Pathogenesis of Isospora amphiboluri in Bearded Dragons (Pogona vitticeps).

Isospora amphiboluri is a common coccidian found in captive bearded dragons (Pogona vitticeps). To minimize the impact of this parasite, it is important to characterize its pathogenesis so that we can develop appropriate methods for diagnosis and treatment. Forty-five juvenile bearded dragons were used for this two-part study. In the first part, ten bearded dragons were infected with 20,000 oocysts per os, while a control group of five animals received only water. Feces were collected over 45 days and screened for oocysts. In the second part, thirty bearded dragons were used to characterize the pathogenesis of I. amphiboluri. Twenty-five bearded dragons were infected as described previously, while five animals served as controls. Five infected bearded dragons and one control were humanely euthanized on days 4, 8, 12, 16, and 20 post-infection for complete necropsies. The pre-patent period for I. amphiboluri was found to be 18.6 ± 1.9 days (range 15-22 days). Histopathology confirmed that I. amphiboluri follows a homoxenous life cycle. Infections begin in the duodenum and progress to the colon over time. The findings of this study can be used to develop better quarantine and treatment protocols for captive bearded dragons.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) loss causes neurodegeneration by altering protein turnover in the first postnatal weeks.

Ubiquitin C-terminal hydrolase L1 (UCH-L1) is one of the most abundant and enigmatic enzymes of the CNS. Based on existing UCH-L1 knockout models, UCH-L1 is thought to be required for the maintenance of axonal integrity, but not for neuronal development despite its high expression in neurons. Several lines of evidence suggest a role for UCH-L1 in mUB homeostasis, although the specific in vivo substrate remains elusive. Since the precise mechanisms underlying UCH-L1-deficient neurodegeneration remain unclear, we generated a transgenic mouse model of UCH-L1 deficiency. By performing biochemical and behavioral analyses we can show that UCH-L1 deficiency causes an acceleration of sensorimotor reflex development in the first postnatal week followed by a degeneration of motor function starting at periadolescence in the setting of normal cerebral mUB levels. In the first postnatal weeks, neuronal protein synthesis and proteasomal protein degradation are enhanced, with endoplasmic reticulum stress, and energy depletion, leading to proteasomal impairment and an accumulation of nondegraded ubiquitinated protein. Increased protein turnover is associated with enhanced mTORC1 activity restricted to the postnatal period in UCH-L1-deficient brains. Inhibition of mTORC1 with rapamycin decreases protein synthesis and ubiquitin accumulation in UCH-L1-deficient neurons. Strikingly, rapamycin treatment in the first 8 postnatal days ameliorates the neurological phenotype of UCH-L1-deficient mice up to 16 weeks, suggesting that early control of protein homeostasis is imperative for long-term neuronal survival. In summary, we identified a critical presymptomatic period during which UCH-L1-dependent enhanced protein synthesis results in neuronal strain and progressive loss of neuronal function.

Multiphase computed tomography for localization of parathyroid disease in patients with primary hyperparathyroidism: How many phases do we really need?

BACKGROUND: Multiphase computed tomography (CT) involves multiple cervical CT acquisitions to accurately identify hyperfunctional parathyroid glands, thus increasing radiation exposure to the patient. We hypothesized that only 2 cervical acquisitions, instead of the conventional 4, would provide equivalent localization information and halve the radiation exposure. METHODS: We identified 53 consecutive patients with primary hyperparathyroidism who underwent multiphase CT before parathyroidectomy. All scans were reinterpreted first using 2 phases then using all 4 phases. The accuracies of interpretations were determined with surgical findings serving as the standard of reference. RESULTS: Sixty-four hyperfunctional parathyroid glands were resected with a mean weight of 394.3 mg. Two-phase CT lateralized the hyperfunctional glands in 38 patients with a sensitivity, positive predictive value (PPV), and accuracy of 100%, 71.7%, and 71.7%, respectively. Four-phase CT lateralized the hyperfunctional glands in 39 patients with a sensitivity, PPV, and accuracy of 95.1%, 76.5%, and 73.6%, respectively. For quadrant localization, the accuracy of 2-phase and 4-phase CT was 50.9% and 52.8%, respectively. CONCLUSION: Our results suggest that 2-phase and 4-phase CT provide an equivalent diagnostic accuracy in localizing hyperfunctional parathyroid glands. The reduced radiation exposure to the patient may make 2-phase acquisitions a more acceptable alternative for preoperative localization.

