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Reasons for performing study: Identification of exercise history patterns that are related to catastrophic scapular fracture will facilitate prevention of racehorse fatalities. Objectives: To determine if exercise patterns are associated with scapular fracture in Thoroughbred ( TB) and Quarter Horse ( QH) racehorses. Methods: High-speed exercise histories for 65 TB and 26 QH racehorses that had a complete scapular fracture (cases) and 2 matched control racehorses were retrospectively studied. Exercise variables were created from lifetime race and official timed workout reports. Associations between exercise variables and scapular fracture were investigated using conditional logistic regression. Results: Thoroughbreds with a scapular fracture had a greater number of workouts, events (combined works and races), and mean event distances than QHs with a scapular fracture. Quarter Horses worked less frequently and accumulated distance at a lower rate than TBs. Breed differences were not found for career race number or length, time between races or lay-up variables for horses with ≥1 lay-up. For both breeds, cases had fewer events, lower recent accumulated distance and fewer active days in training than controls; however, a subset of TB cases with >10 events since lay-up had a longer active career than controls. For QHs that had a lay-up, total and mean lay-up times were greater for cases than controls. Multivariable models revealed that odds ratios ( OR) of scapular fracture were greater for TBs that had not yet raced ( OR = 23.19; 95% confidence interval (CI) 3.03-177.38) and lower for QHs with more events ( OR = 0.71; 95% CI 0.54-0.94). Conclusions and clinical relevance: Racehorses that are in early high-speed training but behind that of their training cohort should be examined for signs of scapular stress remodelling. Quarter Horses that had a prolonged lay-up and TBs that have endured high-speed training for a longer duration than that of their training cohort also were at greater risk. [ABSTRACT FROM AUTHOR]

Reasons for performing study: To determine if scapular fractures occur in racehorses with distinctive characteristics. Objectives: To test the hypothesis that Thoroughbred (TB) and Quarter Horse (QH) racehorses with a scapular fracture have similar characteristics that are different from those of their respective racetrack populations. Methods: Necropsy findings, case details, last race information and career earnings for TB and QH racehorses that had a scapular fracture in California between 1990 and 2008 were retrospectively compared between breeds. Horse signalment, career earnings, career starts and race characteristics were obtained for all California racehorses. Comparisons were made between affected horses, other racehorses that died, and all horses that raced, in California during the 19 year period. Results: Seventy-three TB and 28 QH racehorses had a similar, complete comminuted scapular fracture with an articular component, and right forelimb predilection. The QHs had a higher incidence of scapular fracture incurred during racing than TBs (0.98 vs. 0.39/1000 starters). The TB and QH incident rates for musculoskeletal deaths incurred racing were 20.5 and 17.5/1000 starters, respectively; however, a greater proportion of TB musculoskeletal deaths occurred training (40% vs. 8%). Horses with a scapular fracture were more likely to be male and aged 2 or ≥5 years than the racetrack population. Most affected QHs (64%) were 2-year-olds; most TBs (74%) were aged ≥3 years. Scapular fractures occurred more commonly during racing in QHs (70%) than TBs (44%). Race-related scapular fracture was more likely to occur in a Maiden race than in a non-Maiden race. Horses with a scapular fracture had fewer career starts than the racetrack population. Conclusions and potential relevance: Despite breed differences for signalment and exercise distances, both breeds incur a complete scapular fracture that is more likely to occur in the right scapula of young and older, male racehorses, early in their race career or after few races. Quarter Horses sustain a catastrophic scapular fracture more frequently than TBs. [ABSTRACT FROM AUTHOR]

