Resistance gene-guided genome mining reveals the roseopurpurins as inhibitors of cyclin-dependent kinases.

With the significant increase in the availability of microbial genome sequences in recent years, resistance gene-guided genome mining has emerged as a powerful approach for identifying natural products with specific bioactivities. Here, we present the use of this approach to reveal the roseopurpurins as potent inhibitors of cyclin-dependent kinases (CDKs), a class of cell cycle regulators implicated in multiple cancers. We identified a biosynthetic gene cluster (BGC) with a putative resistance gene with homology to human CDK2. Using targeted gene disruption and transcription factor overexpression in Aspergillus uvarum, and heterologous expression of the BGC in Aspergillus nidulans, we demonstrated that roseopurpurin C (1) is produced by this cluster and characterized its biosynthesis. We determined the potency, specificity, and mechanism of action of 1 as well as multiple intermediates and shunt products produced from the BGC. We show that 1 inhibits human CDK2 with a Kiapp of 44 nM, demonstrates selectivity for clinically relevant members of the CDK family, and induces G1 cell cycle arrest in HCT116 cells. Structural analysis of 1 complexed with CDK2 revealed the molecular basis of ATP-competitive inhibition.

Development and Application of a Weighted Gingivitis and Periodontitis (W-G/P) Score System in Cats.

The adaptation for use in cats of a weighted periodontal scoring system developed in dogs is described. The system uses standardized methods to score the extent of gingivitis and periodontitis of buccal tooth surfaces, weighted by size of teeth and adjusted for missing teeth and size of the cat.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Day one core competencies in veterinary dentistry.

This article describes the core competencies recommended for inclusion in the veterinary curriculum for all veterinary graduates based on the American Association of Veterinary Medical Colleges Competency-Based Veterinary Education document. General practice companion animal veterinarians are frequently presented with patients having dental, oral, or maxillofacial pathology, and veterinary graduates will be relied upon for recommendations for the maintenance of oral health, including the prevention of periodontal disease, identification of endodontic disease, and knowledge of developmental defects. These recommendations should be made for all veterinary patients starting at a young age. These core competencies can apply to many companion species, but mainly are focused on the dog and cat. Because periodontal disease is the most common abnormality observed in dogs and cats, the first key step is taking a few seconds during examination of every patient of any age presented for any reason to examine the oral cavity. Although dental, oral, and maxillofacial pathology is often diagnosed after imaging and evaluation under anesthesia, the first step is observation of dentition and gingivae during the conscious exam to assess periodontal health status. The physical exam of the oral cavity may reveal oral behavior (eg, observation of uncomplicated crown fractures due to chewing on hard objects), which will permit recommendations for enhanced prevention by daily oral hygiene or professional treatment. There are now many involved dental and surgical treatments available, some of which require specialist-level instrumentation and expertise. General practitioners should be able to competently perform the following immediately upon graduation from veterinary school: For patients for whom the owner's reason for the veterinary visit is not dental, oral, or maxillofacial disease, obtain a brief (1 or 2 questions) history of the oral health of the patient. On lifting the lip of every patient, recognize presence or absence of accumulated dental plaque or calculus on the crowns of the teeth, presence or absence of gingival inflammation or ulceration, and presence or absence of other dental, oral, and maxillofacial pathology. On anesthetized patients that have dental, oral, and maxillofacial pathology for which professional treatment is indicated, be able to obtain and interpret appropriately positioned and exposed dental radiographs. When the presence of dental, oral, and maxillofacial pathology is recognized, determine whether each tooth present in the mouth does or does not require professional treatment beyond dental subgingival and supragingival scaling and polishing. List the indications for tooth extraction, know indications for potential oral/dental treatments beyond subgingival and supragingival scaling and polishing or extraction, and determine whether the professional treatment that may be indicated, such as root canal treatment or mass resection of oral tissues, requires referral for specialist-level expertise and instrumentation. Complete a thorough periodontal evaluation and therapy with periodontal probing, including professional subgingival and supragingival ultrasonic scaling with polishing under anesthesia. Demonstrate the ability to extract teeth indicated for extraction, using gentle and appropriate techniques that will risk minimal injury to the jaws and oral soft tissues and reduce postoperative patient pain. Provide appropriate postoperative care, including recognition of when postoperative analgesia and possibly antibiotic administration are indicated.

The Relationship Between Periodontal Infection and Systemic and Distant Organ Disease in Dogs.

