Deployment of Aggregation-Sex Pheromones of Longhorned Beetles (Coleoptera: Cerambycidae) Facilitates the Discovery and Identification of their Parasitoids.

Longhorned beetles (Coleoptera: Cerambycidae) include many species that are among the most damaging pests of managed and natural forest ecosystems worldwide. Many species of cerambycids use volatile chemical signals (i.e., pheromones) to locate mates. Pheromones are often used by natural enemies, including parasitoids, to locate hosts and therefore can be useful tools for identifying host-parasitoid relationships. In two field experiments, we baited linear transects of sticky traps with pheromones of cerambycid beetles in the subfamily Cerambycinae. Enantiomeric mixtures of four linear alkanes or four linear alkanes and a ketol were tested separately to evaluate their attractiveness to hymenopteran parasitoids. We hypothesized that parasitoids would be attracted to these pheromones. Significant treatment effects were found for 10 species of parasitoids. Notably, Wroughtonia ligator (Say) (Hymenoptera: Braconidae) was attracted to syn-hexanediols, the pheromone constituents of its host, Neoclytus acuminatus acuminatus (F.) (Coleoptera: Cerambycidae). Location and time of sampling also significantly affected responses for multiple species of parasitoids. These findings contribute to the basic understanding of cues that parasitoids use to locate hosts and suggest that pheromones can be used to hypothesize host relationships between some species of cerambycids and their parasitoids. Future work should evaluate response by known species of parasitoids to the complete blends of pheromones used by the cerambycids they attack, as well as other odors that are associated with host trees of cerambycids.

(2E,6Z,9Z)-2,6,9-Pentadecatrienal as a Male-Produced Aggregation-Sex Pheromone of the Cerambycid Beetle Elaphidion mucronatum.

An increasing body of evidence suggests that the volatile pheromones of cerambycid beetles are much more diverse in structure than previously hypothesized. Here, we describe the identification, synthesis, and field testing of (2E,6Z,9Z)-2,6,9-pentadecatrienal as a male-produced aggregation-sex pheromone of the cerambycid Elaphidion mucronatum (Say) (subfamily Cerambycinae, tribe Elaphidiini). This novel structure is unlike any previously described cerambycid pheromone, and in field bioassays attracted only this species. Males produced about 9 µg of pheromone per 24 h period, and, in field trials, lures loaded with 10, 25, and 100 mg of synthetic pheromone attracted beetles of both sexes, whereas lures loaded with 1 mg of pheromone or less were not significantly attractive. Other typical cerambycine pheromones such as 3-hydroxy-2-hexanone, syn-2,3-hexanediol, and anti-2,3-hexanediol were not attractive to E. mucronatum, and when combined with (2E,6Z,9Z)-2,6,9-pentadecatrienal, the former two compounds appeared to inhibit attraction. Unexpectedly, adults of the cerambycine Xylotrechus colonus (F.) were attracted in significant numbers to a blend of 3-hydroxyhexan-2-one and (2E,6Z,9Z)-2,6,9-pentadecatrienal, even though there is no evidence that this species produces the latter compound. From timed pheromone trap catches, adults of E. mucronatum were determined to be active from dusk until shortly after midnight.

The Rare North American Cerambycid Beetle Dryobius sexnotatus Shares a Novel Pyrrole Pheromone Component with Species in Asia and South America.

The compound 1-(1H-pyrrol-2-yl)-1,2-propanedione ("pyrrole") is an important pheromone component of several Asian and South American species of longhorned beetles in the subfamily Cerambycinae. Here, we report the first confirmed identification of this compound as a pheromone component of a cerambycine species native to North America, the rare beetle Dryobius sexnotatus Linsley. Headspace volatiles from males contained (R)-3-hydroxyhexan-2-one and pyrrole (ratio 1:0.13), neither of which were detected in samples from a female. A field bioassay confirmed that adults of both sexes were attracted only to the binary blend of racemic 3-hydroxyhexan-2-one plus pyrrole, and not by either compound alone. Adults of another cerambycine, Xylotrechus colonus (F.), were attracted by 3-hydroxyhexan-2-one, consistent with this compound being the primary component of the pheromone of this species; attraction was not influenced by the presence of pyrrole. This study attests to the effectiveness of pheromone-baited traps in capturing rarely encountered species of cerambycids. It also provides further evidence that pyrrole represents another conserved pheromone motif within the Cerambycinae, now having been found in representatives of five cerambycid tribes from three continents.

