Discovery proteomics for the detection of putative markers for eradication of infection in an experimental model of equine septic arthritis using LC-MS/MS.

Septic arthritis (SA) is a life-threatening condition in horses, and identifying eradication of infection in equine SA is challenging. This study explored the discovery of putative biomarkers for the eradication of joint infection in horses. We performed proteomics analysis of synovial fluid (SF) and plasma from horses with experimental SA, non-septic lipopolysaccharide-induced arthritis, and controls. The point of eradication of infection in horses with SA was determined previously. We compared spectral intensities between groups as well as before and after the eradication of infection. Twenty-six differentially abundant proteins were identified, which were upregulated in SF of horses with SA compared to the other groups, as well as compared to the same horses post-eradication of infection. In plasma, we did not identify differentially abundant proteins. Differentially abundant proteins in SF were of cellular origin and their biological functions included ubiquitination, signal transduction, apoptosis etc. The difference in their relative abundance between experimental groups was ≥10-fold compared to the abundance expected based on the difference in cell count alone (2-fold). Since most of cells in joints with bacterial infection are neutrophils, we suggest that the variable abundance of neutrophil- and cell-associated proteins represent potential biomarkers of eradication of infection in equine SA. SIGNIFICANCE: Septic arthritis is an important condition in horses, which can be life-threatening. At present, identifying eradication of infection in cases of equine septic arthritis is challenging. In this study, we performed a global proteomics analysis of synovial fluid and plasma in horses with experimental septic arthritis and identified 26 differentially abundant proteins compared to non-septic arthritis and post eradication of infection. The results of this study provide the basis for further characterization of the differentially abundant proteins and identification of clinically relevant biomarkers of septic arthritis in horses.

Fixation of a scapular body fracture in a 5 month old foal using three 4.5/5.0 mm locking compression plates.

OBJECTIVE: To report a surgical technique and an outcome for the repair of a displaced, transverse scapular body fracture with locking compression plates (LCPs) in a colt. ANIMALS: One 5 month old Thoroughbred colt. STUDY DESIGN: Case report. METHODS: A colt sustained an unstable, comminuted, transverse fracture of the scapular body. Three 4.5/5.0 mm LCPs were used with 6.5 mm cancellous screws, 4.5 mm cortex screws, and 5.0 mm locking head screws. Implants were removed 2 months after surgery. RESULTS: Surgical site infection was identified by purulent discharge at the distal aspect of the suture line 3 days after surgery. The surgical site infection resolved with daily lavage within 15 days after surgery. Three months after internal fixation of the scapular body fracture, the colt was sound and was turned out to pasture. One year later, the colt was sound and in training to be a flat racehorse. CONCLUSION: Repair of a scapular body fracture using LCP provided a good outcome with an early return to soundness. The LCP system can therefore be considered for the repair of scapular body fractures in small equids.

Use of serum amyloid A in serum and synovial fluid to detect eradication of infection in experimental septic arthritis in horses.

While serum amyloid A (SAA) has been investigated as a potential marker for septic arthritis in horses, no study has reported on whether SAA can be used to detect eradication of joint infection. Therefore, the objective of this study was to investigate whether the eradication of joint infection in experimentally induced septic arthritis in horses can be detected using serum and synovial fluid SAA. A total of 17 horses were randomly assigned to 3 groups. A middle carpal joint of each horse was injected with saline (control group, n = 3), lipopolysaccharide (LPS) (nonseptic synovitis group, n = 6), or Escherichia coli (septic arthritis group, n = 8) on day 0. Starting on day 1, horses underwent treatment for septic arthritis. Sequential samples of serum and synovial fluid were collected, and quantification of SAA was carried out. Concentrations of serum and synovial fluid SAA were compared among groups and time points. A concurrent study was conducted and determined that infection was eradicated on day 4 in this experimental model of septic arthritis. Concentrations of serum and synovial fluid SAA rapidly increased after inoculation of E. coli and were highest on day 3 and day 4, respectively. Thereafter, both serum and synovial fluid SAA decreased with eradication of joint infection, although they remained significantly increased from baseline until day 9 and day 10, respectively. Serum and synovial fluid SAA did not increase in the control or nonseptic synovitis group. These findings suggest that serial measurements rather than a single measurement of SAA are required to determine eradication of infection from septic arthritis in horses.