Infection in the mouth causes systemic and distant organ changes in dogs. This article summarizes the information available. Reported changes include an increase in liver-generated acute-phase proteins in response to the infectious insult to the body and evidence of microscopic changes in renal, hepatic, and cardiac tissues. Treatment of periodontal infection results in a decrease in the acute-phase protein concentration, which supports the hypothesis that a cause-and-effect relationship exists between periodontal infection and distant organ changes.

Targeted Genome Mining Reveals the Biosynthetic Gene Clusters of Natural Product CYP51 Inhibitors.

Lanosterol 14α-demethylase (CYP51) is an important target in the development of antifungal drugs. The fungal-derived restricticin 1 and related molecules are the only examples of natural products that inhibit CYP51. Here, using colocalizations of genes encoding self-resistant CYP51 as the query, we identified and validated the biosynthetic gene cluster (BGC) of 1. Additional genome mining of related BGCs with CYP51 led to production of the related lanomycin 2. The pathways for both 1 and 2 were identified from fungi not known to produce these compounds, highlighting the promise of the self-resistance enzyme (SRE) guided approach to bioactive natural product discovery.

Anti-cryptococcal activity of preussolides A and B, phosphoethanolamine-substituted 24-membered macrolides, and leptosin C from coprophilous isolates of Preussia typharum.

Cryptococcus neoformans is a serious human pathogen with limited options for treatment. We have interrogated extracts from fungal fermentations to find Cryptococcus-inhibiting natural products using assays for growth inhibition and differential thermosensitivity. Extracts from fermentations of four fungal strains from wild and domestic animal dung from Arkansas and West Virginia, USA were identified as Preussia typharum. The extracts exhibited two antifungal regions. Purification of one region yielded new 24-carbon macrolides incorporating both a phosphoethanolamine unit and a bridging tetrahydrofuran ring. The structures of these metabolites were established mainly by analysis of high-resolution mass spectrometry and 2D NMR data. Relative configurations were assigned using NOESY data, and the structure assignments were supported by NMR comparison with similar compounds. These new metabolites are designated preussolides A and B. The second active region was caused by the cytotoxin, leptosin C. Genome sequencing of the four strains revealed biosynthetic gene clusters consistent with those known to encode phosphoethanolamine-bearing polyketide macrolides and the biosynthesis of dimeric epipolythiodioxopiperazines. All three compounds showed moderate to potent and selective antifungal activity toward the pathogenic yeast C. neoformans.

Identification of the Antifungal Metabolite Chaetoglobosin P From Discosia rubi Using a Cryptococcus neoformans Inhibition Assay: Insights Into Mode of Action and Biosynthesis.

Cryptococcus neoformans is an important human pathogen with limited options for treatments. We have interrogated extracts from fungal fermentations to find Cryptococcus-inhibiting natural products using assays for growth inhibition, differential thermosensitivity, and synergy with existing antifungal drugs. Extracts from fermentations of strains of Discosia rubi from eastern Texas showed anticryptococcal bioactivity with preferential activity in agar zone of inhibition assays against C. neoformans at 37°C versus 25°C. Assay-guided fractionation led to the purification and identification of chaetoglobosin P as the active component of these extracts. Genome sequencing of these strains revealed a biosynthetic gene cluster consistent with chaetoglobosin biosynthesis and ß-methylation of the tryptophan residue. Proximity of genes of the actin-binding protein twinfilin-1 to the chaetoglobosin P and K gene clusters suggested a possible self-resistance mechanism involving twinfilin-1 which is consistent with the predicted mechanism of action involving interference with the polymerization of the capping process of filamentous actin. A C. neoformans mutant lacking twinfilin-1 was hypersensitive to chaetoglobosin P. Chaetoglobosins also potentiated the effects of amphotericin B and caspofungin on C. neoformans.

Acrophiarin (antibiotic S31794/F-1) from Penicillium arenicola shares biosynthetic features with both Aspergillus- and Leotiomycete-type echinocandins.