Chrysosporium anamorph Nannizziopsis vriesii: an emerging fungal pathogen of captive and wild reptiles.

Chrysosporium anamorph Nannizziopsis vriesii is a recent pathogen associated with infections in lizards, snakes, and crocodilians. It seems to be an obligate pathogen. It has been isolated from wild reptiles in addition to captive animals. Affected animals often present with aggressive, pyogranulomatous lesions that can affect the integument and musculoskeletal systems. Diagnosis can be done using culture, histopathology, and polymerase chain reaction assay. Ancillary diagnostic tests can be useful in characterizing the health status of the affected reptile and aid in planning supportive care and therapy. Treatment using antifungals has shown mixed results.

Cervical ribs: identification on MRI and clinical relevance.

To determine the prevalence of cervical ribs on cervical spine MRI and clinical relevance, we reviewed 2500 studies for cervical ribs and compression of neurovascular structures and compared to CT, when available. Brachial plexus or subclavian artery contact by cervical rib was identified on MRI and/or CT in 12 cases with diagnosis of thoracic outlet syndrome in one. Cervical ribs were identified on 1.2% (25/2083) of examinations, lower than on CT (2%), but MRI may offer equivalent anatomic explanation for patient symptoms.

Head and neck cancer.

Cross sectional imaging fills a crucial role in the work up of squamous cell cancer of the head and neck. The radiologist can suggest important considerations in treatment planning and disease prognosis. Key areas of anatomy in radiologic staging are reviewed.

Molecular clustering identifies complement and endothelin induction as early events in a mouse model of glaucoma.

Glaucoma is one of the most common neurodegenerative diseases. Despite this, the earliest stages of this complex disease are still unclear. This study was specifically designed to identify early stages of glaucoma in DBA/2J mice. To do this, we used genome-wide expression profiling of optic nerve head and retina and a series of computational methods. Eyes with no detectable glaucoma by conventional assays were grouped into molecularly defined stages of disease using unbiased hierarchical clustering. These stages represent a temporally ordered sequence of glaucoma states. We then determined networks and biological processes that were altered at these early stages. Early-stage expression changes included upregulation of both the complement cascade and the endothelin system, and so we tested the therapeutic value of separately inhibiting them. Mice with a mutation in complement component 1a (C1qa) were protected from glaucoma. Similarly, inhibition of the endothelin system with bosentan, an endothelin receptor antagonist, was strongly protective against glaucomatous damage. Since endothelin 2 is potently vasoconstrictive and was produced by microglia/macrophages, our data provide what we believe to be a novel link between these cell types and vascular dysfunction in glaucoma. Targeting early molecular events, such as complement and endothelin induction, may provide effective new treatments for human glaucoma.

Evaluating 21-day doxycycline and azithromycin treatments for experimental Chlamydophila psittaci infection in cockatiels (Nymphicus hollandicus).

To determine the efficacy of 21-day therapy with azithromycin and doxycycline in the treatment of experimental infection with Chlamydophila psittaci in cockatiels (Nymphicus hollandicus), 30 birds randomly assigned to 3 treatment groups and 1 control group were inoculated with C psittaci by combined intranasal and ocular routes. Morbidity, mortality, and results of polymerase chain reaction testing confirmed that infection was successful. Birds in group 1 (n = 8) received azithromycin at 40 mg/kg PO q48h for 21 days; in group 2 (n = 8), doxycycline at 35 mg/kg PO q24h for 21 days; in group 3 (n = 8), doxycycline at 35 mg/kg PO q24h for 45 days; and, in group 4 (controls; n = 6), no treatment. Six birds died either before or within 2 days of initiating treatment: 4 in the 3 treatment groups and 2 in the control group. Clinical signs resolved and mortality ceased 2-6 days after treatment was initiated in all treatment groups, whereas birds in the control group exhibited clinical signs for the duration of the study. Plasma doxycycline concentrations were measured during the treatment period and exceeded 1 microg/mL at all time points. The absence of clinical signs and mortality in the treatment groups, even after inducing an immunocompromised state with dexamethasone (3 mg/kg IM q24h for 5 days), starting on day 70 postinoculation, suggested that treatment resulted in elimination of the pathogen. After euthanasia of the remaining 24 birds, 23 of the carcasses were submitted for necropsy. Spleen and liver samples from the birds in all treatment and control groups were polymerase chain reaction negative for C psittaci nucleic acid, and organisms were not detected by Gimenez stain. No gross or histologic differences were observed in the livers and spleens of treated and untreated infected birds. Lesions consistent with avian chlamydiosis (hystiocytosis) were seen in all birds and were considered residual. In this study, a 21-day course of either doxycycline or azithromycin was effective in eliminating C psittaci infection in experimentally inoculated cockatiels. Additional studies are necessary to evaluate the efficacy of these treatments in naturally infected cockatiels as well as other species of birds.