Past researchers suggested that “static instabilities” exist at certain rotational speeds of whirling rods. This paper shows these instabilities are an artefact of the material constitutive laws that are being used well outside their range of applicability. An alternative approach is developed where strains due to rotation are separated from the superimposed vibration. This enables the generally predicted lowering of longitudinal natural frequencies with rotational speed shown to be simply a result of the bulk changes in the geometry of whirling rods. Steady-state equations of whirling rods are formulated in Lagrangian coordinates. Due to the nonlinear nature of the governing equations, an original numerical method is applied to solve the problem. Numerical results are compared with analytical results obtained from the linearized uniaxial model. There is close agreement between these two models at low angular velocities. However, at high angular velocities, discrepancies between them arise, confirming that the nonlinear strain-displacement relationship has significant effect on the results and the inferred “static instabilities.” This approach first solves the “static” problem of the deformed geometry of a highly strained whirling rod before longitudinal natural modes are determined by classical methods. Furthermore, conditions for existence and uniqueness of solutions are derived. [ABSTRACT FROM AUTHOR]

Plasma fibronectin (PFN) is associated with cryoglobulins of the polyclonal type. Cryoglobulins from two patients with essential mixed cryoglobulinaemia were analysed immunoelectrophoretically to determine a possible association between PFN and immunoglobulin complexes in the formation of cryoprecipitates. In cryoglobulins from each patient, distinctive alterations in electrophoretic migration patterns of both PFN and immunoglobulin were associated with the formation of a cryoprecipitate. The observations indicate that PFN binds to some serum component in the process of cryoglobulin formation. That component may be immunoglobulin. [ABSTRACT FROM AUTHOR]

Twenty-four washed cryoglobulin precipitates were examined for the presence of plasma fibronectin, immunoglobulins, complement components Clq and C3, and fibrinogen. Plasma fibronectin was detected in all preparations by immunodiffusion with antifibronectin serum, whereas the other components were found in some but not all of the cryoglobulins. [ABSTRACT FROM AUTHOR]

Secondary metabolites are generally produced by enzymes encoded by genes within a biosynthetic gene cluster. Transcription factor genes are frequently located within these gene clusters. These transcription factors often drive expression of the other genes of the biosynthetic gene cluster, and overexpression of the transcription factor provides a facile approach to express all genes within a gene cluster, resulting in production of downstream metabolite(s). Unfortunately this approach is not always successful, leading us to engineer more effective hybrid transcription factors. Herein, we attempted to activate a putative cryptic biosynthetic gene cluster in Aspergillus nidulans using a combination of transcription factor engineering and overexpression approaches. This resulted in the discovery of a novel secondary metabolite we term triorsellinaldehyde. Surprisingly, deletion of the polyketide synthase gene within the gene cluster did not prevent triorsellinaldehyde production. However, targeted deletion of a polyketide synthase gene elsewhere in the genome revealed its role in triorsellinaldehyde biosynthesis.

Objective: To measure the density of cellular phenotypes in canine caudal cruciate ligament (CaCL), cranial cruciate ligament (CrCL), medial collateral ligament (MCL), and long digital extensor tendon (LDET)., Study Design: Ex-vivo study., Methods: Ten CaCL, CrCL, MCL, and LDET obtained from 1 stifle of 10 dogs with no gross pathology were analyzed histologically. The density of cells with 3 nuclear phenotypes (fusiform, ovoid, and spheroid) was determined within the core region of each specimen., Results: Cells with fusiform nuclei were most dense in the MCL (median [range], 319 [118-538] cells/mm 2 ) and LDET (331 [61-463]), whereas cells with ovoid nuclei were most dense in the CaCL (276 [123-368]) and CrCL (212 [165-420]). The spheroid nuclear phenotype had the lowest density in all structures (31 [5-61] in CaCL, 54 [5-90] in CrCL, 2 [0-14] in MCL, and 5 [0-80] in LDET); however, the CrCL contained a denser population of spheroid cells compared with MCL and LDET (P < .05). Total cell densities did not differ among the 4 structures (P > .05)., Conclusion: Phenotype density varied within the ligaments and tendon tested here. The cell population of CaCL and CrCL differed from that of dense collagenous tissues such as MCL and LDET., Clinical Significance: The relatively higher density of spheroid phenotype in CrCL may reflect a distinctive native cellular population or a cellular transformation secondary to unique mechanical environment or hypoxia. This intrinsic cellular population may explain altered tissue properties prone to pathological rupture or poor healing potential of the canine CrCL., (© 2019 The American College of Veterinary Surgeons.)