Bien que l'amyloïde sérique (SAA) fut étudiée comme marqueur potentiel pour l'arthrite septique chez les chevaux, aucune étude n'a rapporté si SAA peut être utilisée pour détecter l'élimination d'une infection articulaire. Ainsi, l'objectif de la présente étude était d'examiner si l'élimination d'une infection articulaire lors d'arthrite septique induite expérimentalement chez les chevaux peut être détectée en utilisant la SAA du sérum et du liquide synovial. Un total de 17 chevaux fut réparti de manière aléatoire en trois groupes. Une articulation carpienne médiale de chaque cheval fut injectée avec de la saline (groupe témoin, n = 3), du lipopolysaccharide (LPS) (groupe synovite non-septique, n = 6) ou Escherichia coli (groupe arthrite septique, n = 8) au jour 0. En débutant au jour 1, les chevaux furent soumis à un traitement pour arthrite septique. Des échantillons séquentiels de sérum et de liquide synovial furent prélevés et la quantification de SAA effectuée. Les concentrations de SAA dans le sérum et le liquide synovial furent comparées parmi les groupes et à différents temps. Une étude concomitante était menée et a déterminé que l'infection était éliminée au jour 4 dans ce modèle expérimental d'arthrite septique. Les concentrations de SAA dans le sérum et le liquide synovial ont rapidement augmenté après l'inoculation d'E. coli et étaient maximales au jour 3 et au jour 4, respectivement. Par la suite, les concentrations de SAA du sérum et du liquide synovial ont diminué avec l'élimination de l'infection articulaire, bien qu'elles soient demeurées augmentées significativement par rapport au seuil de base jusqu'au jour 9 et jour 10, respectivement. Les concentrations de SAA du sérum et du liquide synovial n'ont pas augmenté dans les groupes témoin et synovite non-septique. Ces résultats suggèrent que des mesures en série plutôt qu'une mesure unique de SAA sont requises pour déterminer l'élimination de l'infection lors d'arthrite septique chez les chevaux.(Traduit par Docteur Serge Messier).

Discovery of ASP5286: A novel non-immunosuppressive cyclophilin inhibitor for the treatment of HCV.

Evidence indicates that hepatitis C virus (HCV) utilizes cellular cyclophilin proteins in its replication, and cyclophilin inhibitors represent a new class of anti-HCV agents. We have established an efficient synthetic methodology to generate FR901459 derivatives via N, O-acyl migration reaction while avoiding total synthesis. Through a detailed structure-activity relationship study, we improved anti-HCV activity while decreasing immunosuppressive activity. Additionally, we discovered the importance of substitution at the 3 position for not only improving anti-HCV activity but also pharmacokinetic profile. Finally, by striking an appropriate balance between potency, solubility, and permeability, we discovered ASP5286 (13) as a potential clinical candidate for anti-HCV therapy.

Development of an efficient semisynthetic modification of FR901459 via a novel regioselective N, O-acyl migration.

HCV utilizes cellular protein cyclophilins in the virus replication cycle and cyclophilin inhibitors have become a new class of anti-HCV agents. In our screening of natural products, we identified a unique cyclosporin analogue, FR901459, as a cyclophilin inhibitor with potent anti-HCV activity. In this work, we developed an efficient synthetic methodology to prepare FR901459 derivatives via an N, O-acyl migration reaction. This method allows us to efficiently manipulate the amino acid residues at the 3 position while avoiding lengthy total synthesis for each compound. By using this methodology, we discovered 4, which has superior anti-HCV activity and decreased immunosuppressive activity compared to FR901459.

