Biodistribution Analysis of Peptide-Coated Magnetic Iron Nanoparticles: A Simple and Quantitative Method.

Biodistribution tracks compounds or molecules of interest in vivo to understand a compound's anticipated efficacy and safety. Nanoparticles deliver nucleic acid and drug payloads and enhance tumor permeability due to multiple properties such as high surface area to volume ratio, surface functionalization, and modifications. Studying the in vivo biodistribution of nanoparticles documents the effectiveness and safety of nanoparticles and facilitates a more application-driven approach for nanoparticle development that allows for more successful translation into clinical use. In this study, we present a relatively simple method to determine the biodistribution of magnetic iron nanoparticles in mice. In vitro, cells take up branched amphiphilic peptide-coated magnetic nanobeads (BAPc-MNBs) like their counterparts, i.e., branched amphiphilic peptide capsules (BAPCs) with a hollow water-filled core. Both BAPc-MNBs and BAPCs have widespread applications as a nanodelivery system. We evaluated the BAPc-MNBs tissue distribution in wild-type mice injected intravenously (i.v.), intraperitoneally (i.p.), or orally gavaged to understand the biological interactions and to further the development of branched amphiphilic peptide-based nanoparticles. The magnetic nanoparticles allowed collection of the BAPc-MNBs from multiple organs by magnetic bead sorting, followed by a high-throughput screening for iron content. When injected i.v., nanoparticles were distributed widely to various organs before elimination from the system via the intestines in feces. The spleen accumulated the highest amount of BAPc-MNBs in mice administered NPs via i.v. and i.p. but not via oral gavage. Taken together, these data demonstrate that the magnetic sorting not only allowed quantification of the BAPc-MNBs but also identified the distribution of BAPc-MNBs after distinct administration methods.

Understanding the influence of experimental factors on bio-interactions of nanoparticles: Towards improving correlation between in vitro and in vivo studies.

Bionanotechnology has developed rapidly over the past two decades, owing to the extensive and versatile, functionalities and applicability of nanoparticles (NPs). Fifty-one nanomedicines have been approved by FDA since 1995, out of the many NPs based formulations developed to date. The general conformation of NPs consists of a core with ligands coating their surface, that stabilizes them and provides them with added functionalities. The physicochemical properties, especially the surface composition of NPs influence their bio-interactions to a large extent. This review discusses recent studies that help understand the nano-bio interactions of iron oxide and gold NPs with different surface compositions. We discuss the influence of the experimental factors on the outcome of the studies and, thus, the importance of standardization in the field of nanotechnology. Recent studies suggest that with careful selection of experimental parameters, it is possible to improve the positive correlation between in vitro and in vivo studies. This provides a fundamental understanding of the NPs which helps in assessing their potential toxic side effects and may aid in manipulating them further to improve their biocompatibility and biosafety.

A Study of the Cellular Uptake of Magnetic Branched Amphiphilic Peptide Capsules.

Understanding cellular uptake mechanisms of nanoparticles with therapeutic potential has become critical in the field of drug delivery. Elucidation of cellular entry routes can aid in the dissection of the complex intracellular trafficking and potentially allow for the manipulation of nanoparticle fate after cellular delivery (i.e., avoid lysosomal degradation). Branched amphiphilic peptide capsules (BAPCs) are peptide nanoparticles that have been and are being explored as delivery systems for nucleic acids and other therapeutic molecules in vitro and in vivo. In the present study, we determined the cellular uptake routes of BAPCs with and without a magnetic nanobead core (BAPc-MNBs) in two cell lines: macrophages and intestinal epithelial cells. We also studied the influence of size and growth media composition in this cellular process. Substituting the water-filled core with magnetic nanobeads might provide the peptide bilayer nanocapsules with added functionalities, facilitating their use in bio/immunoassays, magnetic field guided drug delivery, and magnetofection among others. Results suggest that BAPc-MNBs are internalized into the cytosol using more than one endocytic pathway. Flow cytometry and analysis of reactive oxygen and nitrogen species (ROS/RNS) demonstrated that cell viability was minimally impacted by BAPc-MNBs. Cellular uptake pathways of peptide vesicles remain poorly understood, particularly with respect to endocytosis and intracellular trafficking. Outcomes from these studies provide a fundamental understanding of the cellular uptake of this peptide-based delivery system which will allow for strengthening of their delivery capabilities and expanding their applications both in vitro and in vivo.

Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread.