Fungal secondary metabolites (SMs) include medically valuable compounds as well as compounds that are toxic, carcinogenic, and/or contributors to fungal pathogenesis. It is consequently important to understand the regulation of fungal secondary metabolism. McrA is a recently discovered transcription factor that negatively regulates fungal secondary metabolism. Deletion of mcrA ( mcrA Δ), the gene encoding McrA, results in upregulation of many SMs and alters the expression of more than 1000 genes. One gene strongly upregulated by the deletion of mcrA is llmG , a putative methyl transferase related to LaeA, a major regulator of secondary metabolism. We artificially upregulated llmG by replacing its promoter with strong constitutive promoters in strains carrying either wild-type mcrA or mcrA Δ. Upregulation of llmG on various media resulted in increased production of the important toxin sterigmatocystin and compounds from at least six major SM pathways. llmG is, thus, a master SM regulator. mcrA Δ generally resulted in greater upregulation of SMs than upregulation of llmG , indicating that the full effects of mcrA on secondary metabolism involve genes in addition to llmG . However, the combination of mcrA Δ and upregulation of llmG generally resulted in greater compound production than mcrA Δ alone (in one case more than 460 times greater than the control). This result indicates that deletion of mcrA and/or upregulation of llmG can likely be combined with other strategies for eliciting SM production to greater levels than can be obtained with any single strategy.

Fungi are a major source of valuable bioactive secondary metabolites (SMs). These compounds are synthesized by enzymes encoded by genes that are clustered in the genome. The vast majority of SM biosynthetic gene clusters are not expressed under normal growth conditions, and their products are unknown. Developing methods for activation of these silent gene clusters offers the potential for discovering many valuable new fungal SMs. While a number of useful approaches have been developed, they each have limitations, and additional tools are needed. One approach, upregulation of SM gene cluster-specific transcription factors that are associated with many SM gene clusters, has worked extremely well in some cases, but it has failed more often than it has succeeded. Taking advantage of transcription factor domain modularity, we developed a new approach. We fused the DNA-binding domain of a transcription factor associated with a silent SM gene cluster with the activation domain of a robust SM transcription factor, AfoA. Expression of this hybrid transcription factor activated transcription of the genes in the target cluster and production of the antibiotic (+)-asperlin. Deletion of cluster genes confirmed that the cluster is responsible for (+)-asperlin production, and we designate it the aln cluster. Separately, coinduction of expression of two aln cluster genes revealed the pathway intermediate (2 Z,4 Z,6 E)-octa-2,4,6-trienoic acid, a compound with photoprotectant properties. Our findings demonstrate the potential of our novel synthetic hybrid transcription factor strategy to discover the products of other silent fungal SM gene clusters.

Mechanical testing of femora brings valuable insights into understanding the contribution of clinically-measureable variables such as bone mineral density distribution and geometry on the femoral mechanical properties. Currently, there is no standard protocol for mechanical testing of such geometrically complex bones to measure strength, and stiffness. To address this gap we have developed a protocol to test cadaveric femora to fracture and to measure their biomechanical parameters. This protocol describes a set of adaptable fixtures to accommodate the various load magnitudes and directions accounting for possible bone orientations in a fall on the hip configuration, test speed, bone size, and left leg-right leg variations. The femora were prepared for testing by cleaning, cutting, scanning, and potting the distal end and greater trochanter contact surfaces in poly(methyl methacrylate) (PMMA) as presented in a different protocol. The prepared specimens were placed in the testing fixture in a position mimicking a sideways fall on the hip and loaded to fracture. During testing, two load cells measured vertical forces applied to the femoral head and greater trochanter, a six-axis load cell measured forces and moments at the distal femoral shaft, and a displacement sensor measured differential displacement between the femoral head and trochanter contact supports. High speed video cameras were used to synchronously record the sequence of fracture events during testing. The reduction of this data allowed us to characterize the strength, stiffness, and fracture energy for nearly 200 osteoporotic, osteopenic, and normal cadaveric femora for further development of engineering-based diagnostic tools for osteoporosis research.

