Incidence and risk factors for complications associated with equine general anaesthesia for elective magnetic resonance imaging.

BACKGROUND: Complications are a practical consideration for elective magnetic resonance imaging (MRI) studies performed under general anaesthesia but relatively little is known about their distribution and risk factors. OBJECTIVES: To describe the incidence of complications associated with MRI performed under general anaesthesia at a large referral facility and evaluate potential risk factors for these complications. STUDY DESIGN: Retrospective case-control study. METHODS: Patient information and details of the MRI procedure were collected retrospectively from medical records of all horses that had undergone an MRI under general anaesthesia at the University of Pennsylvania, New Bolton Center, between September 2005 and April 2012. Complications and categorical variables were examined by chi-squared or Fisher's exact tests as appropriate. A mixed-effects logistic regression approach was used to evaluate associations between explanatory variables and the outcome variable (complications or pyrexia). A univariable screen was used to select variables (likelihood ratio test p < 0.2) for inclusion in the multivariable analysis. Statistical significance was inferred when p ≤ 0.05. RESULTS: Complications were noted after MRI in 51 (17.4%) of 293 events eligible for inclusion. Complications included pyrexia (n = 35), pneumonia (n = 14), colic (n = 10), facial/nerve paralysis (n = 6), diarrhoea (n = 4), and other (n = 3). The odds of developing a post-anaesthetic complication were significantly decreased in horses that received peri-anaesthetic antimicrobials (OR 0.29, 95% CI 0.14-0.63, p = 0.002). Increased age (OR 0.87, 95% CI, 0.76-0.99, p = 0.03) and peri-anaesthetic antimicrobial administration (OR 0.23, 95% CI 0.08-0.65, p = 0.005) were associated with a decreased odds of developing pyrexia. MAIN LIMITATIONS: Single centre retrospective design. CONCLUSIONS: Potential complications including pyrexia, pneumonia and colic should be recognised when pursuing MRI under general anaesthesia. The administration of peri-anaesthetic antimicrobials decreased the odds of a complication and warrants consideration, particularly in horses that might be classified as high risk.

Using Vertically Aligned Carbon Nanofiber Arrays on Rigid or Flexible Substrates for Delivery of Biomolecules and Dyes to Plants.

The delivery of biomolecules and impermeable dyes to intact plants is a major challenge. Nanomaterials are up-and-coming tools for the delivery of DNA to plants. As exciting as these new tools are, they have yet to be widely applied. Nanomaterials fabricated on rigid substrate (backing) are particularly difficult to successfully apply to curved plant structures. This study describes the process for microfabricating vertically aligned carbon nanofiber arrays and transferring them from a rigid to a flexible substrate. We detail and demonstrate how these fibers (on either rigid or flexible substrates) can be used for transient transformation or dye (e.g., fluorescein) delivery to plants. We show how VACNFs can be transferred from rigid silicon substrate to a flexible SU-8 epoxy substrate to form flexible VACNF arrays. To overcome the hydrophobic nature of SU-8, fibers in the flexible film were coated with a thin silicon oxide layer (2-3 nm). To use these fibers for delivery to curved plant organs, we deposit a 1 µL droplet of dye or DNA solution on the fiber side of VACNF films, wait 10 min, place the films on the plant organ and employ a swab with a rolling motion to drive fibers into plant cells. With this method, we have achieved dye and DNA delivery in plant organs with curved surfaces.

Efficacy of the oral supplement, Equine Omega Complete, for the prevention of gastric ulcers and alpha-tocopherol supplementation in horses.

BACKGROUND: Omega-3 fatty acid and alpha-tocopherol supplementation reduces gastric ulcer formation in humans and rodents; however, efficacy of prevention in horses is unknown. Equine Omega Complete (EOC) is an oral supplement containing omega-3 fatty acids and alpha-tocopherol. HYPOTHESIS/OBJECTIVE: Determine if EOC supplementation prevents gastric ulcers and increases serum alpha-tocopherol concentrations in healthy horses. ANIMALS: Nine thoroughbred geldings; 5-13 years old. METHODS: Prospective randomized block design, repeated in crossover model. Horses were administered EOC, omeprazole, or water PO for 28 days. Horses underwent an established gastric ulcer induction protocol from days 21-28 via intermittent feed deprivation. Gastroscopies were performed on days 0, 21, and 28. Serum alpha-tocopherol concentrations were measured on days 0 and 28. The effects of treatment and time on ulcer grades were assessed with ordinal logistic regression, with significance at P-value <.05. RESULTS: Ulcer grades increased during ulcer induction in control and EOC but not omeprazole groups (P = .02). Grades increased in EOC-treated horses after ulcer induction from a median of 1 [95% confidence interval 0-2.5] (day 0) to 2.5 [1.5-3.5] (day 28) and were similar to the control group (P = .54). Serum alpha-tocopherol increased in EOC-treated horses from day 0 to day 28 (mean 2.2 ± 0.43 µg/mL to 2.96 ± 0.89 µg/mL; P < .001) with high individual variation; this increase was not different from omeprazole or control groups. CONCLUSION AND CLINICAL IMPORTANCE: Supplementation with EOC for 28 days did not prevent gastric ulcer formation nor increase alpha-tocopherol concentrations relative to the control group.