UNLABELLED: Intercellular nanotube connections have been identified as an alternative pathway for cellular spreading of certain viruses. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nanotubes were observed connecting two distant cells with contiguous membranes, with the core infectious viral machinery (viral RNA, certain replicases, and certain structural proteins) present in/on the intercellular nanotubes. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2. In MARC-145 cells expressing PRRSV receptors, GFP-nsp2 moved from one cell to another through nanotubes in the presence of virus-neutralizing antibodies. Intercellular transport of viral proteins did not require the PRRSV receptor as it was observed in receptor-negative HEK-293T cells after transfection with an infectious clone of GFP-PRRSV. In addition, GFP-nsp2 was detected in HEK-293T cells cocultured with recombinant PRRSV-infected MARC-145 cells. The intercellular nanotubes contained filamentous actin (F-actin) with myosin-associated motor proteins. The F-actin and myosin IIA were identified as coprecipitates with PRRSV nsp1ß, nsp2, nsp2TF, nsp4, nsp7-nsp8, GP5, and N proteins. Drugs inhibiting actin polymerization or myosin IIA activation prevented nanotube formation and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response. IMPORTANCE: Extracellular virus particles transmit infection between organisms, but within infected hosts intercellular infection can be spread by additional mechanisms. In this study, we describe an alternative pathway for intercellular transmission of PRRSV in which the virus uses nanotube connections to transport infectious viral RNA, certain replicases, and certain structural proteins to neighboring cells. This process involves interaction of viral proteins with cytoskeletal proteins that form the nanotube connections. Intercellular viral spread through nanotubes allows the virus to escape the neutralizing antibody response and may contribute to the pathogenesis of viral infections. The development of strategies that interfere with this process could be critical in preventing the spread of viral infection.

Branched Amphipathic Peptide Capsules: Different Ratios of the Two Constituent Peptides Direct Distinct Bilayer Structures, Sizes, and DNA Transfection Efficiency.

Branched amphipathic peptide capsules (BAPCs) are biologically derived, bilayer delimited, nanovesicles capable of being coated by or encapsulating a wide variety of solutes. The vesicles and their cargos are readily taken up by cells and become localized in the perinuclear region of cells. When BAPCs are mixed with DNA, the BAPCs act as cationic nucleation centers around which DNA winds. The BAPCs-DNA nanoparticles are capable of delivering plasmid DNA in vivo and in vitro yielding high transfection rates and minimal cytotoxicity. BAPCs share several biophysical properties with lipid vesicles. They are however considerably more stable-resisting disruption in the presence of chaotropes such as urea and guanidinium chloride, anionic detergents, proteases, and elevated temperature (∼95 °C). To date, all of our published results have utilized BAPCs that are composed of equimolar concentrations of the two branched sequences (Ac-FLIVI)2-K-K4-CO-NH2 and (Ac-FLIVIGSII)2-K-K4-CO-NH2. The mixture of sizes was utilized to relieve potential curvature strain in the spherical capsule. In this article, different molar ratios of the two peptides were studied to test whether alternate ratios produced BAPCs with different biological and biophysical properties. Additionally, preparation (annealing) temperature was included as a second variable.

A review of solute encapsulating nanoparticles used as delivery systems with emphasis on branched amphipathic peptide capsules.

Various strategies are being developed to improve delivery and increase the biological half-lives of pharmacological agents. To address these issues, drug delivery technologies rely on different nano-sized molecules including: lipid vesicles, viral capsids and nano-particles. Peptides are a constituent of many of these nanomaterials and overcome some limitations associated with lipid-based or viral delivery systems, such as tune-ability, stability, specificity, inflammation, and antigenicity. This review focuses on the evolution of bio-based drug delivery nanomaterials that self-assemble forming vesicles/capsules. While lipid vesicles are preeminent among the structures; peptide-based constructs are emerging, in particular peptide bilayer delimited capsules. The novel biomaterial-Branched Amphiphilic Peptide Capsules (BAPCs) display many desirable properties. These nano-spheres are comprised of two branched peptides-bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK, designed to mimic diacyl-phosphoglycerides in molecular architecture. They undergo supramolecular self-assembly and form solvent-filled, bilayer delineated capsules with sizes ranging from 20 nm to 2 µm depending on annealing temperatures and time. They are able to encapsulate different fluorescent dyes, therapeutic drugs, radionuclides and even small proteins. While sharing many properties with lipid vesicles, the BAPCs are much more robust. They have been analyzed for stability, size, cellular uptake and localization, intra-cellular retention and, bio-distribution both in culture and in vivo.

