Feasibility, indications, and radiographically confirmed diagnoses of standing extremity cone beam computed tomography in the horse.

OBJECTIVE: To report on the feasibility, indications, and diagnostic yield of cone beam computed tomography (CBCT) of horses' extremities performed under standing sedation. STUDY DESIGN: Retrospective clinical case series. SAMPLE POPULATION: Fifty-nine CBCT examinations in 58 horses. METHODS: Examinations were categorized for indications for CBCT dependent on a suspicion, presence, or absence of a diagnosis prior to CBCT. The number of acquisitions per examination, total time for the examination, diagnostic score of each acquisition (diagnostic, diagnostic-compromised, nondiagnostic), and additional diagnostic information regarding preexisting diagnostic information were recorded. RESULTS: Three (median) acquisitions were performed per examination in a median study time of 14 minutes. In 24 of 33 cases with a suspected diagnosis, this diagnosis was confirmed or definitively refuted; in seven of 33 cases, the suspected diagnosis was refuted without a new diagnosis; and, in two of 33 cases, the suspected diagnosis could not be confirmed nor could a new diagnosis be made. In five of nine cases without a preexisting diagnosis, a diagnosis was established. In 16 cases with a diagnosis prior to CBCT, additional information was recorded, or a surgical plan was prepared. In 14 of 18 cases in which additional contrast techniques were used, additional information was gained. CONCLUSION: Standing CBCT of the horses' extremities is feasible and can produce diagnostic information in a timely fashion. CLINICAL SIGNIFICANCE: The results provide evidence of the practicality and diagnostic potential of standing CBCT of horses' extremities.

Novel Potent Capsid Assembly Modulators Regulate Multiple Steps of the Hepatitis B Virus Life Cycle.

The assembly of hepatitis B virus (HBV) core protein (HBc) into capsids represents a critical step of viral replication. HBc has multiple functions during the HBV life cycle, which makes it an attractive target for antiviral therapies. Capsid assembly modulators (CAMs) induce the formation of empty capsid or aberrant capsid devoid of pregenomic RNA (pgRNA) and finally block relaxed circular DNA neosynthesis and virion progeny. In this study, the novel CAMs JNJ-827 and JNJ-890 were found to be potent inhibitors of HBV replication with respective half-maximal effective concentrations of 4.7 and 66 nM, respectively, in HepG2.117 cells. Antiviral profiling in differentiated HepaRG (dHepaRG) cells and primary human hepatocytes revealed that these compounds efficiently inhibited HBV replication, as well as de novo establishment of covalently closed circular DNA (cccDNA). In addition to these two known effects of CAMs, we observed for the first time that a CAM, here JNJ-827, when added postinfection for a short-term period, significantly reduced hepatitis B e antigen (HBeAg) secretion without affecting the levels of cccDNA amount, transcription, and hepatitis B surface antigen (HBsAg) secretion. This inhibitory activity resulted from a direct effect of JNJ-827 on HBeAg biogenesis. In a long-term treatment condition using persistently infected dHepaRG cells, JNJ-827 and JNJ-890 reduced HBsAg concomitantly with a decrease in viral total RNA and pgRNA levels. Altogether, these data demonstrate that some CAMs could interfere with multiple functions of HBc in the viral life cycle.

Characterization of a dengue NS4B inhibitor originating from an HCV small molecule library.

Dengue is the most important mosquito-transmitted viral disease and a major global health concern. Over the last decade, dengue virus (DENV) drug discovery and development has intensified, however, this has not resulted in approved DENV-specific antiviral treatments yet. DENV and hepatitis C virus (HCV) belong to the same Flaviviridae family and, in contrast to DENV, antiviral treatments for HCV have been licensed. Therefore, applying the knowledge gained on anti-HCV drugs may foster the discovery and development of dengue antiviral drugs. Here, we screened a library of compounds with established anti-HCV activity in a DENV-2 sub-genomic replicon inhibition assay and selected compounds with single-digit micromolar activity. These compounds were advanced into a hit-to-lead medicinal chemistry program resulting in lead compound JNJ-1A, which inhibited the DENV-2 sub-genomic replicon at 0.7 µM, in the absence of cytotoxicity. In addition, JNJ-1A showed equipotent antiviral activity against DENV serotypes 1, 2, and 4. In vitro resistance selection experiments with JNJ-1A induced mutation T108I in non-structural protein 4B (NS4B), pointing towards a mechanism of action linked to this protein. Collectively, we described the discovery and characterization of a novel DENV inhibitor potentially targeting NS4B.