Evaluating the Safety of Intra-Articular Mitotherapy in the Equine Model: A Potential Novel Treatment for Osteoarthritis.

No current treatments available halt osteoarthritis progression in horses or humans. Intra-articular injection of mitochondria is a novel treatment that has the potential to improve cell metabolism and decrease inflammation, but safety of this treatment has yet to be established in the horse. Autologous blood-derived mitochondria isolated using a commercially available kit were injected into the left carpus joint of 3 horses which were monitored for 28 days. Horses received physical examinations, video recorded gait evaluations, joint diameter measurement, synovial fluid collection, and blood collection on day 0 (baseline prior to mitotherapy, day of mitochondria injection), 1, 3, 7, 14, and 28. Systemic inflammation was assessed via complete blood count, fibrinogen, and plasma serum amyloid A (SAA). Local inflammation was assessed via synovial fluid cytology and physical examination parameters. Physical exam parameters remained stable and no joint swelling was observed after mitotherapy. No change was noted in video recorded gait evaluations as determined by a blinded evaluator. Complete blood counts revealed no significant increase in white blood cells. SAA only increased mildly in 1 horse. Fibrinogen became slightly elevated above reference range in 2 horses at day 7, but later normalized. Mild increases in synovial fluid nucleated cell counts and total protein occurred on day 1 and 3, but resolved within 7 days without intervention. Autologous mitochondria injection into the equine intercarpal joint was well tolerated with no signs of inflammation. This safety information allows for future studies evaluating mitotherapy efficacy.

Changes in Heart Rate Variability with Induction of Gastric Ulcers in Adult Horses.

Gastric ulceration can be induced by athletic training and is a significant welfare concern. The objective of this study was to evaluate the effect of gastric ulcer induction on heart rate variability (HRV) in the horse. We hypothesized that induction of gastric ulcers would decrease HRV and increase low frequency fluctuations, consistent with increased sympathetic tone. A convenience sample of 8 horses in a larger study were enrolled. Horses were randomly assigned to receive water or 2 mg/kg omeprazole orally once daily for 28 days. Gastric ulcers were induced through intermittent feed withholding on days 21 to 28. Gastroscopy was performed and gastric ulcers were graded (0-IV) by three blinded reviewers on days 21 and 28. Continuous electrocardiograms were obtained for one hour at the start and end of ulcer induction. HRV was assessed in 1-hour recordings for time domain variables and 5 minute sections for frequency domain analysis. HRV and ulcer grade across treatments were compared by a mixed effect model, with treatment and time as fixed effects and horse as a random effect. Gastric ulcer grade increased with induction protocol (P < .0001) and decreased with omeprazole treatment (P = .0007). Omeprazole treatment increased R-R intervals (P = .01) and decreased ratio of low frequency/high frequency signal (P = .008) as compared to horses receiving water. This was attributable to decreasing low frequency fluctuations (P = .05). While limited by the small sample size (four horses/treatment), this study suggests that omeprazole treatment decreases heart rate, and LF/HF ratio during ulcer induction, consistent with a decrease in sympathetic tone.

An efficient and broadly applicable method for transient transformation of plants using vertically aligned carbon nanofiber arrays.