Incidental enchondromas of the knee.

OBJECTIVE: The purpose of our study was to determine the prevalence of incidental enchondromas on routine MR knee imaging. MATERIALS AND METHODS: We retrospectively reviewed 449 consecutive routine knee MR examinations for the presence of enchondromas. MRI was considered positive when a focal geographic area of lobular marrow replacement (nonsubchondral) was identified on T1 weighting and high signal intensity was seen on T2 weighting. Patients with enchondromas were further evaluated for demographics; lesion site, size, and relationship to the physeal plate; aggressive imaging features described with chondrosarcoma; concurrent internal derangement; and study indication. RESULTS: The prevalence of incidental enchondromas was 2.9% on routine knee MR examinations. The prevalence was highest in the distal femur (2.0%), followed by the proximal tibia (0.7%) and the proximal fibula (0.2%). The average lesion size was 1.9 x 1.2 x 1.3 cm (57% of lesions were < 1 cm). Most lesions were located in the metaphysis (71%) or diaphysis (21%). Enchondromas were within 1.5 cm of the physeal plate in 72% of cases. No aggressive imaging features to suggest chondrosarcoma were seen. All patients had evidence of internal derangement as the cause of symptoms and the request for imaging. CONCLUSION: Incidental enchondromas can be identified on 2.9% of routine MR knee examinations, most frequently in the distal femur (2.0%). This significant prevalence is much higher than in an autopsy series (0.2%), likely reflecting the increased sensitivity of MRI for detecting small lesions, and is important to recognize to avoid confusion with other pathologic entities.

Tracheal resection and anastomosis in a mallard duck (Anas platyrhynchos) with traumatic segmental tracheal collapse.

A male mallard duck (Anas platyrhynchos) presented for examination for acute respiratory distress and lethargy. The duck had experienced recurrent episodes of respiratory distress since being attacked by a raccoon the previous year, resulting in neck lacerations. Diagnostic tests, including a complete blood count, plasma biochemical analysis, radiography, and tracheoscopy, revealed a collapsed trachea. Surgical correction of the collapsed tracheal segment resulted in resection of 9% of the total tracheal length and subsequent anastomosis. Tracheoscopy performed 2 and 3 months after surgery revealed a healthy mucosa, minimal reduction of the tracheal lumen in the area of anastomosis, and minimal suture granuloma formation.

Uncoupling and turnover in a Cl-/H+ exchange transporter.

The CLC-family protein CLC-ec1, a bacterial homologue of known structure, stoichiometrically exchanges two Cl(-) for one H(+) via an unknown membrane transport mechanism. This study examines mutations at a conserved tyrosine residue, Y445, that directly coordinates a Cl(-) ion located near the center of the membrane. Mutations at this position lead to "uncoupling," such that the H(+)/Cl(-) transport ratio decreases roughly with the volume of the substituted side chain. The uncoupled proteins are still able to pump protons uphill when driven by a Cl(-) gradient, but the extent and rate of this H(+) pumping is weaker in the more uncoupled variants. Uncoupling is accompanied by conductive Cl(-) transport that is not linked to counter-movement of H(+), i.e., a "leak." The unitary Cl(-) transport rate, measured in reconstituted liposomes by both a conventional initial-velocity method and a novel Poisson dilution approach, is approximately 4,000 s(-1) for wild-type protein, and the uncoupled mutants transport Cl(-) at similar rates.

Biomarker discovery by CE-MS enables sequence analysis via MS/MS with platform-independent separation.

