chondrocytic behavior. As a result, gel-cell constructs prepared with HLT2, cultured under static in vitro conditions, contained more GAG and type II collagen resulting in mechanically superior constructs. Chondrocytes delivered in the more electropositive MAX8 gel experienced a greater degree of cell death during encapsulation and delivery and the remaining viable cells were less prone to maintain their phenotype. As a result, MAX8 gel-cell constructs had fewer cells, of which a limited number were capable of laying down cartilage-specific ECM. [Copyright &y& Elsevier]

Abstract: Curcumin, a hydrophobic polyphenol, is an extract of turmeric root with antioxidant, anti-inflammatory and anti-tumorigenic properties. Its lack of water solubility and relatively low bioavailability set major limitations for its therapeutic use. In this study, a self-assembling peptide hydrogel is demonstrated to be an effective vehicle for the localized delivery of curcumin over sustained periods of time. The curcumin-hydrogel is prepared in-situ where curcumin encapsulation within the hydrogel network is accomplished concurrently with peptide self-assembly. Physical and in vitro biological studies were used to demonstrate the effectiveness of curcumin-loaded β-hairpin hydrogels as injectable agents for localized curcumin delivery. Notably, rheological characterization of the curcumin-loaded hydrogel before and after shear flow have indicated solid-like properties even at high curcumin payloads. In vitro experiments with a medulloblastoma cell line confirm that the encapsulation of the curcumin within the hydrogel does not have an adverse effect on its bioactivity. Most importantly, the rate of curcumin release and its consequent therapeutic efficacy can be conveniently modulated as a function of the concentration of the MAX8 peptide. [Copyright &y& Elsevier]

Abstract: Hydrogel materials are promising vehicles for the delivery of protein therapeutics. Proteins can impart physical interactions, both steric and electrostatic in nature, that influence their release from a given gel network. Here, model proteins of varying hydrodynamic diameter and charge are directly encapsulated and their release studied from electropositive fibrillar hydrogels prepared from the self-assembling peptide, MAX8. Hydrogelation of MAX8 can be triggered in the presence of proteins for their direct encapsulation with neither effect on protein structure nor the hydrogel’s mechanical properties. Bulk release of the encapsulated proteins from the hydrogels was assessed for a month time period at 37 °C before and after syringe delivery of the loaded gels to determine the influence of the protein structure on release. Release of positively charged and neutral proteins was largely governed by the sterics imposed by the network. Conversely, negatively charged proteins interacted strongly with the positively charged fibrillar network, greatly restricting their release to <10% of the initial protein load. Partition and retention studies indicated that electrostatic interactions dictate the amount of protein available for release. Importantly, when protein encapsulated gels were delivered via syringe, the release profiles of the macromolecules show the similar trends as those observed for non-sheared gels. This study demonstrates that proteins can be directly encapsulated in self assembled MAX8 hydrogels, which can then be syringe delivered to a site where subsequent release is controlled by protein structure. [Copyright &y& Elsevier]

Abstract: A peptide-based hydrogel has been designed that directs the formation of hydroxyapatite. MDG1, a twenty-seven residue peptide, undergoes triggered folding to form an unsymmetrical β-hairpin that self-assembles in response to an increase in solution ionic strength to yield a mechanically rigid, self supporting hydrogel. The C-terminal portion of MDG1 contains a heptapeptide (MLPHHGA) capable of directing the mineralization process. Circular dichroism spectroscopy indicates that the peptide folds and assembles to form a hydrogel network rich in β-sheet secondary structure. Oscillatory rheology indicates that the hydrogel is mechanically rigid (G′ ˜ 2500Pa) before mineralization. In separate experiments, mineralization was induced both biochemically and with cementoblast cells. Mineralization-domain had little effect on the mechanical rigidity of the gel. SEM and EDXS show that MDG1 gels are capable of directing the formation of hydroxapatite. Control hydrogels, prepared by peptides either lacking the mineral-directing portion or reversing its sequence, indicated that the heptapeptide is necessary and its actions are sequence specific. [ABSTRACT FROM AUTHOR]

