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Azoxystrobin is the only synthetic, systemic fungicide labeled in the United States for management of frogeye leaf spot (FLS) of tobacco ( Nicotiana tabacum L.), caused by Cercospora nicotianae . Though traditionally considered a minor disease in the United States, FLS has recently become yield and quality limiting. In 2016 and 2017, 100 C. nicotianae isolates were collected from symptomatic tobacco from eight counties in Kentucky, United States. Prior to azoxystrobin sensitivity testing, some C. nicotianae isolates were found to utilize the alternative oxidase pathway and, after assay comparisons, conidial germination was utilized to evaluate sensitivity in C. nicotianae as opposed to mycelial growth. Azoxystrobin sensitivity was determined by establishing the effective concentration to inhibit 50% conidial germination (EC 50 ) for 47 (in 2016) and 53 (in 2017) C. nicotianae isolates. Distributions of C. nicotianae EC 50 values indicated three qualitative levels of sensitivity to azoxystrobin. Partial cytochrome b sequence, encompassing the F129L and G143A mutation sites, indicated single-nucleotide polymorphisms (SNPs) conferring the F129L mutation in C. nicotianae of moderate resistance (azoxystrobin at 0.177 ≤ EC 50 ≤ 0.535 µg/ml) and the G143A mutation in isolates with an azoxystrobin-resistant phenotype (azoxystrobin EC 50 > 1.15 µg/ml). Higher frequencies of resistant isolates were identified from greenhouse transplant (4 of 17) and conventionally produced (58 of 62) tobacco samples, as compared with field-grown tobacco (<4 weeks prior to harvest; 4 of 62) or organically produced samples (1 of 7), respectively. Together, these results suggest that resistance to azoxystrobin in C. nicotianae occurs broadly in Kentucky, and generate new hypotheses about selection pressure affecting resistance mutation frequencies.

Ligaments and tendons are comprised of aligned, crimped collagen fibrils that provide tissue-specific mechanical properties with non-linear extension behaviour, exhibiting low stress at initial strain (toe region behaviour). To approximate this behaviour, we report fibrous scaffolds with sinusoidal patterns by melt electrowriting (MEW) below the critical translation speed (CTS) by exploitation of the natural flow behaviour of the polymer melt. More specifically, we synthesised photopolymerizable poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) (p(LLA-co-ε-CL-co-AC)) and poly(ε-caprolactone-co-acryloyl carbonate) (p(ε-CL-co-AC)) by ring-opening polymerization (ROP). Single fibre (fØ = 26.8 ± 1.9 µm) tensile testing revealed a customisable toe region with Young's Moduli ranging from E = 29 ± 17 MPa for the most crimped structures to E = 314 ± 157 MPa for straight fibres. This toe region extended to scaffolds containing multiple fibres, while the sinusoidal pattern could be influenced by printing speed. The synthesized polymers were cytocompatible and exhibited a tensile strength of σ = 26 ± 7 MPa after 10 4 cycles of preloading at 10% strain while retaining the distinct toe region commonly observed in native ligaments and tendon tissue., Statement of Significance: Damaged tendons and ligaments are serious and frequently occurring injuries worldwide. Recent therapies, including autologous grafts, still have severe disadvantages leading to a demand for synthetic alternatives. Materials envisioned to induce tendon and ligament regeneration should be degradable, cytocompatible and mimic the ultrastructural and mechanical properties of the native tissue. Specifically, we utilised photo-cross-linkable polymers for additive manufacturing (AM) with MEW. In this way, we were able to direct-write cytocompatible fibres of a few micrometres thickness into crimp-structured elastomer scaffolds that mimic the non-linear biomechanical behaviour of tendon and ligament tissue., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Polymerase chain reaction-based detection of plant-associated microbes depends on physical disruption of tissues of the host and microorganism in order to liberate nucleic acids during extraction. Using six types of plant tissues as well as an oospore preparation of Phytophthora capsici, we evaluated the use of pressure-cycling technology (PCT) compared with several common techniques for physical tissue disruption. With all tissues tested, bead-beating provided excellent yields of amplifiable nucleic acid, with a few inconsistent exceptions. The use of PCT did not consistently improve nucleic acid yields or "amplifiability". The use of a mortar and pestle to physically disrupt plant tissue also provided good results at low cost, though it was not consistently as effective as the bead-beater. Furthermore, handling of ground tissues in an open mortar may present more challenges in minimizing cross-contamination than working with tissues pulverized in a bead-beater tube.