CE-MS is a successful proteomic platform for the definition of biomarkers in different body fluids. Besides the biomarker defining experimental parameters, CE migration time and molecular weight, especially biomarker's sequence identity is an indispensable cornerstone for deeper insights into the pathophysiological pathways of diseases or for made-to-measure therapeutic drug design. Therefore, this report presents a detailed discussion of different peptide sequencing platforms consisting of high performance separation method either coupled on-line or off-line to different MS/MS devices, such as MALDI-TOF-TOF, ESI-IT, ESI-QTOF and Fourier transform ion cyclotron resonance, for sequencing indicative peptides. This comparison demonstrates the unique feature of CE-MS technology to serve as a reliable basis for the assignment of peptide sequence data obtained using different separation MS/MS methods to the biomarker defining parameters, CE migration time and molecular weight. Discovery of potential biomarkers by CE-MS enables sequence analysis via MS/MS with platform-independent sample separation. This is due to the fact that the number of basic and neutral polar amino acids of biomarkers sequences distinctly correlates with their CE-MS migration time/molecular weight coordinates. This uniqueness facilitates the independent entry of different sequencing platforms for peptide sequencing of CE-MS-defined biomarkers from highly complex mixtures.

Predicting the clinical outcome of congenital unilateral ureteropelvic junction obstruction in newborn by urinary proteome analysis.

We analyzed urinary polypeptides from individuals with neonatal ureteropelvic junction (UPJ) obstruction to predict which individuals with this condition will evolve toward obstruction that needs surgical correction. We identified polypeptides that enabled diagnosis of the severity of obstruction and validated these biomarkers in urine collected in a prospective blinded study. Using these noninvasive biomarkers, we were able to predict, several months in advance and with 94% precision, the clinical evolution of neonates with UPJ obstruction.

Discovery and validation of new protein biomarkers for urothelial cancer: a prospective analysis.

BACKGROUND: Non-invasive methods for diagnosis of urothelial carcinoma have reduced specificity in patients with non-malignant genitourinary disease or other disorders. We aimed to use mass spectrometry and bioinformatics to define and validate a cancer-specific proteomic pattern. METHODS: We used capillary-electrophoresis-coupled mass spectrometry to obtain polypeptide patterns from urine samples of 46 patients with urothelial carcinoma and 33 healthy volunteers. From signatures of polypeptide mass, we established a model for predicting the presence of cancer. The model was refined further by use of 366 urine samples obtained from other healthy volunteers and patients with malignant and non-malignant genitourinary disease. We estimated the proportion of correct classifications from the refined model by applying it to a masked group containing 31 patients with urothelial carcinoma, 11 healthy individuals, and 138 patients with non-malignant genitourinary disease. We also sequenced several diagnostic polypeptides for urothelial carcinoma. FINDINGS: We identified a diagnostic urothelial-carcinoma pattern of 22 polypeptide masses. On masked assessment, prediction models based on these polypeptides correctly classified all samples of urothelial carcinoma (sensitivity 100% [95% CI 87-100) and all healthy samples (specificity 100% [84-100]). Correct identification of patients with urothelial carcinoma from those with other malignant and non-malignant genitourinary disease ranged from 86% to 100%. A prominent polypeptide from the diagnostic pattern for urothelial carcinoma was identified as fibrinopeptide A-a known biomarker of ovarian cancer and gastric cancer. INTERPRETATION: Validation of a highly specific biomarker pattern for urothelial carcinoma in a large group of patients with various urological disorders could be used in the diagnosis of other diseases that are identified in urine samples or in other body fluids.

Effects of oral vitamin C supplementation in hemodialysis patients: a proteomic assessment.

Evidence indicates that oxidative stress is present in dialysis patients, and is associated with vitamin C deficiency. Limited data are available regarding the effects of vitamin C supplementation on oxidative stress and inflammation markers in these patients. Moreover, there are no data available on plasma polypeptide fingerprints by proteome analysis before and after vitamin C supplementation. Therefore, we analyzed plasma samples from a prospective, randomized, open-labeled trial to assess the effects of oral vitamin C supplementation (250 mg three times per week), to define the plasma polypeptide pattern in hemodialysis patients. Our results reveal that more than 30 polypeptides show significant changes in the dialysis patients in comparison to controls with normal renal function, and that several polypeptides are affected/normalized by oral vitamin C supplementation. These results underline the remarkable potential for proteomics to recognize specific peptide profiles in different pathological situations, which might not be detected by classical methods.

Separate ion pathways in a Cl-/H+ exchanger.