Injectable ECM Scaffolds for Cardiac Repair.

Injectable biomaterials have been developed as potential minimally invasive therapies for treating myocardial infarction (MI) and heart failure. Christman et al. first showed that the injection of a biomaterial alone into rat myocardium can improve cardiac function after MI. More recently, hydrogel forms of decellularized extracellular matrix (ECM) materials have shown substantial promise. Here, we present the methods for fabricating an injectable cardiac-specific ECM biomaterial with demonstrated positive outcomes in small and large animal models for cardiac repair as well as initial safety in a Phase I clinical trial. This chapter also covers the methods for the injection of a biomaterial into rat myocardium using a surgical approach through the diaphragm. Although the methods shown here are for injection of an acellular biomaterial, cells or other therapeutics could also be added to the injection for testing other regenerative medicine strategies.

Tailoring material properties of a nanofibrous extracellular matrix derived hydrogel.

In the native tissue, the interaction between cells and the extracellular matrix (ECM) is essential for cell migration, proliferation, differentiation, mechanical stability, and signaling. It has been shown that decellularized ECMs can be processed into injectable formulations, thereby allowing for minimally invasive delivery. Upon injection and increase in temperature, these materials self-assemble into porous gels forming a complex network of fibers with nanoscale structure. In this study we aimed to examine and tailor the material properties of a self-assembling ECM hydrogel derived from porcine myocardial tissue, which was developed as a tissue specific injectable scaffold for cardiac tissue engineering. The impact of gelation parameters on ECM hydrogels has not previously been explored. We examined how modulating pH, temperature, ionic strength, and concentration affected the nanoscale architecture, mechanical properties, and gelation kinetics. These material characteristics were assessed using scanning electron microscopy, rheometry, and spectrophotometry, respectively. Since the main component of the myocardial matrix is collagen, many similarities between the ECM hydrogel and collagen gels were observed in terms of the nanofibrous structure and modulation of properties by altering ionic strength. However, variation from collagen gels was noted for the gelation temperature along with varied times and rates of gelation. These discrepancies when compared to collagen are likely due to the presence of other ECM components in the decellularized ECM based hydrogel. These results demonstrate how the material properties of ECM hydrogels could be tailored for future in vitro and in vivo applications.

The Role of Minor Pheromone Components in Segregating 14 Species of Longhorned Beetles (Coleoptera: Cerambycidae) of the Subfamily Cerambycinae.

We present research on the chemical ecology of 14 species of longhorned beetles (Coleoptera: Cerambycidae), in four tribes of the subfamily Cerambycinae, conducted in east-central Illinois over 8 yr. Adult males produce aggregation-sex pheromones that attract both sexes. Twenty independent field bioassays explored the pheromone chemistry of the species and tested the possible attractive or antagonistic effects of compounds that are not produced by a given species, but are pheromone components of other species. Analyses of beetle-produced volatiles revealed compounds that had not been reported previously from several of the species. The most common pheromone component was (R)-3-hydroxyhexan-2-one, but pheromones of some species included isomers of the related 2,3-hexanediols. Males of the congeners Phymatodes amoenus (Say) and Phymatodes testaceus (L.) produced pure (R)-2-methylbutan-1-ol. Enantiomers of 2-methylbutan-1-ol also proved to be powerful synergists for Megacyllene caryae (Gahan), Sarosesthes fulminans (F.), and Xylotrechus colonus (F.). The major components of pheromone blends were consistently present in collections of headspace volatiles from male beetles, and only the major components were inherently attractive to a subset of species when tested as single components. Minor components of some species acted as powerful synergists, but in other cases appeared not to influence attraction. Among the minor components identified in headspace extracts from males, 2,3-hexanedione and 2-hydroxyhexan-3-one appeared to be analytical artifacts or biosynthetic by-products, and were neither attractants nor synergists. The antagonistic effects of minor compounds produced by heterospecific males suggest that these compounds serve to maintain prezygotic reproductive isolation among some species that share pheromone components.