Capsid Assembly Modulators Have a Dual Mechanism of Action in Primary Human Hepatocytes Infected with Hepatitis B Virus.

Hepatitis B virus (HBV) capsid assembly is a critical step in the propagation of the virus and is mediated by the core protein. Due to its multiple functions in the viral life cycle, core became an attractive target for new antiviral therapies. Capsid assembly modulators (CAMs) accelerate the kinetics of capsid assembly and prevent encapsidation of the polymerase-pregenomic RNA (Pol-pgRNA) complex, thereby blocking viral replication. CAM JNJ-632 is a novel and potent inhibitor of HBV replication in vitro across genotypes A to D. It induces the formation of morphologically intact viral capsids, as demonstrated by size exclusion chromatography and electron microscopy studies. Antiviral profiling in primary human hepatocytes revealed that CAMs prevented formation of covalently closed circular DNA in a dose-dependent fashion when the compound was added together with the viral inoculum, whereas nucleos(t)ide analogues (NAs) did not. This protective effect translated into a dose-dependent reduction of intracellular HBV RNA levels as well as reduced HBe/cAg and HBsAg levels in the cell culture supernatant. The same observation was made with another CAM (BAY41-4109), suggesting that mechanistic rather than compound-specific effects play a role. Our data show that CAMs have a dual mechanism of action, inhibiting early and late steps of the viral life cycle. These effects clearly differentiate CAMs from NAs and may translate into higher functional cure rates in a clinical setting when given alone or in combination with the current standard of care.

Clinicopathologic observations on laryngoplasty failure in a horse.

OBJECTIVE: To report morphologic findings associated with laryngoplasty failure in a horse. STUDY DESIGN: Clinical report. ANIMALS: A 9-year-old Thoroughbred cross gelding. METHODS: Necropsy and histopathology were performed on a horse that died peracutely during anesthetic recovery after correction of a right dorsal displacement of the ascending colon. Three weeks earlier the horse had left laryngoplasty and ventriculocordectomy. RESULTS: Dissection of the larynx revealed that the laryngoplasty suture had pulled through the muscular process of the left arytenoid cartilage, which appeared grossly normal. Histopathology of the arytenoid muscular process revealed cartilage necrosis, granulation tissue, and inflammation around the cartilage and within the cartilage failure line, and small numbers of coccoid bacteria in a minority of cartilage canals. Multifocal cardiomyopathy and pulmonary congestion, edema, and hemorrhage were also observed histologically. CONCLUSION: Death was attributed to peracute pulmonary edema associated with cardiac abnormalities and airway obstruction from laryngoplasty failure. Morphologic changes in the muscular process indicate gradual progression toward laryngoplasty failure, possibly associated with suture-induced pressure necrosis and/or microscopic low-grade postoperative infection.

Development of a high-content screening assay to identify compounds interfering with the formation of the hepatitis C virus replication complex.

The hepatitis C virus (HCV) replicates its genome on a membrane-associated replication complex. These complexes are represented by "dot-like" structures on the endoplasmic reticulum when standard fluorescence microscopy techniques are applied. To screen compound libraries for inhibitors interfering with the formation of the HCV replication complex independent of RNA replication, an image-based high-content screening assay was developed utilizing inducible expression of the HCV non-structural proteins NS3-5B in an U2-OS Tet-On cell line. An eGFP was fused to NS5A for the detection of replication complexes. The cell line was tightly regulated and the eGFP insertion within NS5A did not alter polyprotein processing. The NS5AeGFP signal colocalized with other non-structural proteins in "dot-like" structures. Accompanying image analysis tools were developed enabling the detection of changes in replication complex formation. Finally, the addition of a HCV NS3/4A protease inhibitor resulted in a dose-dependent reduction of "dot-like" structures demonstrating the practicability of the assay.