Transient transformation in plants is a useful process for evaluating gene function. However, there is a scarcity of minimally perturbing methods for gene delivery that can be used on multiple organs, plant species, and non-excised tissues. We pioneered and demonstrated the use of vertically aligned carbon nanofiber (VACNF) arrays to efficiently perform transient transformation of different tissues with DNA constructs in multiple plant species. The VACNFs permeabilize plant tissue transiently to allow molecules into cells without causing a detectable stress response. We successfully delivered DNA into leaves, roots and fruit of five plant species (Arabidopsis, poplar, lettuce, Nicotiana benthamiana, and tomato) and confirmed accumulation of the encoded fluorescent proteins by confocal microscopy. Using this system, it is possible to transiently transform plant cells with both small and large plasmids. The method is successful for species recalcitrant to Agrobacterium-mediated transformation. VACNFs provide simple, reliable means of DNA delivery into a variety of plant organs and species.

Fracture Stabilization and Management in the Field.

Management of fractures in the field starts with successful assessment and stabilization of the patient by the practitioner on the front lines. A careful examination is vital to succesful patient management. This includes identifying the fracture location and severity, evaluating skin integrity and potential contamination of the fracture, and treating any ongoing hemorrhage, hypovolemia or stress. Appropriate application of splints in the field will minimize ongoing tissue damage and improve patient comfort. This ultimately aids further assessment, facilitates referral, and improves opportunities for successful fracture repair.

Cytotoxic Activity of Cunila angustifolia Essential Oil.

The Cunila angustifolia essential oil was obtained from fresh leaves by hydrodistillation and analyzed by GC-FID and GC-MS to determine its chemical composition. The essential oil presented pulegone (29.5 %) and isomenthol (27.0 %) as major components, and other compounds such as menthone (8.6 %), neomenthol (7.2 %), menthyl acetate (2.5 %) and caryophyllene oxide (2.0 %) were identified. The cytotoxic activity of the essential oil was evaluated by MTS assay, with the human cancer cell lines of the lung (A549), breast (MCF-7) and skin melanoma (SK-Mel-28). The assay showed the highest selectivity, to MCF-7 cell lines, with IC50 equal to 34.0 µg mL-1 , low selectivity for SK-Mel-28 cell lines, with IC50 equal to 279.9 µg mL-1 , and no mortality to A549 cell lines.

Effects of acepromazine and xylazine on subjective and objective assessments of forelimb lameness.

BACKGROUND: To facilitate lameness evaluation, sedatives such as xylazine and acepromazine are regularly used in the clinical setting, despite concerns that they may confound lameness assessment. OBJECTIVES: The objective of this study was to determine the effect of low doses of acepromazine and xylazine on subjective and objective lameness assessment. STUDY DESIGN: Randomised, blinded, crossover study. METHODS: Six horses with experimentally induced solar pain were evaluated over a 1-hour period after treatment with intravenous xylazine (0.1 or 0.2 mg/kg), intravenous acepromazine (0.02 or 0.04 mg/kg), intravenous saline (1 mL) or local analgesia (4 mL 2% mepivacaine administered subcutaneously). Lameness was assessed objectively with inertial sensors and subjectively on a scale from 0 to 5. Lameness assessments were compared with logistic regression analysis to account for the repeated measures and cross-over study design (P < .05). RESULTS: Xylazine (0.1 and 0.2 mg/kg) or acepromazine (0.02 and 0.04 mg/kg) did not result in significant differences in objective lameness assessment (vector sum) or average subjective lameness grade. Local analgesia was associated with a decrease in subjective lameness grade (OR 0.32 [0.11-0.92], P = .03). Objective measures of lameness (vector sum) were significantly decreased 45 minutes (vector sum 41.8, P = .04) and 60 minutes (vector sum 47.3, P = .03) following local analgesia administration compared with baseline (vector sum 121.4). MAIN LIMITATIONS: Extrapolation of the experimental model of moderate lameness used in this study to broad range of clinical lameness situations should be performed carefully. CONCLUSIONS: These results support the use of low doses of xylazine or acepromazine to facilitate forelimb lameness evaluation up to 1 hour in duration.

A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos.

BACKGROUND: Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming. RESULTS: Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding. CONCLUSIONS: SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.

An Experimental Pipeline for Initial Characterization of Bacterial Type III Secretion System Inhibitor Mode of Action Using Enteropathogenic Yersinia.