Humanized mouse model for assessing the human immune response to xenogeneic and allogeneic decellularized biomaterials.

Current assessment of biomaterial biocompatibility is typically implemented in wild type rodent models. Unfortunately, different characteristics of the immune systems in rodents versus humans limit the capability of these models to mimic the human immune response to naturally derived biomaterials. Here we investigated the utility of humanized mice as an improved model for testing naturally derived biomaterials. Two injectable hydrogels derived from decellularized porcine or human cadaveric myocardium were compared. Three days and one week after subcutaneous injection, the hydrogels were analyzed for early and mid-phase immune responses, respectively. Immune cells in the humanized mouse model, particularly T-helper cells, responded distinctly between the xenogeneic and allogeneic biomaterials. The allogeneic extracellular matrix derived hydrogels elicited significantly reduced total, human specific, and CD4+ T-helper cell infiltration in humanized mice compared to xenogeneic extracellular matrix hydrogels, which was not recapitulated in wild type mice. T-helper cells, in response to the allogeneic hydrogel material, were also less polarized towards a pro-remodeling Th2 phenotype compared to xenogeneic extracellular matrix hydrogels in humanized mice. In both models, both biomaterials induced the infiltration of macrophages polarized towards a M2 phenotype and T-helper cells polarized towards a Th2 phenotype. In conclusion, these studies showed the importance of testing naturally derived biomaterials in immune competent animals and the potential of utilizing this humanized mouse model for further studying human immune cell responses to biomaterials in an in vivo environment.

Quantification of decellularized human myocardial matrix: A comparison of six patients.

PURPOSE: The purpose of this study was to characterize and quantitatively analyze human cardiac extracellular matrix (ECM) isolated from six different cadaveric donor hearts. EXPERIMENTAL DESIGN: ECM was isolated by decellularization of six human cadaveric donor hearts and characterized by quantifying sulfated glycosaminoglycan content (sGAG) and via PAGE. The protein content was then quantified using ECM-targeted Quantitative conCATamers (QconCAT) by LC-SRM analysis using 83 stable isotope labeled (SIL) peptides representing 48 different proteins. Nontargeted global analysis was also implemented using LC-MS/MS. RESULTS: The sGAG content, PAGE, and QconCAT proteomics analysis showed significant variation between each of the six patient samples. The quantitative proteomics indicated that the majority of the protein content was composed of various fibrillar collagen components. Also, quantification of difficult to remove cellular proteins represented less than 1% of total protein content, which is very low for a decellularized biomaterial. Global proteomics identified over 200 distinct proteins present in the human cardiac ECM. CONCLUSION AND CLINICAL RELEVANCE: In conclusion, quantification and characterization of human myocardial ECM showed significant patient-to-patient variability between the six investigated patients. This is an important outcome for the development of allogeneic derived biomaterials and for increasing our understanding of human myocardial ECM composition.

Fibronectin and Cyclic Strain Improve Cardiac Progenitor Cell Regenerative Potential In Vitro.

Cardiac progenitor cells (CPCs) have rapidly advanced to clinical trials, yet little is known regarding their interaction with the microenvironment. Signaling cues present in the microenvironment change with development and disease. This work aims to assess the influence of two distinct signaling moieties on CPCs: cyclic biaxial strain and extracellular matrix. We evaluate four endpoints for improving CPC therapy: paracrine signaling, proliferation, connexin43 expression, and alignment. Vascular endothelial growth factor A (about 900 pg/mL) was secreted by CPCs cultured on fibronectin and collagen I. The application of mechanical strain increased vascular endothelial growth factor A secretion 2-4-fold for CPCs cultured on poly-L-lysine, laminin, or a naturally derived cardiac extracellular matrix. CPC proliferation was at least 25% higher on fibronectin than that on other matrices, especially for lower strain magnitudes. At 5% strain, connexin43 expression was highest on fibronectin. With increasing strain magnitude, connexin43 expression decreased by as much as 60% in CPCs cultured on collagen I and a naturally derived cardiac extracellular matrix. Cyclic mechanical strain induced the strongest CPC alignment when cultured on fibronectin or collagen I. This study demonstrates that culturing CPCs on fibronectin with 5% strain magnitude is optimal for their vascular endothelial growth factor A secretion, proliferation, connexin43 expression, and alignment.