1,5-benzodiazepines, a novel class of hepatitis C virus polymerase nonnucleoside inhibitors.

The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here, we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies, 4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other viral and human polymerases, as well as BZD receptors.

Congenital retrosternal (Morgagni) diaphragmatic hernias in three horses.

CASE DESCRIPTION: 3 Horses were examined and treated because of sudden onset of signs of abdominal pain. CLINICAL FINDINGS: All horses had a retrosternal (Morgagni) hernia involving the right side of the diaphragm. In each horse, the large colon was incarcerated in a right muscular defect in the diaphragm with a large hernial sac. TREATMENT AND OUTCOME: Definitive surgical repair of the hernia was not performed during the initial celiotomy. The hernia was repaired with mesh herniorrhaphy, but without resection of the hernia sac in 2 horses. For 1 horse, conservative management was applied. In the 2 horses treated with surgical correction, no major postoperative complications developed, and all 3 horses have been free of signs of abdominal pain. CLINICAL RELEVANCE: Horses with retrosternal hernias involving the diaphragm can develop clinical signs of intermittent obstruction of the large colon and chronic colic. In horses, retrosternal diaphragmatic hernias appear to develop exclusively in the right ventral aspect of the diaphragm and could represent an embryologic defect of diaphragm formation. Affected horses can be successfully treated with mesh herniorrhaphy or, in some instances, with conservative management.

A new approach for perineural injection of the lateral palmar nerve in the horse.

OBJECTIVE: To evaluate the accuracy of a new technique for perineural injection of the lateral palmar nerve and to determine frequency of inadvertent injection into the carpal synovial sheath with this technique. STUDY DESIGN: Prospective experimental study. ANIMALS: Thirty equine cadaver forelimbs. METHODS: Each of 3 clinicians injected 0.5 mL of a 1% aqueous solution of new methylene blue as a marker at the medial aspect of the accessory carpal bone of 10 limbs. Immediately after each injection, the lateral palmar nerve was identified by dissection of and inspected for proximity of dye, and the carpal synovial sheath was inspected for the presence of dye. RESULTS: New methylene blue solution was observed to surround the nerve (29 limbs) or to lie within 2 mm of it (1 limb). Dye was not found in the carpal synovial sheath of any specimen. CONCLUSIONS: Using this technique, perineural injection of the lateral palmar nerve can be consistently achieved, and the carpal synovial sheath is unlikely to be penetrated by the needle during the procedure. CLINICAL RELEVANCE: The technique described provides an accurate and simple method for perineural injection of the lateral palmar nerve proximal to the origin of its deep branch. This technique can be used to anesthetize the lateral palmar nerve for diagnosis of pain originating in the palmaroproximal aspect of the metacarpus without risk of inadvertently desensitizing structures within the carpal synovial sheath.

Glutathione and catalase provide overlapping defenses for protection against respiration-generated hydrogen peroxide in Haemophilus influenzae.

Glutathione is an abundant and ubiquitous low-molecular-weight thiol that may play a role in many cellular processes, including protection against the deleterious effects of reactive oxygen species. We address here the role of glutathione in protection against hydrogen peroxide (H2O2) in Haemophilus influenzae and show that glutathione and catalase provide overlapping defense systems. H. influenzae is naturally glutathione deficient and imports glutathione from the growth medium. Mutant H. influenzae lacking catalase and cultured in glutathione-deficient minimal medium is completely devoid of H2O2 scavenging activity and, accordingly, substantial amounts of H2O2 accumulate in the growth medium. H. influenzae generates H2O2 at rates similar to those reported for Escherichia coli, but the toxicity of this harmful metabolite is averted by glutathione-based H2O2 removal, which appears to be the primary system for protection against H2O2 endogenously generated during aerobic respiration. When H2O2 concentrations exceed low micromolar levels, the hktE gene-encoded catalase becomes the predominant scavenger. The requirement for glutathione in protection against oxidative stress is analogous to that in higher and lower eukaryotes but is unlike the situation in other bacteria in which glutathione is dispensable for aerobic growth during both normal and oxidative stress conditions.