Fungal nonribosomal peptide synthetases (NRPSs) are megasynthetases that produce cyclic and acyclic peptides. In Aspergillus nidulans, the NRPS ivoA (AN10576) has been associated with the biosynthesis of grey-brown conidiophore pigments. Another gene, ivoB (AN0231), has been demonstrated to be an N-acetyl-6-hydroxytryptophan oxidase that putatively acts downstream of IvoA. A third gene, ivoC, has also been predicted to be involved in pigment biosynthesis based on publicly available genomic and transcriptomic information. In this paper, we report the replacement of the promoters of the ivoA, ivoB, and ivoC genes with the inducible promoter alcA in a single cotransformation. Co-overexpression of the three genes resulted in the production of a dark-brown pigment in hyphae. In addition, overexpression of each of the Ivo genes, ivoA-C, individually or in combination, allowed us to isolate intermediates and confirm the function of each gene. IvoA was found to be the first known NRPS to carry out the acetylation of the amino acid, tryptophan., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Cadaveric fracture testing is routinely used to understand factors that affect proximal femur strength. Because ex vivo biological tissues are prone to lose their mechanical properties over time, specimen preparation for experimental testing must be performed carefully to obtain reliable results that represent in vivo conditions. For that reason, we designed a protocol and a set of fixtures to prepare the femoral specimens such that their mechanical properties experienced minimal changes. The femora were kept in a frozen state except during preparation steps and mechanical testing. The relevant clinical measures of total hip and femoral neck bone mineral density (BMD) were obtained with a clinical dual X-ray absorptiometry (DXA) bone densitometer, and the 3D geometry and distribution of bone mineral were obtained using CT with a calibration phantom for quantitative estimations based on the greyscale values. Any possible bone disease, fracture, or the presence of implants or artifacts affecting the bone structure, was ruled out with X-ray scans. For preparation, all bones were carefully cleaned of excess soft tissue, and were cut and potted at the internal rotation angle of interest. A cutting fixture allowed the distal end of the bone to be cut off leaving the proximal femur at a desired length. To allow positioning of the femoral neck at prescribed angles during later CT scanning and mechanical testing, the proximal femoral shafts were potted in polymethylmethacrylate (PMMA) using a fixture designed specifically for desired orientations. The data collected from our experiments were then used for validation of quantitative computed tomography (QCT)-based finite element analysis (FEA), as described in a different protocol. In this manuscript, we present the protocol for the precise bone preparation for mechanical testing and subsequent QCT/FEA modeling. The current protocol was successfully applied to prepare about 200 cadaveric femora over a 6-year time period.

Fungal secondary metabolites (SMs) are extremely important in medicine and agriculture, but regulation of their biosynthesis is incompletely understood. We have developed a genetic screen in Aspergillus nidulans for negative regulators of fungal SM gene clusters and we have used this screen to isolate mutations that upregulate transcription of the non-ribosomal peptide synthetase gene required for nidulanin A biosynthesis. Several of these mutations are allelic and we have identified the mutant gene by genome sequencing. The gene, which we designate mcrA, is conserved but uncharacterized, and it encodes a putative transcription factor. Metabolite profiles of mcrA deletant, mcrA overexpressing, and parental strains reveal that mcrA regulates at least ten SM gene clusters. Deletion of mcrA stimulates SM production even in strains carrying a deletion of the SM regulator laeA, and deletion of mcrA homologs in Aspergillus terreus and Penicillum canescens alters the secondary metabolite profile of these organisms. Deleting mcrA in a genetic dereplication strain has allowed us to discover two novel compounds as well as an antibiotic not known to be produced by A. nidulans. Deletion of mcrA upregulates transcription of hundreds of genes including many that are involved in secondary metabolism, while downregulating a smaller number of genes., Competing Interests: The authors declare no conflict of interests., (© 2016 John Wiley & Sons Ltd.)