Sinocutaneous fistula repair with a masseter muscle transposition flap combined with wound matrix and cancellous bone graft in a horse: A new technique.

OBJECTIVE: To describe a new technique to repair a sinocutaneous fistula with a masseter muscle transposition flap. STUDY DESIGN: Case report. ANIMAL: One 13-year-old thoroughbred stallion. METHODS: One 13-year-old stallion with a 3.5 × 6-cm sinocutaneous fistula over the right caudal maxillary sinus was treated with a transpositional masseter muscle flap. This repair consisted of a commercial wound matrix dressing placed directly over the hole in the maxilla and secured with suture material; a cancellous bone graft collected from the right tuber coxa placed on the dressing; and a portion of the superficial layer of the masseter muscle, with its pedicle at the facial crest, transposed dorsally over the bone graft, followed by a rotational skin flap with skin rostral to the fistula to close the defect. RESULTS: Seroma formation and dehiscence of the skin flap occurred, but the transposed muscle flap survived, and the technique resulted in successful closure of the sinocutaneous fistula with excellent cosmetic and functional outcome. CONCLUSION: A chronic maxillary sinocutaneous fistula was successfully treated by using a transposition flap of the masseter muscle and a rotational skin flap with minor complications. CLINICAL IMPACT: Transposition of the superficial layer of the masseter muscle should be considered for a repair of large maxillary sinocutaneous fistulas in horses.

Sacrococcygeal luxation and complete tail amputation following a tail pull injury in a horse.

A 17-year-old Quarter horse mare was presented because of traumatic luxation of the fifth sacral and first coccygeal vertebrae resulting in loss of sensation, motor function, and perfusion of the tail. The case was complicated by an associated tail head hematoma. Due to the severity of the injury, tail amputation was performed at the level of the luxation. Tail amputations in horses at the sacrococcygeal junction following a suspected tail pull injury are infrequently reported in the literature.

Luxation sacrococcygienne et amputation complète de la queue à la suite d'une blessure par traction de la queue chez un cheval. Une jument Quarter horse âgée de 17 ans fut présentée pour cause de luxation traumatique de la cinquième vertèbre sacrée et de la première vertèbre coccygienne résultant en une perte de sensation, de fonction moteur, et de perfusion de la queue. Le cas était compliqué par l'association d'un hématome de la tête de la queue. Compte tenu de la sévérité de la blessure, l'amputation de la queue fut effectuée au site de la luxation. Les amputations de la queue chez les chevaux à la jonction sacrococcygienne à la suite d'une blessure suspectée causée par traction de la queue ne sont rapportées que peu fréquemment dans la littérature.(Traduit par Dr Serge Messier).

Discovery and Biological Evaluation of Potent and Orally Active Human 11ß-Hydroxysteroid Dehydrogenase Type 1 Inhibitors for the Treatment of Type 2 Diabetes Mellitus.

We synthesized and evaluated novel 5-[2-(thiophen-2-yl)propan-2-yl]-4H-1,2,4-triazole derivatives as 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitors. Optimization of the thiophene ring and the substituents on the 1,2,4-triazole ring produced 3,4-dicyclopropyl-5-{2-[3-fluoro-5-(trifluoromethyl)thiophen-2-yl]propan-2-yl}-4H-1,2,4-triazole monohydrochloride (9a), which showed potent and selective inhibitory activity against human 11ß-HSD1. Compound 9a was also metabolically stable against human and mouse liver microsomes. Oral administration of 9a to diabetic ob/ob mice lowered corticosterone levels in adipose tissue, and thereby reduced plasma glucose and insulin levels in a dose-dependent manner.

Use of standard diagnostic techniques to determine eradication of infection in experimental equine septic arthritis.