The antifungal echinocandin lipopeptide, acrophiarin, was circumscribed in a patent in 1979. We confirmed that the producing strain NRRL 8095 is Penicillium arenicola and other strains of P. arenicola produced acrophiarin and acrophiarin analogues. Genome sequencing of NRRL 8095 identified the acrophiarin gene cluster. Penicillium arenicola and echinocandin-producing Aspergillus species belong to the family Aspergillaceae of the Eurotiomycetes, but several features of acrophiarin and its gene cluster suggest a closer relationship with echinocandins from Leotiomycete fungi. These features include hydroxy-glutamine in the peptide core instead of a serine or threonine residue, the inclusion of a non-heme iron, α-ketoglutarate-dependent oxygenase for hydroxylation of the C3 of the glutamine, and a thioesterase. In addition, P. arenicola bears similarity to Leotiomycete echinocandin-producing species because it exhibits self-resistance to exogenous echinocandins. Phylogenetic analysis of the genes of the echinocandin biosynthetic family indicated that most of the predicted proteins of acrophiarin gene cluster exhibited higher similarity to the predicted proteins of the pneumocandin gene cluster of the Leotiomycete Glarea lozoyensis than to those of the echinocandin B gene cluster from A. pachycristatus. The fellutamide gene cluster and related gene clusters are recognized as relatives of the echinocandins. Inclusion of the acrophiarin gene cluster into a comprehensive phylogenetic analysis of echinocandin gene clusters indicated the divergent evolutionary lineages of echinocandin gene clusters are descendants from a common ancestral progenitor. The minimal 10-gene cluster may have undergone multiple gene acquisitions or losses and possibly horizontal gene transfer after the ancestral separation of the two lineages.

A biosensor-based approach reveals links between efflux pump expression and cell cycle regulation in pleiotropic drug resistance of yeast.

Multidrug resistance is highly conserved in mammalian, fungal, and bacterial cells, is characterized by resistance to several unrelated xenobiotics, and poses significant challenges to managing infections and many cancers. Eukaryotes use a highly conserved set of drug efflux transporters that confer pleiotropic drug resistance (PDR). To interrogate the regulation of this critical process, here we developed a small molecule-responsive biosensor that couples transcriptional induction of PDR genes to growth rate in the yeast Saccharomyces cerevisiae Using diverse PDR inducers and the homozygous diploid deletion collection, we applied this biosensor system to genome-wide screens for potential PDR regulators. In addition to recapitulating the activity of previously known factors, these screens identified a series of genes involved in a variety of cellular processes with significant but previously uncharacterized roles in the modulation of yeast PDR. Genes identified as down-regulators of the PDR included those encoding the MAD family of proteins involved in the mitotic spindle assembly checkpoint (SAC) complex. Of note, we demonstrated that genetic disruptions of the mitotic spindle assembly checkpoint elevate expression of PDR-mediating efflux pumps in response to exposure to a variety of compounds that themselves have no known influence on the cell cycle. These results not only establish our biosensor system as a viable tool for investigating PDR in a high-throughput fashion, but also uncover critical control mechanisms governing the PDR response and a previously uncharacterized link between PDR and cell cycle regulation in yeast.

HEx: A heterologous expression platform for the discovery of fungal natural products.

For decades, fungi have been a source of U.S. Food and Drug Administration-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.

Reversible Product Release and Recapture by a Fungal Polyketide Synthase Using a Carnitine Acyltransferase Domain.

Fungal polyketides have significant biological activities, yet the biosynthesis by highly reducing polyketide synthases (HRPKSs) remains enigmatic. An uncharacterized group of HRPKSs was found to contain a C-terminal domain with significant homology to carnitine O-acyltransferase (cAT). Characterization of one such HRPKS (Tv6-931) from Trichoderma virens showed that the cAT domain is capable of esterifying the polyketide product with polyalcohol nucleophiles. This process is readily reversible, as confirmed through the holo ACP-dependent transesterification of the released product. The methyltransferase (MT) domain of Tv6-931 can perform two consecutive α-methylation steps on the last ß-keto intermediate to yield an α,α-gem-dimethyl product, a new programing feature among HRPKSs. Recapturing of the released product by cAT domain is suggested to facilitate complete gem-dimethylation by the MT.

Odontogenic Fibromyxoma in a Cat: First Confirmed Case in This Species.

An inflammatory gingival mass surrounding resorbing teeth was diagnosed via biopsy in a 9-year-old domestic shorthair cat. A dorsal rim excision was performed to remove the entire mass with associated teeth and bone. Histopathological diagnosis of the en bloc tissue revealed an odontogenic fibromyxoma. Extensive literature review revealed few case reports of companion animals with this neoplasm, and none in a feline patient. This report documents the clinical presentation, diagnostic differentials, surgical therapy, and long-term follow-up of an odontogenic fibromyxoma in a cat.

Comprehensive curation and analysis of fungal biosynthetic gene clusters of published natural products.