To reduce the secondary metabolite background in Aspergillus nidulans and minimize the rediscovery of compounds and pathway intermediates, we created a "genetic dereplication" strain in which we deleted eight of the most highly expressed secondary metabolite gene clusters (more than 244,000 base pairs deleted in total). This strain allowed us to discover a novel compound that we designate aspercryptin and to propose a biosynthetic pathway for the compound. Interestingly, aspercryptin is formed from compounds produced by two separate gene clusters, one of which makes the well-known product cichorine. This raises the exciting possibility that fungi use differential regulation of expression of secondary metabolite gene clusters to increase the diversity of metabolites they produce., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aggregation of the microtubule-associated protein tau is a significant event in many neurodegenerative diseases including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activity. We have screened Aspergillus nidulans secondary metabolites containing aromatic ring structures for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol and the previously identified aggregation inhibitor emodin as a positive control. While several compounds showed some activity, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde were potent aggregation inhibitors as determined by both a filter trap assay and electron microscopy. In this study, these three compounds were stronger inhibitors than emodin, which has been shown in a prior study to inhibit the heparin induction of tau aggregation with an IC50 of 1-5 µM. Additionally, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde reduced, but did not block, tau stabilization of microtubules. 2,ω-Dihydroxyemodin and asperthecin have similar structures to previously identified tau aggregation inhibitors, while asperbenzaldehyde represents a new class of compounds with tau aggregation inhibitor activity. Asperbenzaldehyde can be readily modified into compounds with strong lipoxygenase inhibitor activity, suggesting that compounds derived from asperbenzaldehyde could have dual activity. Together, our data demonstrates the potential of 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde as lead compounds for further development as therapeutics to inhibit tau aggregation in Alzheimer's disease and neurodegenerative tauopathies., (Georg Thieme Verlag KG Stuttgart · New York.)

Aspernidine A is a prenylated isoindolinone alkaloid isolated from the model fungus Aspergillus nidulans. A genome-wide kinase knockout library of A. nidulans was examined, and it was found that a mitogen-activated protein kinase gene, mpkA, deletion strain produces aspernidine A. Targeted gene deletions were performed in the kinase deletion background to identify the gene cluster for aspernidine A biosynthesis. Intermediates were isolated from mutant strains which provided information about the aspernidine A biosynthesis pathway.

Fungal secondary metabolites (SMs) are an important source of medically valuable compounds. Genome projects have revealed that fungi have many SM biosynthetic gene clusters that are not normally expressed. To access these potentially valuable, cryptic clusters, we have developed a heterologous expression system in Aspergillus nidulans . We have developed an efficient system for amplifying genes from a target fungus, placing them under control of a regulatable promoter, transferring them into A. nidulans , and expressing them. We have validated this system by expressing nonreducing polyketide synthases of Aspergillus terreus and additional genes required for compound production and release. We have obtained compound production and release from six of these nonreducing polyketide synthases and have identified the products. To demonstrate that the procedure allows transfer and expression of entire secondary metabolite biosynthetic pathways, we have expressed all the genes of a silent A. terreus cluster and demonstrate that it produces asperfuranone. Further, by expressing the genes of this pathway in various combinations, we have clarified the asperfuranone biosynthetic pathway. We have also developed procedures for deleting entire A. nidulans SM clusters. This allows us to remove clusters that might interfere with analyses of heterologously expressed genes and to eliminate unwanted toxins.

Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.