Septic arthritis is an important disease in horses, necessitating aggressive and prolonged therapy. In order to guide therapy, reliable methods of detecting the eradication of infection are needed. Therefore, the objective of this study was to investigate detection of eradication of infection in an experimental model of equine septic arthritis using standard diagnostic techniques. For this purpose, 17 adult horses were assigned to 3 experimental groups. The middle carpal joint of each horse was injected with Escherichia coli (Septic group, n = 8), lipopolysaccharide (LPS) (LPS group, n = 6), or sterile saline (Control group, n = 3) at day 0. Contralateral joints were not injected. Standard therapy was applied to all joints except non-injected joints in the Control group at day 1. Sequential samples of synovial fluid (SF) were collected for bacterial culture using 3 culture media [Columbia blood agar (CBA), brain heart infusion broth (BHI), and Signal blood culture medium] and for cytological evaluation [percentage neutrophils (PN), total nucleated cell count (TNCC), and total protein (TP)]. Escherichia coli-specific polymerase chain reaction (PCR) was carried out to detect E. coli DNA in synovial fluid. Culture and PCR were positive for E. coli in all joints injected with E. coli at day 1 and 1 joint was positive on BHI at day 4. Based on the results of bacterial culture, PCR, and TNCC, the elimination of infection in our experimental model occurred by day 4 post-infection in 6 out of 7 cases. Total protein (TP) and PN remained elevated at clinical threshold used for diagnosis of septic arthritis until day 14. In our experimental model of E. coli-induced arthritis, we conclude that TP and PN may not be good indicators for detecting the eradication of bacterial infection caused by E. coli from infected and subsequently treated joints.

L'arthrite septique est une pathologie importante chez les chevaux, nécessitant une thérapie agressive et prolongée. Afin de guider la thérapie, des méthodes fiables pour détecter l'éradication de l'infection sont requises. Ainsi, l'objectif de la présente étude était d'examiner la détection de l'éradication de l'infection dans un modèle expérimental d'arthrite septique équine en utilisant des techniques diagnostiques standards. À cet effet, 17 chevaux adultes ont été assignés à trois groupes expérimentaux. L'articulation carpienne moyenne de chaque cheval a été injectée avec Escherichia coli (groupe septique, n = 8), du lipopolysaccharide (LPS) (groupe LPS, n = 6), ou de la saline stérile (groupe témoin, n = 3) au jour 0. Les articulations contra-latérales n'ont pas été injectées. Au jour 1, une thérapie standard fut appliquée à toutes les articulations sauf les articulations non-injectées dans le groupe témoin. De manière séquentielle des échantillons de liquide synovial (LS) furent prélevés pour culture bactérienne en utilisant trois milieux de culture [gélose au sang Columbia (CBA), bouillon coeur-cerveau (BHI), et hémoculture Signal] et pour évaluation cytologique [pourcentage de neutrophiles (PN), dénombrement total de cellules nucléées (DTCN), et la quantité de protéines totales (PT)]. Une réaction d'amplification en chaîne par la polymérase (ACP) spécifique à E. coli a été réalisée afin de détecter l'ADN d'E. coli dans le LS. La culture et l'ACP étaient positives pour E. coli dans toutes les articulations injectées avec E. coli au jour 1 et une articulation était positive avec le BHI au jour 4. Sur la base des résultats des cultures bactériennes, de l'ACP, et du DTCN, l'élimination de l'infection dans notre modèle expérimental est survenue au jour 4 post-infection dans 6 des 7 cas. Les valeurs de PT et de PN sont demeurées élevées au seuil clinique utilisé pour diagnostiquer une arthrite septique jusqu'au jour 14. Dans notre modèle expérimental d'arthrite induite par E. coli, nous concluons que les valeurs de PT et de PN ne seraient pas de bons indicateurs pour détecter l'éradication de l'infection bactérienne causée par E. coli dans des articulations infectées et subséquemment traitées.(Traduit par Docteur Serge Messier).

ASP2397: a novel antifungal agent produced by Acremonium persicinum MF-347833.