Dozens of Gram negative pathogens use one or more type III secretion systems (T3SS) to disarm host defenses or occupy a beneficial niche during infection of a host organism. While the T3SS represents an attractive drug target and dozens of compounds with T3SS inhibitory activity have been identified, few T3SS inhibitors have been validated and mode of action determined. One issue is the lack of standardized orthogonal assays following high throughput screening. Using a training set of commercially available compounds previously shown to possess T3SS inhibitory activity, we demonstrate the utility of an experiment pipeline comprised of six distinct assays to assess the stages of type III secretion impacted: T3SS gene copy number, T3SS gene expression, T3SS basal body and needle assembly, secretion of cargo through the T3SS, and translocation of T3SS effector proteins into host cells. We used enteropathogenic Yersinia as the workhorse T3SS-expressing model organisms for this experimental pipeline, as Yersinia is sensitive to all T3SS inhibitors we tested, including those active against other T3SS-expressing pathogens. We find that this experimental pipeline is capable of rapidly distinguishing between T3SS inhibitors that interrupt the process of type III secretion at different points in T3SS assembly and function. For example, our data suggests that Compound 3, a malic diamide, blocks either activity of the assembled T3SS or alters the structure of the T3SS in a way that blocks T3SS cargo secretion but not antibody recognition of the T3SS needle. In contrast, our data predicts that Compound 4, a haloid-containing sulfonamidobenzamide, disrupts T3SS needle subunit secretion or assembly. Furthermore, we suggest that misregulation of copy number control of the pYV virulence plasmid, which encodes the Yersinia T3SS, should be considered as a possible mode of action for compounds with T3SS inhibitory activity against Yersinia.

Piericidin A1 Blocks Yersinia Ysc Type III Secretion System Needle Assembly.

The type III secretion system (T3SS) is a bacterial virulence factor expressed by dozens of Gram-negative pathogens but largely absent from commensals. The T3SS is an attractive target for antimicrobial agents that may disarm pathogenic bacteria while leaving commensal populations intact. We previously identified piericidin A1 as an inhibitor of the Ysc T3SS in Yersinia pseudotuberculosis. Piericidins were first discovered as inhibitors of complex I of the electron transport chain in mitochondria and some bacteria. However, we found that piericidin A1 did not alter Yersinia membrane potential or inhibit flagellar motility powered by the proton motive force, indicating that the piericidin mode of action against Yersinia type III secretion is independent of complex I. Instead, piericidin A1 reduced the number of T3SS needle complexes visible by fluorescence microscopy at the bacterial surface, preventing T3SS translocator and effector protein secretion. Furthermore, piericidin A1 decreased the abundance of higher-order YscF needle subunit complexes, suggesting that piericidin A1 blocks YscF needle assembly. While expression of T3SS components in Yersinia are positively regulated by active type III secretion, the block in secretion by piericidin A1 was not accompanied by a decrease in T3SS gene expression, indicating that piericidin A1 may target a T3SS regulatory circuit. However, piericidin A1 still inhibited effector protein secretion in the absence of the T3SS regulator YopK, YopD, or YopN. Surprisingly, while piericidin A1 also inhibited the Y. enterocolitica Ysc T3SS, it did not inhibit the SPI-1 family Ysa T3SS in Y. enterocolitica or the Ysc family T3SS in Pseudomonas aeruginosa. Together, these data indicate that piericidin A1 specifically inhibits Yersinia Ysc T3SS needle assembly. IMPORTANCE The bacterial type III secretion system (T3SS) is widely used by both human and animal pathogens to cause disease yet remains incompletely understood. Deciphering how some natural products, such as the microbial metabolite piericidin, inhibit type III secretion can provide important insight into how the T3SS functions or is regulated. Taking this approach, we investigated the ability of piericidin to block T3SS function in several human pathogens. Surprisingly, piericidin selectively inhibited the Ysc family T3SS in enteropathogenic Yersinia but did not affect the function of a different T3SS within the same species. Furthermore, piericidin specifically blocked the formation of T3SS needles on the bacterial surface without altering the localization of several other T3SS components or regulation of T3SS gene expression. These data show that piericidin targets a mechanism important for needle assembly that is unique to the Yersinia Ysc T3SS.

Cargo rigidity affects the sensitivity of dynein ensembles to individual motor pausing.