Objective: Harvesting osteochondral grafts results in a zone of chondrocyte death (ZCD) in and around the periphery of the graft, creating a barrier for chondrocytes to migrate to the graft periphery, thus limiting cartilage-to-cartilage healing. The purpose of this study was to repopulate the induced ZCD through the combined effects of collagenase treatment and delivery of a chemotactic agent., Design: In bovine cartilage, the ZCD induced by the OATS™ osteochondral harvesting system was determined, followed by a corresponding collagenase treatment to penetrate the developed ZCD. The chemotactic potential of platelet derived growth factor (PDGF-bb), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (bFGF) (2.5-100 ng/mL) was then assessed using a modified Boyden chamber assay to select an appropriate agent to induce chondrocyte migration. Afterwards, the combined effects of collagenase treatment and chondrocyte chemotaxis on the repopulation of an induced ZCD were examined in cartilage explants over a 4-week-period., Results: The OATS™ osteochondral harvesting system induced a significant ZCD (173 μm, 95% CI: [72-274 μm]) in the grafts. Chondrocyte chemotaxis was induced by all agents investigated at concentrations greater than 25 ng/mL. After 4 weeks in culture, collagenase treatment alone reduced the ZCD by approximately 40% relative to untreated explants. Coupling the collagenase treatment with 25 ng/mL IGF-I reduced the ZCD by approximately 80% relative to untreated explants, and 65% relative to explants treated only with collagenase., Conclusion: Treating cartilage explants with collagenase and 25 ng/mL IGF-I resulted in a decreased ZCD after a 4-week-period, and increased chondrocyte density within the induced ZCD., (Copyright © 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

A synthetic peptide fragment of human collagen type I (BCSP-1) was linked to the surface of a commercially available ceramic in an effort to improve the properties of the bone graft substitute to accelerate local healing. BCSP-1 was covalently immobilized on the surface of the ceramic via the linkers 3-aminopropyl-triethoxysilane (APTES) and suberic acid bis-N-hydroxysuccinimide ester (DSS). The chosen chemistry was non-cytotoxic. A rat calvaria cell assay using alkaline phosphatase (ALP) as an osteoblast differentiation marker, showed that modifying the surface of the ceramic was enough to enhance ALP activity, although the total cell population on the surface decreased. A significant increase in ALP activity/cell was noted with serum albumin bound to the surface, however, the BCSP-1 bound surface exhibited an even greater ALP activity that showed a surface concentration dependent trend. An optimal BCSP-1 surface density in the range of 0.87-2.24 nmol/cm2 elicited the maximum ALP activity/cell at day 6 of culture. The peptide bound ceramic generated an ALP activity/cell that was roughly 3-fold higher than the non-modified ceramic and 2-fold higher than the APTES-grafted ceramic.

Protein therapeutics are a rapidly growing drug class, with sales in 2004 in the area of $US 34 billion. They are presently administered primarily by injection, although there is increasing recognition that many proteins would benefit from long-term, localized delivery. Such delivery represents a significant challenge due principally to protein stability concerns. Polymeric delivery systems which rely on osmotic pressure driven drug release may prove to be an effective formulation approach. This paper reviews the evolution of osmotic pressure drug release from polymers, with an emphasis on their potential for protein delivery. It is concluded that osmotic pressure driven release is promising for protein delivery, but there is still a need for in vivo demonstration of protein stability and delivery efficacy.

An elastomer was prepared from biodegradable components as a potential biomaterial for drug delivery and tissue engineering applications. The elastomer was synthesized in two steps. First, a star copolymer (SCP) was manufactured via ring opening polymerization of -caprolactone (epsilon-CL) with D,L-lactide using glycerol as initiator and stannous 2-ethylhexanoate as catalyst. This living SCP was further reacted with different ratios of a crosslinking monomer, 2,2-bis(epsilon-CL-4-yl)-propane in the presence of epsilon-CL as a solvent and co-monomer. The elastomers had very low glass transitions (-32 degrees C), sol contents ranging from 17% to 37%, and were soft and weak with physical properties similar to those of natural elastomers such as elastin. The physical properties decreased in a logarithmic fashion with time when degraded in phosphate buffered saline, indicative of first-order degradation kinetics. The elastomers degraded relatively slowly, with degradation being incomplete after 12 weeks.