Armet is an effector protein mediating aphid-plant interactions.

Aphid saliva is predicted to contain proteins that modulate plant defenses and facilitate feeding. Armet is a well-characterized bifunctional protein in mammalian systems. Here we report a new role of Armet, namely as an effector protein in the pea aphid, Acyrthosiphon pisum. Pea aphid Armet's physical and chemical properties and its intracellular role are comparable to those reported for mammalian Armets. Uniquely, we detected Armet in aphid watery saliva and in the phloem sap of fava beans fed on by aphids. Armet's transcript level is several times higher in the salivary gland when aphids feed on bean plants than when they feed on an artificial diet. Knockdown of the Armet transcript by RNA interference disturbs aphid feeding behavior on fava beans measured by the electrical penetration graph technique and leads to a shortened life span. Inoculation of pea aphid Armet protein into tobacco leaves induced a transcriptional response that included pathogen-responsive genes. The data suggest that Armet is an effector protein mediating aphid-plant interactions.

Organization and Structure of Branched Amphipathic Oligopeptide Bilayers.

A class of self-assembling branched amphiphilic peptide capsules (BAPCs) was recently developed that could serve as a new drug delivery vehicle. BAPCs can encapsulate solutes up to ∼12 kDa during assembly, are unusually stable, and are readily taken up by cells with low cytotoxicity. Coarse-grained simulations have supported that BAPCs are defined by bilayers that resemble those formed by diacyl phospholipids. Here, atomistic simulations were performed to characterize the structure and organization of bilayers formed by three branched amphiphilic peptides (BAPs): bis(Ac-FLIVIGSII)-K-K4-CO-NH2, bis(Ac-CHA-LIVIGSII)-K-K4-CO-NH2, and bis(Ac-FLIVI)-K-K4-CO-NH2. The results show BAPs form a network of intra- and intermolecular backbone hydrogen bonds within the same leaflet in addition to hydrophobic side-chain interactions. The terminal residues of two leaflets form an interdigitation region locking two leaflets together. The phenyl groups in bis(Ac-FLIVIGSII)-K-K4-CO-NH2 and bis(Ac-FLIVI)-K-K4-CO-NH2 are tightly packed near the bilayer center but do not formed ordered structures with specific π-π stacking. Replacing phenyl groups with the cyclohexane side chain only slightly increases the level of disorder in bilayer structures and thus should not significantly affect the stability, consistent with experimental results on bis(Ac-CHA-LIVIGSII)-K-K4-CO-NH2 BAPCs. Self-assembly simulations further suggest that leaflet interdigitation likely occurs at early stages of BAPC formation. Atomistic simulations also reveal that the BAPC bilayers are highly permeable to water. This prediction was validated using fluorescence measurements of encapsulated self-quenching dye upon transferring BAPCs to buffers with different salt concentrations. Improved understanding of the organization and structure of BAPC bilayers at the atomic level will provide a basis for future rational modifications of BAP sequence to improve BAPC properties as a new class of delivery vehicle.

Free energy analysis of conductivity and charge selectivity of M2GlyR-derived synthetic channels.

Significant progresses have been made in the design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the second transmembrane domain of the α-subunit of the glycine receptor (GlyR). The latest designs, including p22 (KKKKP ARVGL GITTV LTMTT QS), are highly soluble in water with minimal aggregation propensity and insert efficiently into cell membranes to form highly conductive ion channels. The last obstacle to a potential lead sequence for channel replacement treatment of CF patients is achieving adequate chloride selectivity. We have performed free energy simulation to analyze the conductance and charge selectivity of M2GlyR-derived synthetic channels. The results reveal that the pentameric p22 pore is non-selective. Moderate barriers for permeation of both K(+) and Cl(-) are dominated by the desolvation cost. Despite previous evidence suggesting a potential role of threonine side chains in anion selectivity, the hydroxyl group is not a good surrogate of water for coordinating these ions. We have also tested initial ideas of introducing additional rings of positive changes to various positions along the pore to increase anion selectivity. The results support the feasibility of achieving anion selectivity by modifying the electrostatic properties of the pore, but at the same time suggest that the peptide assembly and pore topology may also be dramatically modified, which could abolish the effects of modified electrostatics on anion selectivity. This was confirmed by subsequent two-electrode voltage clamp measurements showing that none of the tested mono-, di- and tri-Dap substituted sequences was selective. The current study thus highlights the importance of controlling channel topology besides modifying pore electrostatics for achieving anion selectivity. Several strategies are now being explored in our continued efforts to design an anion selective peptide channel with suitable biophysical, physiological and pharmacological properties as a potential treatment modality for channel replacement therapy. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Branched amphiphilic peptide capsules: cellular uptake and retention of encapsulated solutes.