Cytoplasmic dynein is a minus-end directed microtubule-based motor protein that drives intracellular cargo transport in eukaryotic cells. Although many intracellular cargos are propelled by small groups of dynein motors, the biophysical mechanisms governing ensemble motility remain largely unknown. To investigate the emergent motility of motor ensembles, we have designed a programmable DNA origami synthetic cargo "chassis" enabling us to control the number of dynein motors in the ensemble and vary the rigidity of the cargo chassis itself. Using total internal reflection fluorescence microscopy, we have observed dynein ensembles transporting these cargo chassis along microtubules in vitro. We find that ensemble motility depends on cargo rigidity: as the number of motors increases, ensembles transporting flexible cargos move comparatively faster than a single motor, whereas ensembles transporting rigid cargos move slower than a single motor. To explain this, we show that ensembles connected through flexible cargos are less sensitive to the pauses of individual motors within the ensemble. We conclude that cargo rigidity plays an important role in communicating and coordinating the states of motors, and consequently in the subsequent mechanisms of collective motility. The insensitivity of ensemble-driven cargos to the pausing of individual motors may contribute to the robustness and versatility of intracellular cargo transport. © 2016 Wiley Periodicals, Inc.

Regulation of tenascin expression in bone.

Tenascins regulate cell interaction with the surrounding pericellular matrix. Within bone, tenascins C and W influence osteoblast adhesion and differentiation, although little is known about the regulation of tenascin expression. In this study we examined the effect of osteogenic differentiation, bone morphogenetic protein (BMP) and Wnt growth factors, and mechanical loading on tenascin expression in osteogenic cells. Osteogenic differentiation increased tenascin C (TnC), and decreased tenascin W (TnW), expression. Both growth factors and mechanical loading increased both TnC and TnW expression, albeit via distinct signaling mechanisms. Both BMP-2 and Wnt5a induction of tenascin expression were mediated by MAP kinases. These data establish a role for BMP, Wnts, and mechanical loading in the regulation of tenascin expression in osteoblasts.

Src is sufficient, but not necessary, for osteopontin induction in osteoblasts.

The ability of bone cells to detect and transduce mechanical signals is central to the mechanism whereby bone adapts to mechanical load and maintains healthy bone mass. Src, a non-receptor tyrosine kinase, is located in focal adhesions, highly specialized and localized sites of attachment, that are thought to be a primary site of mechanotransduction. While Src is activated by mechanical loads in other cell types, its role in osteoblast mechanotransduction is unclear. In this study we examined whether oscillatory fluid flow influenced Src phosphorylation, and Src's role in the flow-induced osteopontin response. Additionally, we investigated the effect of constitutively active Src on osteopontin expression. Oscillatory fluid flow induced a statistically significant increase in phosphorylation of Src at tyrosine residue 416 after a 15 min exposure. Transfection with constitutively-active Src resulted in an increase in Src-Y416 phosphorylation and an increase in osteopontin mRNA transcript under static conditions. However, inhibition of Src activity had no effect on oscillatory fluid flow-stimulated osteopontin expression or ERK1/2 phosphorylation. These data suggest that although Src activity regulates osteopontin expression under static conditions, and is induced under conditions of shear stress, it is not required for load-induced osteopontin expression.

Hoof accelerations and ground reaction forces of Thoroughbred racehorses measured on dirt, synthetic, and turf track surfaces.

OBJECTIVE: To compare hoof acceleration and ground reaction force (GRF) data among dirt, synthetic, and turf surfaces in Thoroughbred racehorses. ANIMALS: 3 healthy Thoroughbred racehorses. PROCEDURES: Forelimb hoof accelerations and GRFs were measured with an accelerometer and a dynamometric horseshoe during trot and canter on dirt, synthetic, and turf track surfaces at a racecourse. Maxima, minima, temporal components, and a measure of vibration were extracted from the data. Acceleration and GRF variables were compared statistically among surfaces. RESULTS: The synthetic surface often had the lowest peak accelerations, mean vibration, and peak GRFs. Peak acceleration during hoof landing was significantly smaller for the synthetic surface (mean + or - SE, 28.5g + or - 2.9g) than for the turf surface (42.9g + or - 3.8g). Hoof vibrations during hoof landing for the synthetic surface were < 70% of those for the dirt and turf surfaces. Peak GRF for the synthetic surface (11.5 + or - 0.4 N/kg) was 83% and 71% of those for the dirt (13.8 + or - 0.3 N/kg) and turf surfaces (16.1 + or - 0.7 N/kg), respectively. CONCLUSIONS AND CLINICAL RELEVANCE: The relatively low hoof accelerations, vibrations, and peak GRFs associated with the synthetic surface evaluated in the present study indicated that synthetic surfaces have potential for injury reduction in Thoroughbred racehorses. However, because of the unique material properties and different nature of individual dirt, synthetic, and turf racetrack surfaces, extending the results of this study to encompass all track surfaces should be done with caution.