Purification and characterization of a chimeric enzyme from Haemophilus influenzae Rd that exhibits glutathione-dependent peroxidase activity.

While belonging to the same family of antioxidant enzymes, members of the peroxiredoxins do not necessarily employ one and the same method for their reduction. Most representatives become reduced with the aid of thioredoxin, whereas some members use AhpF, tryparedoxin, or cyclophilin A. Recent research on a new peroxiredoxin isoform (type C) from Populus trichocarpa has shown that these particular types may also use glutaredoxin instead of thioredoxin. This finding is supported by the occurrence of chimeric proteins composed of a peroxiredoxin and glutaredoxin region. A gene encoding such a fusion protein is enclosed in the Haemophilus influenzae Rd genome. We expressed the H. influenzae protein, denoted here as PGdx, in Escherichia coli and purified the recombinant enzyme. In vitro assays demonstrate that PGdx, in the presence of dithiothreitol or glutathione, is able to protect supercoiled DNA against the metal ion-catalyzed oxidation-system. Enzymatic assays did, indeed, characterize PGdx as a peroxidase, requiring the glutathione redox cycle for the reduction of hydrogen peroxide (k(cat)/K(m) 5.01 x 10(6) s(-1) m(-1)) as well as the small organic hydroperoxide tert-butylhydroperoxide (k(cat)/K(m) 5.67 x 10(4) s(-1) m(-1)). Enzymatic activity as function of the glutathione concentration deviated from normal Michaelis-Menten kinetics, giving a sigmoidal pattern with an apparent Hill coefficient of 2.9. Besides the formation of a disulfide-linked PGdx dimer, it was also shown by mass spectrometric analysis that cysteine 49, which is equivalent to the active site cysteine of the peroxiredoxins, undergoes glutathionylation during purification under nonreducing conditions. Based on these results, we propose a model for the catalytic mechanism.

Exogenous glutathione completes the defense against oxidative stress in Haemophilus influenzae.

Since they are equipped with several strategies by which they evade the antimicrobial defense of host macrophages, it is surprising that members of the genus Haemophilus appear to be deficient in common antioxidant systems that are well established to protect prokaryotes against oxidative stress. Among others, no genetic evidence for glutathione (gamma-Glu-Cys-Gly) (GSH) biosynthesis or for alkyl hydroperoxide reduction (e.g., the Ahp system characteristic or enteric bacteria) is apparent from the Haemophilus influenzae Rd genome sequence, suggesting that the organism relies on alternative systems to maintain redox homeostasis or to reduce small alkyl hydroperoxides. In this report we address this apparent paradox for the nontypeable H. influenzae type strain NCTC 8143. Instead of biosynthesis, we could show that this strain acquires GSH by importing the thiol tripeptide from the growth medium. Although such GSH accumulation had no effect on growth rates, the presence of cellular GSH protected against methylglyoxal, tert-butyl hydroperoxide (t-BuOOH), and S-nitrosoglutathione toxicity and regulated the activity of certain antioxidant enzymes. H. influenzae NCTC 8143 extracts were shown to contain GSH-dependent peroxidase activity with t-BuOOH as the peroxide substrate. The GSH-mediated protection against t-BuOOH stress is most probably catalyzed by the product of open reading frame HI0572 (Prx/Grx), which we isolated from a genomic DNA fragment that confers wild-type resistance to t-BuOOH toxicity in the Ahp-negative Escherichia coli strain TA4315 and that introduces GSH-dependent alkyl hydroperoxide reductase activity into naturally GSH peroxidase-negative E. coli. Finally, we demonstrated that cysteine is an essential amino acid for growth and that cystine, GSH, glutathione amide, and cysteinylglycine can be catabolized in order to complement cysteine deficiency.