Non-Invasive Techniques Reveal Heifer Response to Fescue Endophyte Type in Grazing Studies.

Cattle grazing tall fescue (Schedonorus arundinaceous) infected with wild-type endophytes (WE) leads to a syndrome commonly known as fescue toxicosis. Replacing WE tall fescue with a novel endophyte-infected (NE) tall fescue can mitigate this problem but adoption of this technology has been limited. This study measured and determined the physiological and behavioral responses of heifers that grazed either WE or NE tall fescue, utilizing relatively non-invasive techniques including hair cortisol, thermography (for extremity temperatures), small loggers for intravaginal temperature, and remote observation of in-field behavior. Heifers that grazed WE had greater (p < 0.0001) hair cortisol levels, lower extremity temperatures (p ≤ 0.0075), and 0.3-0.9 °C greater (p ≤ 0.02) intravaginal temperatures (particularly during the daytime) than heifers that grazed NE. From 1200 h-1700 h each day, heifers on WE pastures spent 1.5 more (p = 0.0003) hours standing up and 0.9 fewer (p = 0.0402) hours lying down than heifers on NE pastures. Differences (p = 0.0160) in ADG were small (0.1 kg d-1) and were only observed in the first year of these 8-week studies. However, even in the mild environment of the study site, grazing NE tall fescue provided clear welfare benefits as evidenced by heifer behavioral changes, temperature differentials, and hair cortisol levels. This study underscores the potential utility of non-invasive techniques, such as thermographic imaging and hair cortisol analysis, for evaluating animal responses to stress in extensive grazing systems.

Viral Nucleases from Herpesviruses and Coronavirus in Recombination and Proofreading: Potential Targets for Antiviral Drug Discovery.

In this review, we explore recombination in two very different virus families that have become major threats to human health. The Herpesviridae are a large family of pathogenic double-stranded DNA viruses involved in a range of diseases affecting both people and animals. Coronaviridae are positive-strand RNA viruses (CoVs) that have also become major threats to global health and economic stability, especially in the last two decades. Despite many differences, such as the make-up of their genetic material (DNA vs. RNA) and overall mechanisms of genome replication, both human herpes viruses (HHVs) and CoVs have evolved to rely heavily on recombination for viral genome replication, adaptation to new hosts and evasion of host immune regulation. In this review, we will focus on the roles of three viral exonucleases: two HHV exonucleases (alkaline nuclease and PolExo) and one CoV exonuclease (ExoN). We will review the roles of these three nucleases in their respective life cycles and discuss the state of drug discovery efforts against these targets.

Two-Metal Ion-Dependent Enzymes as Potential Antiviral Targets in Human Herpesviruses.

The majority of drug discovery efforts against herpesviruses have focused on nucleoside analogs that target viral DNA polymerases, agents that are associated with dose-limiting toxicity and/or a narrow spectrum of activity. We are pursuing a strategy based on targeting two-metal ion-dependent (TMID) viral enzymes. This family of enzymes consists of structurally related proteins that share common active sites containing conserved carboxylates predicted to coordinate divalent cations essential for catalysis. Compounds that target TMID enzymes, such as HIV integrase and influenza endoribonuclease, have been successfully developed for clinical use. HIV integrase inhibitors have been reported to inhibit replication of herpes simplex virus (HSV) and other herpesviruses; however, the molecular targets of their antiviral activities have not been identified. We employed a candidate-based approach utilizing several two-metal-directed chemotypes and the potential viral TMID enzymatic targets in an effort to correlate target-based activity with antiviral potency. The panel of compounds tested included integrase inhibitors, the anti-influenza agent baloxavir, three natural products previously shown to exhibit anti-HSV activity, and two 8-hydroxyquinolines (8-HQs), AK-157 and AK-166, from our in-house program. The integrase inhibitors exhibited weak overall anti-HSV-1 activity, while the 8-HQs were shown to inhibit both HSV-1 and cytomegalovirus (CMV). Target-based analysis demonstrated that none of the antiviral compounds acted by inhibiting ICP8, contradicting previous reports. On the other hand, baloxavir inhibited the proofreading exonuclease of HSV polymerase, while AK-157 and AK-166 inhibited the alkaline exonuclease UL12. In addition, AK-157 also inhibited the catalytic activity of the HSV polymerase, which provides an opportunity to potentially develop dual-targeting agents against herpesviruses. IMPORTANCE Human herpesviruses (HHVs) establish lifelong latent infections, which undergo periodic reactivation and remain a major cause of morbidity and mortality, especially in immunocompromised individuals. Currently, HHV infections are treated primarily with agents that target viral DNA polymerase, including nucleoside analogs; however, long-term treatment can be complicated by the development of drug resistance. New therapies with novel modes of action would be important not only for the treatment of resistant viruses but also for use in combination therapy to reduce dose-limiting toxicities and potentially eliminate infection. Since many essential HHV proteins are well conserved, inhibitors of novel targets would ideally exhibit broad-spectrum activity against multiple HHVs.

lmplementation of the prolyl hydroxylase inhibitor Roxadustat (FG-4592) and its main metabolites into routine doping controls.