Extracellular Matrix Hydrogel Promotes Tissue Remodeling, Arteriogenesis, and Perfusion in a Rat Hindlimb Ischemia Model.

OBJECTIVE: This study aimed to examine acellular extracellular matrix based hydrogels as potential therapies for treating peripheral artery disease (PAD). We tested the efficacy of using a tissue specific injectable hydrogel, derived from decellularized porcine skeletal muscle (SKM), compared to a new human umbilical cord derived matrix (hUC) hydrogel, which could have greater potential for tissue regeneration because of its young tissue source age. BACKGROUND: The prevalence of PAD is increasing and can lead to critical limb ischemia (CLI) with potential limb amputation. Currently there are no therapies for PAD that effectively treat all of the underlying pathologies, including reduced tissue perfusion and muscle atrophy. METHODS: In a rodent hindlimb ischemia model both hydrogels were injected 1-week post-surgery and perfusion was regularly monitored with laser speckle contrast analysis (LASCA) to 35 days post-injection. Histology and immunohistochemistry were used to assess neovascularization and muscle health. Whole transcriptome analysis was further conducted on SKM injected animals on 3 and 10 days post-injection. RESULTS: Significant improvements in hindlimb tissue perfusion and perfusion kinetics were observed with both biomaterials. End point histology indicated this was a result of arteriogenesis, rather than angiogenesis, and that the materials were biocompatible. Skeletal muscle fiber morphology analysis indicated that the muscle treated with the tissue specific, SKM hydrogel more closely matched healthy tissue morphology. Short term histology also indicated arteriogenesis rather than angiogenesis, as well as improved recruitment of skeletal muscle progenitors. Whole transcriptome analysis indicated that the SKM hydrogel caused a shift in the inflammatory response, decreased cell death, and increased blood vessel and muscle development. CONCLUSION: These results show the efficacy of an injectable ECM hydrogel alone as a potential therapy for treating patients with PAD. Our results indicate that the SKM hydrogel improved functional outcomes through stimulation of arteriogenesis and muscle progenitor cell recruitment.

Injectable ECM scaffolds for cardiac repair.

Injectable biomaterials have been developed as potential minimally invasive therapies for treating myocardial infarction (MI) and heart failure. Christman et al. first showed that the injection of a biomaterial alone into rat myocardium can improve cardiac function after MI (Christman et al. Tissue Eng 10:403-409, 2004). More recently, hydrogel forms of decellularized extracellular matrix (ECM) materials have shown substantial promise. Here we present the methods for fabricating an injectable cardiac specific ECM biomaterial shown to already have positive outcomes in small and large animal models for cardiac repair (Singelyn et al. Biomaterials 30:5409-5416, 2009; Singelyn et al. J Am Coll Cardiol 59:751-763, 2012; Seif-Naraghi et al. Sci Transl Med 5:173ra25, 2013). Also covered are the methods for the injection of a biomaterial into rat myocardium using a surgical approach through the diaphragm. Although the methods shown here are for injection of an acellular biomaterial, cells or other therapeutics could also be added to the injection for testing other regenerative medicine strategies.

Human versus porcine tissue sourcing for an injectable myocardial matrix hydrogel.

