Transcription Factor Engineering in Leads to the Discovery of an Orsellinaldehyde Derivative Produced via an Unlinked Polyketide Synthase Gene.

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).

Overexpression of an LaeA-like Methyltransferase Upregulates Secondary Metabolite Production in .

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.

Comparative histomorphometric analysis of cellular phenotype in canine stifle ligaments and tendon.

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.

Hybrid Transcription Factor Engineering Activates the Silent Secondary Metabolite Gene Cluster for (+)-Asperlin in Aspergillus nidulans.

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.

Method and Instrumented Fixture for Femoral Fracture Testing in a Sideways Fall-on-the-Hip Position.

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.

Proximal Cadaveric Femur Preparation for Fracture Strength Testing and Quantitative CT-based Finite Element Analysis.

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.

Overexpression of a three-gene conidial pigment biosynthetic pathway in Aspergillus nidulans reveals the first NRPS known to acetylate tryptophan.

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.

Discovery of McrA, a master regulator of Aspergillus secondary metabolism.

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.

Development of Genetic Dereplication Strains in Aspergillus nidulans Results in the Discovery of Aspercryptin.

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.

Inhibition of Tau aggregation by three Aspergillus nidulans secondary metabolites: 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde.

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.

Molecular genetic characterization of the biosynthesis cluster of a prenylated isoindolinone alkaloid aspernidine A in Aspergillus nidulans.

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.

An efficient system for heterologous expression of secondary metabolite genes in Aspergillus nidulans.

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 .

Case-control study of high-speed exercise history of Thoroughbred and Quarter Horse racehorses that died related to a complete scapular fracture.

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.

Mechanical and morphological properties of trabecular bone samples obtained from third metacarpal bones of cadavers of horses with a bone fragility syndrome and horses unaffected by that syndrome.

Objective: To determine morphological and mechanical properties of trabecular bone of horses with a bone fragility syndrome (BFS; including silicate-associated osteoporosis).

Sample: Cylindrical trabecular bone samples from the distal aspects of cadaveric third metacarpal bones of 39 horses (19 horses with a BFS [BFS bone samples] and 20 horses without a BFS [control bone samples]).

Procedures: Bone samples were imaged via micro-CT for determination of bone volume fraction; apparent and mean mineralized bone densities; and trabecular number, thickness, and separation.

Identification and molecular genetic analysis of the cichorine gene cluster in .

We recently demonstrated that the phytotoxin cichorine is produced by . Through a set of targeted deletions, we have found a cluster of seven genes that are required for its biosynthesis. Two of the deletions yielded molecules that give information about the biosynthesis of this metabolite.

Differentiating between models of epothilone binding to microtubules using tubulin mutagenesis, cytotoxicity, and molecular modeling.

Microtubule stabilizers are powerful antimitotic compounds and represent a proven cancer treatment strategy. Several classes of compounds in clinical use or trials, such as the taxanes and epothilones, bind to the same region of β-tubulin. Determining how these molecules interact with tubulin and stabilize microtubules is important both for understanding the mechanism of action and enhancing chemotherapeutic potential, for example, minimizing side effects, increasing solubility, and overcoming resistance.

Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis.

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.

Illuminating the diversity of aromatic polyketide synthases in Aspergillus nidulans.

Genome sequencing has revealed that fungi have the ability to synthesize many more natural products (NPs) than are currently known, but methods for obtaining suitable expression of NPs have been inadequate. We have developed a successful strategy that bypasses normal regulatory mechanisms. By efficient gene targeting, we have replaced, en masse, the promoters of nonreducing polyketide synthase (NR-PKS) genes, key genes in NP biosynthetic pathways, and other genes necessary for NR-PKS product formation or release.

Two separate gene clusters encode the biosynthetic pathway for the meroterpenoids austinol and dehydroaustinol in Aspergillus nidulans.

Meroterpenoids are a class of fungal natural products that are produced from polyketide and terpenoid precursors. An understanding of meroterpenoid biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has previously been found to produce two meroterpenoids, austinol and dehydroaustinol.

Biomechanical evaluation of screw-in femoral implant in cementless total hip system.

Objective: To compare (1) proximal femoral axial strains, (2) femoral head deflection, and (3) failure mechanical properties, between Helica head and neck prosthesis implanted femora and normal femora.

Study Design: In vitro study.

Sample Population: Cadaveric canine femora (n = 5 pair).

Characteristics of Thoroughbred and Quarter Horse racehorses that sustained a complete scapular fracture.

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.

Genome-based deletion analysis reveals the prenyl xanthone biosynthesis pathway in Aspergillus nidulans.

Xanthones are a class of molecules that bind to a number of drug targets and possess a myriad of biological properties. An understanding of xanthone biosynthesis at the genetic level should facilitate engineering of second-generation molecules and increasing production of first-generation compounds. The filamentous fungus Aspergillus nidulans has been found to produce two prenylated xanthones, shamixanthone and emericellin, and we report the discovery of two more, variecoxanthone A and epishamixanthone.

Vascular distribution in ruptured canine cranial cruciate ligament.

Objective: To (1) determine the microanatomic vascular distribution in ruptured canine cranial cruciate ligaments (CCL) using specific vascular immunohistochemical techniques, and (2) compare vessel density between ruptured and intact canine CCL and between different areas of interest in ruptured CCL using histomorphometric analysis.

Study Design: In vitro study.

Animals: Dogs (n=41) admitted for surgical treatment of ruptured CCL and 19 dogs euthanatized for nonorthopedic conditions.

Immunohistochemical and histomorphometric evaluation of vascular distribution in intact canine cranial cruciate ligament.

Objectives: To (1) describe vascular distribution in the grossly intact canine cranial cruciate ligament (CCL) using immunohistochemical techniques specific to 2 components of blood vessels (factor VIII for endothelial cells, laminin for basement membrane); and (2) compare the vascularity in different areas of interest (craniomedial versus caudolateral bands; core versus epiligamentous regions; and proximal versus middle versus distal portions) in the intact normal canine CCL.

Study Design: In vitro study.

Animals: Large, mature dogs (n=7) of breeds prone to CCL disease that were euthanatized for nonorthopedic conditions.