Microorganisms produce a wide range of natural products (NPs) with clinically and agriculturally relevant biological activities. In bacteria and fungi, genes encoding successive steps in a biosynthetic pathway tend to be clustered on the chromosome as biosynthetic gene clusters (BGCs). Historically, "activity-guided" approaches to NP discovery have focused on bioactivity screening of NPs produced by culturable microbes. In contrast, recent "genome mining" approaches first identify candidate BGCs, express these biosynthetic genes using synthetic biology methods, and finally test for the production of NPs. Fungal genome mining efforts and the exploration of novel sequence and NP space are limited, however, by the lack of a comprehensive catalog of BGCs encoding experimentally-validated products. In this study, we generated a comprehensive reference set of fungal NPs whose biosynthetic gene clusters are described in the published literature. To generate this dataset, we first identified NCBI records that included both a peer-reviewed article and an associated nucleotide record. We filtered these records by text and homology criteria to identify putative NP-related articles and BGCs. Next, we manually curated the resulting articles, chemical structures, and protein sequences. The resulting catalog contains 197 unique NP compounds covering several major classes of fungal NPs, including polyketides, non-ribosomal peptides, terpenoids, and alkaloids. The distribution of articles published per compound shows a bias toward the study of certain popular compounds, such as the aflatoxins. Phylogenetic analysis of biosynthetic genes suggests that much chemical and enzymatic diversity remains to be discovered in fungi. Our catalog was incorporated into the recently launched Minimum Information about Biosynthetic Gene cluster (MIBiG) repository to create the largest known set of fungal BGCs and associated NPs, a resource that we anticipate will guide future genome mining and synthetic biology efforts toward discovering novel fungal enzymes and metabolites.

Effect of Frequency of Brushing Teeth on Plaque and Calculus Accumulation, and Gingivitis in Dogs.

The efficacy of brushing the teeth of beagle dogs in a randomized, controlled, blinded study design using a clearly-defined brushing technique was evaluated for 4 brushing frequencies: brushing daily, brushing every other day, brushing weekly and brushing every other week, compared with no brushing in a control group of dogs. All dogs were fed a standard dry kibble diet during the study. Standard plaque, calculus, and gingivitis indices were used to score the teeth. A 'clean tooth' model was used. No gingival or non-gingival lacerations or other signs of injury to oral tissues were found at the end of the 28 day trial period. Brushing more frequently had greater effectiveness in retarding accumulation of plaque and calculus, and reducing the severity of pre-existing gingivitis. Brushing daily or every other day produced statistically significant improved results compared with brushing weekly or every other week. Based on the results of this study, daily brushing is recommended.

Precursor directed biosynthesis of an orthogonally functional erythromycin analogue: selectivity in the ribosome macrolide binding pocket.

The macrolide antibiotic erythromycin A and its semisynthetic analogues have been among the most useful antibacterial agents for the treatment of infectious diseases. Using a recently developed chemical genetic strategy for precursor-directed biosynthesis and colony bioassay of 6-deoxyerythromycin D analogues, we identified a new class of alkynyl- and alkenyl-substituted macrolides with activities comparable to that of the natural product. Further analysis revealed a marked and unexpected dependence of antibiotic activity on the size and degree of unsaturation of the precursor. Based on these leads, we also report the precursor-directed biosynthesis of 15-propargyl erythromycin A, a novel antibiotic that not only is as potent as erythromycin A with respect to its ability to inhibit bacterial growth and cell-free ribosomal protein biosynthesis but also harbors an orthogonal functional group that is capable of facile chemical modification.

Validation of use of subsets of teeth when applying the total mouth periodontal score (TMPS) system in dogs.

A total mouth periodontal score (TMPS) system in dogs has been described previously. Use of buccal and palatal/lingual surfaces of all teeth requires observation and recording of 120 gingivitis scores and 120 periodontitis scores. Although the result is a reliable, repeatable assessment of the extent of periodontal disease in the mouth, observing and recording 240 data points is time-consuming. Using data from a previously reported study of periodontal disease in dogs, correlation analysis was used to determine whether use of any of seven different subsets of teeth can generate TMPS subset gingivitis and periodontitis scores that are highly correlated with TMPS all-site, all-teeth scores. Overall, gingivitis scores were less highly correlated than periodontitis scores. The minimal tooth set with a significant intra-class correlation (> or = 0.9 of means of right and left sides) for both gingivitis scores and attachment loss measurements consisted of the buccal surface of the maxillary third incisor canine, third premolar fourth premolar; and first molar teeth; and, the mandibular canine, third premolar, fourth premolar and first molar teeth on one side (9 teeth, 15 root sites). Use of this subset of teeth, which reduces the number of data points per dog from 240 to 30 for gingivitis and periodontitis at each scoring episode, is recommended when calculating the gingivitis and periodontitis scores using the TMPS system.