Branched amphiphilic peptide capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble to form bilayer delimited capsules. In two recent publications we described the lipid analogous characteristics of our BAPCs, examined their initial assembly, mode of fusion, solute encapsulation, and resizing and delineated their capability to be maintained at a specific size by storing them at 4°C. In this report we describe the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs in vivo. The ability of our constructs to retain alpha particle emitting radionuclides without any apparent leakage and their persistence in the peri-nuclear region of the cell for extended periods of time, coupled with their ease of preparation and potential tune-ability, makes them attractive as biocompatible carriers for targeted cancer therapy using particle emitting radioisotopes. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.

Branched amphiphilic cationic oligopeptides form peptiplexes with DNA: a study of their biophysical properties and transfection efficiency.

Over the past decade, peptides have emerged as a new family of potential carriers in gene therapy. Peptides are easy to synthesize and quite stable. Additionally, sequences shared by the host proteome are not expected to be immunogenic or trigger inflammatory responses, which are commonly observed with viral approaches. We recently reported on a new class of branched amphiphilic peptide capsules (BAPCs) that self-assemble into extremely stable nanospheres. These capsules are capable of retaining and delivering alpha-emitting radionuclides to cells. Here we report that, in the presence of double stranded plasmid DNA, BAPCs are unable to form. Instead, depending of the peptide/DNA ratios, the peptides either coat the plasmid surface forming nanofibers (high peptide to DNA ratio) or condense the plasmid into nanometer-sized compacted structures (at low peptide to DNA ratios). Different gene delivery efficiencies are observed for the two types of assemblies. The compacted nanometer-sized structures display much higher transfection efficiencies in HeLa cells. This level of transfection is greater than that observed for a lipid-based reagent when the total number of viable transfected cells is taken into account.

Thermally induced conformational transitions in nascent branched amphiphilic peptide capsules.

Branched amphiphilic peptide capsules (BAPCs) are biocompatible, bilayer delimited polycationic nanospheres that spontaneously form at room temperature through the coassembly of two amphiphilic branched peptides: bis(FLIVI)-K-K4 and bis(FLIVIGSII)-K-K4. BAPCs are readily taken up by cells in culture, where they escape and/or evade the endocytic pathway and accumulate in the perinuclear region, persisting there without apparent degradation or extravasation. Drugs, small proteins, and solutes as well as α particle emitting radionuclides are stably encapsulated for extended periods of time. BAPC formation at room temperature proceeds via a fusogenic process and after 48 h a range of BAPCs sizes are observed, from 50 nm to a few microns in diameter. It was previously reported that cooling BAPCs from 25 to 4 °C and then back to 25 °C eliminated their fusogenic property. In this report, biophysical techniques reveal that BAPCs undergo thermosensitive conformational transitions as a function of both time and temperature and that the properties of BAPCs vary based on the temperature of assembly. The solvent dissociation properties of BAPCs were studied as well as the contributions of specific amino acid residues to the observed conformations. The roles of the potential stabilizing forces present within the bilayer that bestow the unusal stability of the BAPCs are discussed. Ultimately this study presents revised assembly protocols for preparing BAPCs with discrete sizes and solvent-induced extravasation properties.

Formation of rigid, non-flight forewings (elytra) of a beetle requires two major cuticular proteins.

Insect cuticle is composed primarily of chitin and structural proteins. To study the function of structural cuticular proteins, we focused on the proteins present in elytra (modified forewings that become highly sclerotized and pigmented covers for the hindwings) of the red flour beetle, Tribolium castaneum. We identified two highly abundant proteins, TcCPR27 (10 kDa) and TcCPR18 (20 kDa), which are also present in pronotum and ventral abdominal cuticles. Both are members of the Rebers and Riddiford family of cuticular proteins and contain RR2 motifs. Transcripts for both genes dramatically increase in abundance at the pharate adult stage and then decline quickly thereafter. Injection of specific double-stranded RNAs for each gene into penultimate or last instar larvae had no effect on larval-larval, larval-pupal, or pupal-adult molting. The elytra of the resulting adults, however, were shorter, wrinkled, warped, fenestrated, and less rigid than those from control insects. TcCPR27-deficient insects could not fold their hindwings properly and died prematurely approximately one week after eclosion, probably because of dehydration. TcCPR18-deficient insects exhibited a similar but less dramatic phenotype. Immunolocalization studies confirmed the presence of TcCPR27 in the elytral cuticle. These results demonstrate that TcCPR27 and TcCPR18 are major structural proteins in the rigid elytral, dorsal thoracic, and ventral abdominal cuticles of the red flour beetle, and that both proteins are required for morphogenesis of the beetle's elytra.