The novel antifungal agent ASP2397 (Vical's compound ID VL-2397) is produced by the fungal strain MF-347833 that was isolated from Malaysian leaf litter and is identified here as an Acremonium species based on its morphology, physiological properties and 28S ribosomal DNA sequence. Because of its potential importance for producing novel antifungal agents, we determined the taxonomic and biologic properties of MF-347833. We show here that ASP2397 is a cyclic hexapeptide that chelates aluminum ion and is therefore similar to ferrichrome, a hydroxamate siderophore. However, ASP2397 differs structurally from licensed antifungal agents such as amphotericin B, triazoles and echinocandins. To understand the relationship between chemical structure and biological function, we isolated certain ASP2397 derivatives from the culture broth, and we further chemically converted the metal-free form to other derivatives.

Discovery of an 8-methoxytetrahydroisoquinoline derivative as an orally active N-type calcium channel blocker for neuropathic pain without CYP inhibition liability.

In lead optimization efforts starting from the tetrahydroisoquinoline (S)-1, we identified 2-{[(2R)-2-hydroxypropyl]amino}-1-[(1S)-8-methoxy-1-phenyl-3,4-dihydroisoquinolin-2(1H)-yl]ethanone ((1S)-8t) as a novel orally active small-molecule N-type calcium channel blocker without CYP inhibition liability. CYP3A4 inhibition profile was improved by reducing the lipophilicity of compound (S)-1. Moreover, introduction of a methoxy group to the C-8 position of tetrahydroisoquinoline led to identification of (1S)-8t, which eliminated CYP2D6 inhibition liability. Oral administration of (1S)-8t exerted efficacy in a rat spinal nerve ligation (SNL) model of neuropathic pain with an ED50 value of 2.8 mg/kg.

Bioconversion of FR901459, a novel derivative of cyclosporin A, by Lentzea sp. 7887.

FR901459, a product of the fungus Stachybotrys chartarum No. 19392, is a derivative of cyclosporin A (CsA) and a powerful immunosuppressant that binds cyclophilin. Recently, it was reported that CsA was effective against hepatitis C virus (HCV). However, FR901459 lacks active moieties, which are essential for synthesizing more potent and safer derivatives of this anti-HCV agent. Here we identified an actinomycete strain (designated 7887) that was capable of efficient bioconversion of FR901459. Structural elucidation of the isolated bioconversion products (1-7) revealed that compounds 1-4 were mono-hydroxylated at the position of 1-MeBmt or 9-MeLeu, whereas compounds 5-7 were bis-hydroxylated at both positions. The results of morphological and chemical characterization, as well as phylogenetic analysis of 16S ribosomal DNA (rDNA), suggested that strain 7887 belonged to the genus Lentzea. Comparison of the FR901459 conversion activity of strain 7887 with several other Lentzea strains revealed that although all examined strains metabolized FR901459, strain 7887 had a characteristic profile with respect to bioconversion products. Taken together, these findings suggest that strain 7887 can be used to derivative FR901459 to produce a chemical template for further chemical modifications that may provide more effective and safer anti-HCV drugs.

[A case of primary hepatic actinomycosis].

Primary hepatic actinomycosis is relatively rare, but it should be remembered in the differential diagnosis of liver masses. A 66-year-old woman with right hypochondralgia was admitted and for detailed examination and treatment of a liver tumor. Abdominal ultrasonography revealed a hypoechoic lesion 55 mm in diameter in the anterior segment. Enhanced CT showed a deep-stained tumor in the early phase and a low density area in the late phase. The feeding arteries were the right hepatic artery and right inferior phrenic artery on abdominal angiography. The patient was given a diagnosis of hepatocellular carcinoma with invasion of the inferior lobe of the right lung. We performed a central bisegmentectomy of the liver and partial resection of the inferior lobe of the right lung. Microscopic findings of the resected specimen revealed sulfur granules and the tumor was diagnosed as primary hepatic actinomycosis.

Role of a cysteine residue in the active site of ERK and the MAPKK family.

Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGFbeta-induced AP-1-dependent luciferase expression with respective IC50 values of 0.08 and 0.05 microM. FR265083 (1'-2' dihydro form) and FR263574 (1'-2' and 7'-8' tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the alpha,beta-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding site of ERK2, involving a covalent bond to Sgamma of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, Nzeta of Lys114, backbone C=O of Ser153, Ndelta2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPKalpha/beta/gamma/delta which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades.

The novel gluconeogenesis inhibitor FR225654 that originates from Phoma sp. no. 00144. I. Taxonomy, fermentation, isolation and physico-chemical properties.

FR225654, a novel gluconeogenesis inhibitor, was isolated from the culture broth of Phoma sp. No. 00144 and purified by adsorptive resin and reverse-phase column chromatography. This compound is a potent inhibitor of gluconeogenesis and is a promising candidate of anti-diabetic agent.

The novel gluconeogenesis inhibitor FR225654 that originates from Phoma sp. no. 00144. III. Structure determination.

A novel gluconeogenesis inhibitor, FR225654 was isolated from the culture broth of Phoma sp. No. 00144. Spectroscopic analysis concluded that FR225654 has a highly oxygenated trans-decalin ring and beta-keto-enol in its main part, and has a characteristic side chain possessing a conjugated carboxylic acid and tri-substituted olefin.

FR207944, an antifungal antibiotic from Chaetomium sp. No. 217 II. Isolation and structure elucidation.

We discovered FR207944 produced by Chaetomium sp. No. 217 in the course of screening for antifungal antibiotics from natural products. FR207944 is identical with fuscoatroside, described in the preceding paper as an anti-Aspergillus flavus agent. Determination of the relative stereochemistry of fuscoatroside was made formally by comparison with WF11605 (16-Oxo-FR207944). We confirmed the stereochemistry on the basis of single crystal X-ray analysis.

FR177391, a new anti-hyperlipidemic agent from Serratia. I. Taxonomy, fermentation, isolation, physico-chemical properties, structure elucidation and biological activities.

In the course of screening for a new anti-hyperlipidemic agent from microbial products, we found that FR177391, produced by Serratia liquefaciens No. 1821, alleviated the decrease in lipid droplet formation in differentiated 3T3-L1 adipocyte cells, induced by the addition of tumor necrosis factor-alpha. Structural elucidation by spectroscopic methods and X-ray crystallographic analysis of its propylamide derivative revealed that FR177391 was a chlorinated macrocyclic lactone.

FR177391, a new anti-hyperlipidemic agent from Serratia. III. Microbial conversion of FR177391 and synthesis of FR177391 derivatives for its target protein screening by chemical genetic approaches.

FR177391 produced by Serratia liquefaciens No. 1821 enhances differentiation of mouse 3T3-L1 fibroblasts to adipocytes and reduces the circulating levels of triglyceride in C57BL/KsJ-db/bd mice, an obese non-insulin-dependent diabetes mellitus animal model, although its mechanism of actions remained to be unknown. Its active derivative, 20-hydroxy FR177391, and its inactive derivative, 3-hydroxy FR177391 were produced by microbial conversion of FR177391, and biotin-labeled FR177391 was synthesized from 20-hydroxy FR177391 as an active affinity ligand to identify target molecules of FR177391 by chemical genetic approaches.

FR182876, a new microtubule modulator with high water-solubility, from a Streptomyces.

In the course of seeking new anti-tumor drugs, a new microtubule modulator with high water-solubility, FR182876, was isolated from a Streptomyces which also produces FR182877. Even modern spectroscopic methods could not solve the structure of FR182876 due to its structural complexity and chemical instability. Thus, we have combined chemical correlations with spectroscopic methods and determined its structure, which features a highly fused ring system and 3-methylhistidine. The latter is believed to contribute to both solubility in water and activity in promoting tubulin polymerization. FR182876 showed potent cytotoxicity against a panel of cancer cells at concentrations of 28-75 ng/ml.