Heart failure (HF) after myocardial infarction (MI) is a leading cause of death in the western world with a critical need for new therapies. A previously developed injectable hydrogel derived from porcine myocardial matrix (PMM) has had successful results in both small and large animal MI models. In this study, we sought to evaluate the impact of tissue source on this biomaterial, specifically comparing porcine and human myocardium sources. We first developed an analogous hydrogel derived from human myocardial matrix (HMM). The biochemical and physical properties of the PMM and HMM hydrogels were then characterized, including residual dsDNA, protein content, sulfated glycosaminoglycan (sGAG) content, complex viscosity, storage and loss moduli, and nano-scale topography. Biochemical activity was investigated with in vitro studies for the proliferation of vascular cells and differentiation of human cardiomyocyte progenitor cells (hCMPCs). Next, in vivo gelation and material spread were confirmed for both PMM and HMM after intramyocardial injection. After extensive comparison, the matrices were found to be similar, yet did show some differences. Because of the rarity of collecting healthy human hearts, the increased difficulty in processing the human tissue, shifts in ECM composition due to aging, and significant patient-to-patient variability, these studies suggest that the HMM is not a viable option as a scalable product for the clinic; however, the HMM has potential as a tool for in vitro cell culture.

Injectable hydrogel therapies and their delivery strategies for treating myocardial infarction.

INTRODUCTION: Heart failure following myocardial infarction (MI) impacts millions of people each year in the US. The field of tissue engineering has developed several potential therapies for treating MI including injectable acellular hydrogels. These injectable biomaterials can either be synthetic or naturally derived, and have the potential to be delivered minimally invasively. AREAS COVERED: This review covers the different methods of delivery and presents the initial work on the use of injectable biomaterial scaffolds alone to improve cardiac function post-MI. Several naturally derived materials including alginate, collagen, chitosan, decellularized tissues, fibrin, hyaluronic acid, keratin, and Matrigel, as well as a few synthetic materials have shown promise on their own without the addition of therapeutics such as cells or growth factors. These biomaterials can be potentially delivered via endocardial, epicardial, or intracoronary injections and some can even utilize current catheter technology, indicating a potential for avoiding invasive surgical procedures. Once injected into the wall of the heart, these hydrogels create a scaffold that provides biochemical and structural cues, and the ability for cellular infiltration and remodeling of the local environment. EXPERT OPINION: Injectable biomaterials have several crucial challenges that should be over come to design optimal therapies for MI and heart failure, including optimizing material properties, methods of injection and understanding the mechanisms of action. But, studies in both small and large animals have shown significant improvement in important parameters including wall thickness, vascularization of the ischemic region, left ventricular volumes, and cardiac function. Thus, the application of injectable biomaterials shows promise for developing into new therapies to treat MI, potentially improving millions of lives.

Catheter-deliverable hydrogel derived from decellularized ventricular extracellular matrix increases endogenous cardiomyocytes and preserves cardiac function post-myocardial infarction.

OBJECTIVES: This study evaluated the use of an injectable hydrogel derived from ventricular extracellular matrix (ECM) for treating myocardial infarction (MI) and its ability to be delivered percutaneously. BACKGROUND: Injectable materials offer promising alternatives to treat MI. Although most of the examined materials have shown preserved or improved cardiac function in small animal models, none have been specifically designed for the heart, and few have translated to catheter delivery in large animal models. METHODS: We have developed a myocardial-specific hydrogel, derived from decellularized ventricular ECM, which self-assembles when injected in vivo. Female Sprague-Dawley rats underwent ischemia reperfusion followed by injection of the hydrogel or saline 2 weeks later. The implantation response was assessed via histology and immunohistochemistry, and the potential for arrhythmogenesis was examined using programmed electrical stimulation 1 week post-injection. Cardiac function was analyzed with magnetic resonance imaging 1 week pre-injection and 4 weeks post-MI. In a porcine model, we delivered the hydrogel using the NOGA-guided MyoStar catheter (Biologics Delivery Systems, Irwindale, California), and utilized histology to assess retention of the material. RESULTS: We demonstrate that injection of the material in the rat MI model increases endogenous cardiomyocytes in the infarct area and maintains cardiac function without inducing arrhythmias. Furthermore, we demonstrate feasibility of transendocardial catheter injection in a porcine model. CONCLUSIONS: To our knowledge, this is the first in situ gelling material to be delivered via transendocardial injection in a large animal model, a critical step towards the translation of injectable materials for treating MI in humans. Our results warrant further study of this material in a large animal model of MI and suggest this may be a promising new therapy for treating MI.