Small ß2-glycoprotein I peptides protect from intestinal ischemia reperfusion injury.

Intestinal ischemic events, which are followed by reperfusion, induce significant tissue damage and frequently result in multiple organ failure, with >70% mortality. Upon reperfusion, excessive inflammation leads to exacerbated tissue damage. Previous studies indicated that binding of the serum protein, ß2-glycoprotein I, to the endothelium initiates a cascade of inflammatory molecules that is required for damage. We hypothesized that peptides derived from the binding domain (domain V) of ß2-glycoprotein I would attenuate ischemia/reperfusion-induced damage and inflammation in a therapeutic manner. Using a mouse model of intestinal ischemia/reperfusion, we administered peptides either prior to ischemia or at clinically relevant time points during reperfusion and evaluated intestinal tissue damage and inflammation after 2 h of reperfusion. We demonstrate that multiple peptides attenuate injury and inflammation in a dose-dependent manner and, perhaps more significantly, are efficacious when administered up to 30 min after the onset of reperfusion. In addition, an all D-amino acid retro-inverso peptide was biologically active. Thus, the ß2-glycoprotein I-derived peptides attenuate injury and inflammation when administered in a therapeutic manner in intestinal ischemia/reperfusion injury.

Effect of diaminopropionic acid (Dap) on the biophysical properties of a modified synthetic channel-forming peptide.

Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was nonimmunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17, and 20 line the pore of assembled p22-T19R, S22W, and here we used 2,3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap substitutions. To gain further insights into the effects of Dap residues on the properties of the putative pore, we performed two-electrode voltage clamp electrophysiology on Xenopus oocytes exposed to p22-T19R, S22W or its Dap-modified analogues. We observed that Dap-substituted peptides also induced significantly larger voltage-dependent currents than the parent compound, but there was no apparent change in reversal potential upon replacement of external Na+, Cl- or K+, indicating that these currents remained nonselective. These results suggest that the introduction of positively charged side chains in predicted pore-lining residues does not improve anion-to-cation selectivity, but results in higher conductance, perhaps due to higher oligomerization numbers.

Branched oligopeptides form nanocapsules with lipid vesicle characteristics.

In a recent article (Gudlur et al. PLOS ONE, 2012, 7 (9) e45374), we described the special properties of a mixed branched peptide assembly in which equimolar bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK self-associate to form bilayer delimited capsules capable of trapping solutes. These polycationic vesicle-like capsules are readily taken up by epithelial cells in culture, escape or evade the endocytic pathway, and accumulate in the perinuclear region where they persist without any apparent degradation. In this report, we examine the lipidlike properties of this system including initial assembly; solute encapsulation and washing; fusion and resizing by membrane extrusion through polycarbonate filters with defined pore sizes. The resized peptide capsules have uniform diameters in nm size ranges. Once resized, the capsules can be maintained at the new size by storing them at 4 °C. Having the ability to prepare stable uniform nanoscale capsules of desired sizes makes them potentially attractive as biocompatible delivery vehicles for various solutes/drugs.

Biodistribution Analysis of Peptide-Coated Magnetic Iron Nanoparticles: A Simple and Quantitative Method.