CLC-ec1 is a prokaryotic CLC-type Cl(-)/H+ exchange transporter. Little is known about the mechanism of H+ coupling to Cl-. A critical glutamate residue, E148, was previously shown to be required for Cl(-)/H+ exchange by mediating proton transfer between the protein and the extracellular solution. To test whether an analogous H+ acceptor exists near the intracellular side of the protein, we performed a mutagenesis scan of inward-facing carboxyl-bearing residues and identified E203 as the unique residue whose neutralization abolishes H+ coupling to Cl- transport. Glutamate at this position is strictly conserved in all known CLCs of the transporter subclass, while valine is always found here in CLC channels. The x-ray crystal structure of the E203Q mutant is similar to that of the wild-type protein. Cl- transport rate in E203Q is inhibited at neutral pH, and the double mutant, E148A/E203Q, shows maximal Cl- transport, independent of pH, as does the single mutant E148A. The results argue that substrate exchange by CLC-ec1 involves two separate but partially overlapping permeation pathways, one for Cl- and one for H+. These pathways are congruent from the protein's extracellular surface to E148, and they diverge beyond this point toward the intracellular side. This picture demands a transport mechanism fundamentally different from familiar alternating-access schemes.

Combined top-down and bottom-up mass spectrometric approach to characterization of biomarkers for renal disease.

Here we describe a mass spectrometry (MS) approach for biomarker discovery and structural characterization, based on both top-down and bottom-up analyses. Capillary electrophoresis (CE) coupled to electrospray ionization (ESI) time-of-flight (TOF) MS serves to separate and mass-measure the thousands of polypeptides contained in human urine. Statistical analysis of the differences between healthy control samples and patients with focal-segmental glomerulosclerosis, membranous glomerulonephritis, minimal change disease, IgA nephropathy, and diabetic nephropathy validates multiple biomarkers for the control and each of the diseases. To identify those biomarkers, we employ preparative CE, enabling direct infusion ESI MS analysis, followed by sample manipulation and reanalysis where necessary. We show how tandem Fourier transform ion cyclotron resonance (FT-ICR) MS identifies these sometimes large (>8 kDa) biomarkers. Critically, we maintain connectivity between the CE TOF MS data and the ICR data used for biomarker identification.

Detection of acute tubulointerstitial rejection by proteomic analysis of urinary samples in renal transplant recipients.

This study investigates proteomic analysis of urinary samples as a non-invasive method to detect acute rejection of renal allografts. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to analyze urinary samples in 19 patients with different grades of subclinical or clinical acute rejection (BANFF Ia to IIb), 10 patients with urinary tract infection and 29 patients without evidence of rejection or infection. A distinct urinary polypeptide pattern identified 16 out of 17 cases of acute tubolointerstitial rejection, but was absent in two cases of vascular rejection. Urinary tract infection resulted in a different polypeptide pattern that allowed to differentiate between infection and acute rejection in all cases. Potentially confounding variables such as acute tubular lesions, tubular atrophy, tubulointerstitial fibrosis, calcineurin inhibitor toxicity, proteinuria, hematuria, allograft function and different immunosuppressive regimens did not interfere with test results. Blinded analysis of samples with and without rejection showed correct diagnosis by CE-MS in the majority of cases. Detection of acute rejection by CE-MS offers a promising non-invasive tool for the surveillance of renal allograft recipients. Further investigation is needed to establish polypeptide patterns in vascular rejection and to explore whether changes in the urinary proteome occur before the onset of histologically discernible rejection.

Pilot study of capillary electrophoresis coupled to mass spectrometry as a tool to define potential prostate cancer biomarkers in urine.

We describe the use of capillary electrophoresis (CE) coupled with mass spectrometry (MS) to identify single polypeptides and patterns of polypeptides specific for prostate cancer (CaP) in human urine. Using improved sample preparation methods that enable enhanced comparability between different samples, we examined samples from 47 patients who underwent prostate biopsy. Of this group, 21 patients had benign pathology and 26 with CaP, and these were used to define potential biomarkers, which allow discrimination between these two states. In addition, CE-MS data from these 47 urine samples were compared to that of 41 young men (control) without known or suspected clinical CaP to further confirm the polypeptides indicative for CaP. Upon crossvalidation of the same samples, several polypeptides were selected that enabled correct classification of the CaP patients with 92% sensitivity and 96% specificity. We then examined an additional 474 samples from patients with renal disease enrolled in other studies and found that 14 (3%) had polypeptides suggestive of CaP possibly indicating that they harbor clinical CaP. In conclusion, this early pilot study suggests that CE-MS of urine warrants further investigation as a tool that can identify putative biomarkers for CaP.