The utility of hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors as a therapeutic means of treating patients suffering from anaemia has been demonstrated for various clinical settings. However, besides this intended use, HIF stabilizers can be the subject of misuse in amateur and elite sports due to their erythropoietic properties, as recently proven by several cases of adverse analytical findings in doping control testing. Consequently, to allow for adequate and comprehensive test methods, knowledge of the drug candidates' metabolism and analytical options enabling appropriate detection windows in sports drug testing samples (i.e., blood and urine) is essential to doping control laboratories. In the present study, a novel HIF prolyl hydroxylase inhibitor referred to as Roxadustat (FG-4592) and main plasma- and urine-derived metabolites were investigated in the context of routine doping control analytical approaches. Liquid chromatography-mass spectrometry-based test methods were used to study the target analytes' dissociation pathways following electrospray ionization and collision-induced dissociation. Diagnostic precursor-product ion pairs were selected to enable the implementation of the intact drug Roxadustat and selected metabolites into multi-analyte initial testing procedures for plasma and urine specimens. The assays were validated in accordance to guidelines of the World Anti-Doping Agency (WADA) and results demonstrated the suitability (fitness-for-purpose) of the employed analytical methods with detection limits ranging from 0.05 to 1 ng/mL and 1 to 5 ng/mL for urine and plasma, respectively. Subsequently, elimination study plasma and urine samples collected up to 167 h post-administration were analyzed using the validated methods, which suggested the use of different target analytes for blood and urine analyses with FG-4592 and its glucuronide, respectively, for optimal detection windows. Additionally, a light-induced rearrangement product (photoisomer) of Roxadustat resulted in the formation of an additional compound of identical mass. Copyright © 2017 John Wiley & Sons, Ltd.

Fabrication and characterization of three-dimensional electrospun scaffolds for bone tissue engineering.

When traumatic injury, tumor removal, or disease results in significant bone loss, reconstructive surgery is required. Bone grafts are used in orthopedic reconstructive procedures to provide mechanical support and promote bone regeneration. In this study, we applied a heat sintering technique to fabricate 3D electrospun scaffolds that were used to evaluate effects of mineralization and fiber orientation on scaffold strength. We electrospun PLLA/gelatin scaffolds with a layer of PDLA and heat sintered them into three-dimensional cylindrical scaffolds. Scaffolds were mineralized by incubation in 10× simulated body fluid for 6, 24, and 48 h to evaluate the effect of mineralization on scaffolds compressive mechanical properties. The effects of heat sintering hydroxyapatite (HA) microparticles directly to the scaffolds on mineral deposition, distribution and mechanical properties of the scaffolds were also evaluated. We found that orientation of the fibers had little effect on the compressive mechanical properties of the scaffolds. However, increasing the mineralization times resulted in an increase in compressive mechanical properties. Also, the direct addition of HA microparticles had no effect on the scaffold mechanical properties, but had a significant effect on the mineral deposition on PLLA/gelatin scaffolds.

Rapid mineralization of electrospun scaffolds for bone tissue engineering.

We investigated different techniques to enhance calcium phosphate mineral precipitation onto electrospun poly((L)-lactide) (PLLA) scaffolds when incubated in concentrated simulated body fluid (SBF), 10×SBF. The techniques included the use of vacuum, pre-treatment with 0.1 M NaOH and electrospinning gelatin/PLLA blends as means to increase overall mineral precipitation and distribution throughout the scaffolds. Mineral precipitation was evaluated using environmental scanning electron microscopy, energy dispersive spectroscopy mapping and the determination of the mineral weight percents. In addition we evaluated the effect of the techniques on mechanical properties, cellular attachment and cellular proliferation on scaffolds. Two treatments, pre-treatment with NaOH and incorporation of 10% gelatin into PLLA solution, both in combination with vacuum, resulted in significantly higher degrees of mineralization (16.55 and 15.14%, respectively) and better mineral distribution on surfaces and through the cross-sections after 2 h of exposure to 10×SBF. While both scaffold groups supported cell attachment and proliferation, 10% gelatin/PLLA scaffolds had significantly higher yield stress (1.73 vs 0.56 MPa) and elastic modulus (107 vs 44 MPa) than NaOH-pre-treated scaffolds.