Abstract: Self-assembling peptide hydrogels are used to directly encapsulate and controllably release model FITC–dextran macromolecules of varying size and hydrodynamic diameters. MAX1 and MAX8 are two peptide sequences with different charge states that have been designed to intramolecularly fold and self assemble into hydrogels at physiological buffer conditions (pH 7.4, 150mm NaCl). When self-assembly is initiated in the presence of dextran or protein probes, these macromolecules are directly encapsulated in the gel. Self-diffusion studies using fluorescence recovery after photobleaching (FRAP) and bulk release studies indicate that macromolecule mobility within, and release out of, these gels can be modulated by varying the hydrogel mesh size. The average mesh size can be modulated by simply varying the concentration of a given peptide used to construct the gel or by altering the peptide sequence. In addition, results suggest that electrostatic interactions between the macromolecules and the peptide network influence mobility and release. Depending on probe size, release half-lives can be varied from 8h to over a month. [Copyright &y& Elsevier]

Abstract: The pro-inflammatory potential of β-hairpin peptide hydrogels (MAX1 and MAX8) was assessed in vitro by measuring the cellular response of J774 mouse peritoneal macrophages cultured on the hydrogel surfaces. An enzyme-linked immunosorbent assay (ELISA) was used to measure the level of TNF-α, a pro-inflammatory cytokine, secreted by cells cultured on the gel surfaces. Both bulk and thin films of gels did not elicit TNF-α secretion from the macrophages. In addition, live/dead assays employing laser scanning confocal microscopy (LSCM) and phase-contrast light micrographs show the hydrogel surfaces are non-cytotoxic toward the macrophages and allow the cells to adopt healthy morphologies. When macrophages were activated with lipopolysaccharide (LPS), a known bacterial pathogen that activates an innate immune response, an increase in the TNF-α titers by two orders of magnitude was observed. On LPS induction, macrophages displayed a decrease in cell density, enlarged nuclei, and an increase in cytoplasmic granularity, all characteristics of activated macrophages indicating that the cells are still capable of reacting to insult. The data presented herein indicate that MAX1 and MAX8 gels do not elicit macrophage activation in vitro and suggest that these materials are excellent candidates for in vivo assessment in appropriate animal models. [Copyright &y& Elsevier]

Abstract: MAX1 is a 20 amino acid peptide that undergoes triggered self-assembly to form a rigid hydrogel. When dissolved in aqueous solutions, this peptide exists in an ensemble of random coil conformations rendering it fully soluble. The addition of an exogenous stimulus results in peptide folding into β-hairpin conformation. This folded structure undergoes rapid assembly into a highly crosslinked hydrogel network. DMEM cell culture media is one stimulus able to initiate folding and consequent self-assembly of MAX1. The cytocompatibility of this gel towards NIH 3T3 murine fibroblasts is demonstrated. Gels were shown to be non-toxic to the fibroblast cells. MAX1 hydrogels also foster the ability of the cells to attach to the hydrogel scaffold in the absence or presence of serum proteins. Additionally MAX1 hydrogels were able to support fibroblast proliferation to confluency with little effect on the rheological properties of the scaffold. MAX1 hydrogels meet the preliminary mechanical and cytocompatibiltiy requirements of a tissue engineering scaffold. [Copyright &y& Elsevier]

Aim: The IMGT/HLA Database provides a specialist database for sequences of the human major histocompatibility complex, known as HLA and includes the official sequences for the WHO Nomenclature Committee For Factors of the HLA System. The database currently provides exports of the data in a variety of formats; this is been expanded to include Extensible Markup Language (XML). Methods: The XML format defines a set of rules for encoding documents in a format that is both human and machine-readable. XML is becoming a common format for exporting database contents for dissemination over the World Wide Web. A collaborative working group has provided an XML export of the data contained within the IMGT/HLA Database. This export format would be supported by tools for the utilisation of this dataset into different database formats. Results: The XML format combines the data included in the sequence alignments with the data available in the individual allele reports. This combination of data has not previously been available in a single format. The XML format will enable users to identify the regions within the DNA sequence, such as exons, as well as reconstruct the sequence alignments. This machine-readable format provides a standardised format for importing data from the reference database into a variety of programs. In addition the collaborative project has developed a suite of tools for importing the data into different database schema for allowing incorporation into different laboratory systems. Conclusions: The XML files will be regularly updated as part of the quarterly releases of the IMGT/HLA Database. A beta test version of the XML format and associated tools will shortly be available from the hla.alleles.org and the National Marrow Donor Program websites. [Copyright &y& Elsevier]