Biodistribution is the tracking of compounds or molecules of interest in the subject which is integral to understanding their anticipated efficacy and safety. Nanoparticles are highly desirable delivery systems which have the ability to deliver higher nucleic acid and drug payloads and they have enhanced tumor permeability due to their unique properties such as high surface area to volume ratio. Studying the biodistribution of nanoparticles is crucial to understand their effectiveness and safety in vivo, facilitate a more application driven approach for nanoparticle development which will lead to their successful translation into clinical use. In this study, we present a relatively simple method to determine the biodistribution of magnetic iron nanoparticles in mice. Branched Amphiphilic Peptide coated Magnetic Nanobeads BAPc-MNBs like their counterpart i.e., Branched Amphiphilic Peptide capsules (BAPCs) with a hollow water-filled core, are readily taken up by cells in vitro and have widespread application as a nanodelivery systems. We evaluated the BAPc-MNBs tissue distribution in wildtype mice injected intravenously (i.v.), intraperitoneally (i.p.) or orally gavaged to understand the biological interactions of the peptide nanoparticles and to further the development of branched amphiphilic peptides-based nanoparticles. BAPc-MNBs were distributed widely to various organs when injected i.v. and were eliminated from the system via the intestines in feces. The spleen was found to accumulate the highest amount of BAPc-MNBs in mice administered the NPs i.v. and i.p. while they were not absorbed into the system via oral gavage. This study not only presents a relatively simple quantification method to determine in vivo biodistribution of magnetic iron nanoparticles that can be widely applied but also demonstrates the potential of Branched Amphiphilic Peptides in the form of BAPCs or BAPc-MNBs as a delivery system.

Proteomic and transcriptomic analyses of rigid and membranous cuticles and epidermis from the elytra and hindwings of the red flour beetle, Tribolium castaneum.

The insect cuticle is a composite biomaterial made up primarily of chitin and proteins. The physical properties of the cuticle can vary greatly from hard and rigid to soft and flexible. Understanding how different cuticle types are assembled can aid in the development of novel biomimetic materials for use in medicine and technology. Toward this goal, we have taken a combined proteomics and transcriptomics approach with the red flour beetle, Tribolium castaneum, to examine the protein and gene expression profiles of the elytra and hindwings, appendages that contain rigid and soft cuticles, respectively. Two-dimensional gel electrophoresis analysis revealed distinct differences in the protein profiles between elytra and hindwings, with four highly abundant proteins dominating the elytral cuticle extract. MALDI/TOF mass spectrometry identified 19 proteins homologous to known or hypothesized cuticular proteins (CPs), including a novel low complexity protein enriched in charged residues. Microarray analysis identified 372 genes with a 10-fold or greater difference in transcript levels between elytra and hindwings. CP genes with higher expression in the elytra belonged to the Rebers and Riddiford family (CPR) type 2, or cuticular proteins of low complexity (CPLC) enriched in glycine or proline. In contrast, a majority of the CP genes with higher expression in hindwings were classified as CPR type 1, cuticular proteins analogous to peritrophins (CPAP), or members of the Tweedle family. This research shows that the elyra and hindwings, representatives of rigid and soft cuticles, have different protein and gene expression profiles for structural proteins that may influence the mechanical properties of these cuticles.

Comparative proteomics uncovers the signature of natural selection acting on the ejaculate proteomes of two cricket species isolated by postmating, prezygotic phenotypes.

Two of the most well-supported patterns to have emerged over the past two decades of research in evolutionary biology are the occurrence of divergent natural selection acting on many male and female reproductive tract proteins and the importance of postmating, prezygotic phenotypes in reproductively isolating closely related species. Although these patterns appear to be common across a wide variety of taxa, the link between them remains poorly documented. Here, we utilize comparative proteomic techniques to determine whether or not there is evidence for natural selection acting on the ejaculate proteomes of two cricket species (Allonemobius fasciatus and A. socius) which are reproductively isolated primarily by postmating, prezygotic phenotypes. In addressing this question, we compare the degree of within-species polymorphism and between-species divergence between the ejaculate and thorax proteomes of these two species. We found that the ejaculate proteomes are both less polymorphic and more divergent than the thorax proteomes. Additionally, we assessed patterns of nucleotide variation for two species-specific ejaculate proteins and found evidence for both reduced levels of variation within species and positive selection driving divergence between species. In contrast, non-species-specific proteins exhibited higher levels of within-species nucleotide variation and no signatures of positive selection. Nucleotide and putative functional data for the two species-specific proteins, along with data for a third protein (ejaculate serine protease), suggest that all three of these genes are candidate speciation genes in need of further study. Overall, these patterns of proteome and nucleotide divergence provide support for the hypothesis that there is a causative link between selection-driven divergence of male ejaculate proteins and the evolution of postmating, prezygotic barriers to gene flow within Allonemobius.

Domain V peptides inhibit beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue damage and inflammation.

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, ß2-glycoprotein I as an initiating Ag for Ab recognition and ß2-glycoprotein I (ß2-GPI) peptides as a therapeutic for mesenteric IR. The time course of ß2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of ß2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti-ß2-GPI Abs into Rag-1(-/-) mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein ß2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.