Tissue engineering of the anterior cruciate ligament: the viscoelastic behavior and cell viability of a novel braid-twist scaffold.

The anterior cruciate ligament (ACL) is the most commonly injured ligament of the knee; it also contributes to normal knee function and stability. Due to its poor healing potential severe ACL damage requires surgical intervention, ranging from suturing to complete replacement. Current ACL replacements have a host of limitations that prevent their extensive use. Investigators have begun to utilize tissue-engineering techniques to create new options for ACL repair, regeneration and replacement. In this study we tested novel braid-twist scaffolds, as well as braided scaffolds, twisted fiber scaffolds and aligned fiber scaffolds, for use as ACL replacements composed of poly(L-lactic acid) fibers. Scaffolds were examined using stress relaxation tests, cell viability assays and scanning electron microscopy. The behaviors of the braid-twist scaffolds were modeled with Maxwell and quasi-linear viscoelastic (QLV) models. In stress relaxation tests, the braid-twist scaffolds behaved similarly to native ACL tissue, with final normalized stresses of 87% and 83% after an 8 N load. There was agreement between the experimental data and the Maxwell model when the model included an element for each structural element in the scaffold. There was also agreement between the experimental data and QLV model, scaffolds with similar braiding angles shared constants. In cell proliferation studies no differences were found between fibroblast growth on the braided scaffolds and the braid-twist scaffolds. SEM images showed the presence of new extracellular matrix. Data from this and previous tensile studies demonstrate that the braid-twist scaffold design may be effective in scaffolds for ACL tissue regeneration.

Mechanical property, degradation rate, and bone cell growth of chitosan coated titanium influenced by degree of deacetylation of chitosan.

Chitosan has shown promise as a coating for dental/craniofacial and orthopaedic implants. However, the effects of degree of deacetylation (DDA) of chitosan on coating bond strength, degradation, and biological performance is not known. The aim of this project was to evaluate bonding, degradation, and bone cell growth on titanium coated with chitosans of different DDA and from different manufacturers. Three different chitosans, 80.6%, 81.7%, and 92.3% DDA were covalently bonded to titanium coupons via silane-glutaraldehyde molecules. Bond strengths were evaluated in mechanical tensile tests, and degradation, over 5 weeks, was conducted in cell culture medium with and without 100 microg/mL lysozyme. Cytocompatibility was evaluated for 10 days using UMR 106 osteoblastic cells. Results showed that mean chitosan coating bond strengths ranged from 2.2-3.8 MPa, and that there was minimal affect of DDA on coating bond strengths. The coatings exhibited little dissolution over 5 weeks in medium with or without lysozyme. However, the molecular weight (MW) of the chitosan coatings remaining on the titanium samples after 5 weeks decreased by 69-85% with the higher DDA chitosan coatings exhibiting less percent change in MW than the lower DDA materials. The growth of the UMR 106 osteoblast cells on the 81.7% DDA chitosan coating was lower on days 3 and 5, as compared with the other two coatings, but by day 10, there were no differences in growth among three coatings or to the uncoated titanium controls. Differences in growth were attributed to differences in manufacturer source material, though all coatings were judged to be osteocompatible in vitro.

Guanidinium rich peptide transporters and drug delivery.

The use of peptide or peptidomimetic transporters to enable or enhance the uptake of drugs or probe molecules into cells and tissues has received increasing research attention and clinical interest over the past 10 years. This review summarizes a class of transporters that have been studied and focuses on the variation and use of guanidinium based transporters to facilitate the uptake of various types of molecules into cells and tissues. Lead conjugates in this area are currently in clinical trials.

Arginine-rich molecular transporters for drug delivery: role of backbone spacing in cellular uptake.

Short oligomers of arginine, either alone or when conjugated to therapeutic agents or large biopolymers, have been shown to cross readily a variety of biological barriers (e.g., lipid bilayers and epithelial tissue). Molecular modeling suggests that only a subset of the side chain guanidinium groups of these transporters might be required for transport involving contact with a common surface such as a plasma membrane or cell surface receptor. To evaluate this hypothesis, a series of decamers were prepared that incorporated seven arginines and three nonarginine residues. Several of these mixed decamers were comparable to the all arginine decamer in their ability to enter cells. More significantly, these decamers containing seven arginines performed almost without exception better than heptaarginine itself, suggesting that spacing between residues is also important for transport. The influence of spacing was more fully evaluated with a library of oligomers incorporating seven arginines separated by one or more nonconsecutive, non-alpha-amino acids. This study led to the identification of a new series of highly efficient molecular transporters.