Extraneuronal pathology in a canine model of CLN2 neuronal ceroid lipofuscinosis after intracerebroventricular gene therapy that delays neurological disease progression.

CLN2 neuronal ceroid lipofuscinosis is a hereditary lysosomal storage disease with primarily neurological signs that results from mutations in TPP1, which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Studies using a canine model for this disorder demonstrated that delivery of TPP1 enzyme to the cerebrospinal fluid (CSF) by intracerebroventricular administration of an AAV-TPP1 vector resulted in substantial delays in the onset and progression of neurological signs and prolongation of life span. We hypothesized that the treatment may not deliver therapeutic levels of this protein to tissues outside the central nervous system that also require TPP1 for normal lysosomal function. To test this hypothesis, dogs treated with CSF administration of AAV-TPP1 were evaluated for the development of non-neuronal pathology. Affected treated dogs exhibited progressive cardiac pathology reflected by elevated plasma cardiac troponin-1, impaired cardiac function and development of histopathological myocardial lesions. Progressive increases in the plasma activity levels of alanine aminotransferase and creatine kinase indicated development of pathology in the liver and muscles. The treatment also did not prevent disease-related accumulation of lysosomal storage bodies in the heart or liver. These studies indicate that optimal treatment outcomes for CLN2 disease may require delivery of TPP1 systemically as well as directly to the central nervous system.

Microbial Phosphite Oxidation and Its Potential Role in the Global Phosphorus and Carbon Cycles.

Phosphite [Formula: see text] is a highly soluble, reduced phosphorus compound that is often overlooked in biogeochemical analyses. Although the oxidation of phosphite to phosphate is a highly exergonic process (Eo'=-650mV), phosphite is kinetically stable and can account for 10-30% of the total dissolved P in various environments. There is also evidence that phosphite was more prevalent under the reducing conditions of the Archean period and may have been involved in the development of early life. Its role as a phosphorus source for a variety of extant microorganisms has been known since the 1950s, and the pathways involved in assimilatory phosphite oxidation have been well characterized. More recently, it was demonstrated that phosphite could also act as an electron donor for energy metabolism in a process known as dissimilatory phosphite oxidation (DPO). The bacterium described in this study, Desulfotignum phosphitoxidans strain FiPS-3, was isolated from brackish sediments and is capable of growing by coupling phosphite oxidation to the reduction of either sulfate or carbon dioxide. FiPS-3 remains the only isolated organism capable of DPO, and the prevalence of this metabolism in the environment is still unclear. Nonetheless, given the widespread presence of phosphite in the environment and the thermodynamic favorability of its oxidation, microbial phosphite oxidation may play an important and hitherto unrecognized role in the global phosphorus and carbon cycles.

Pharmacokinetics and dynamics of mycophenolate mofetil after single-dose oral administration in juvenile dachshunds.

Mycophenolate mofetil (MMF) is recommended as an alternative/complementary immunosuppressant. Pharmacokinetic and dynamic effects of MMF are unknown in young-aged dogs. We investigated the pharmacokinetics and pharmacodynamics of single oral dose MMF metabolite, mycophenolic acid (MPA), in healthy juvenile dogs purpose-bred for the tripeptidyl peptidase 1 gene (TPP1) mutation. The dogs were heterozygous for the mutation (nonaffected carriers). Six dogs received 13 mg/kg oral MMF and two placebo. Pharmacokinetic parameters derived from plasma MPA were evaluated. Whole-blood mitogen-stimulated T-cell proliferation was determined using a flow cytometric assay. Plasma MPA Cmax (mean ± SD, 9.33 ± 7.04 µg/ml) occurred at <1 hr. The AUC0-∞ (mean ± SD, 12.84±6.62 hr*µg/ml), MRTinf (mean ± SD, 11.09 ± 9.63 min), T1/2 (harmonic mean ± PseudoSD 5.50 ± 3.80 min), and k/d (mean ± SD, 0.002 ± 0.001 1/min). Significant differences could not be detected between % inhibition of proliferating CD5+ T lymphocytes at any time point (p = .380). No relationship was observed between MPA concentration and % inhibition of proliferating CD5+ T lymphocytes (R = .148, p = .324). Pharmacodynamics do not support the use of MMF in juvenile dogs at the administered dose based on existing therapeutic targets.

Fecal Sterol and Runoff Analysis for Nonpoint Source Tracking.

Fecal pollution source identification is needed to quantify risk, target installation of source controls, and assess performance of best management practices in impaired surface waters. Sterol analysis is a chemical method for fecal source tracking that allows for differentiation between several fecal pollution sources. The objectives of this study were to use these chemical tracers for quantifying human fecal inputs in a mixed-land-use watershed without point sources of pollution and to determine the relationship between land use and sterol ratios. Fecal sterol analysis was performed on bed and suspended sediment from impaired streams. Human fecal signatures were found at sites with sewer overflow and septic inputs. Different sterol ratios used to indicate human fecal pollution varied in their sensitivity. Next, geospatial data was used to determine the runoff volumes associated with each land-use category in the watersheds. Fecal sterol ratios were compared between sampling locations and correlations were tested between ratio values and percentage of runoff for a given land-use category. Correlation was not observed between percentage of runoff from developed land and any of the five tested human-indicating sterol ratios in streambed sediments, confirming that human fecal inputs were not evenly distributed across the urban landscape. Several practical considerations for adopting this chemical method for microbial source tracking in small watersheds are discussed. Results indicate that sterol analysis is useful for identifying the location of human fecal nonpoint-source inputs.

Heterologous expression and purification of a multiheme cytochrome from a Gram-positive bacterium capable of performing extracellular respiration.

Microbial electrochemical technologies are emerging as environmentally friendly biotechnological processes. Recently, a thermophilic Gram-positive bacterium capable of electricity production in a microbial fuel cell was isolated. Thermincola potens JR contains several multiheme c-type cytochromes that were implicated in the process of electricity production. In order to understand the molecular basis by which Gram-positive bacteria perform extracellular electron transfer, the relevant proteins need to be characterized in detail. Towards this end, a chimeric gene containing the signal peptide from Shewanella oneidensis MR-1 small tetraheme cytochrome c (STC) and the gene sequence of the target protein TherJR_0333 was constructed. This manuscript reports the successful expression of this chimeric gene in the Gram-negative bacterium Escherichia coli and its subsequent purification and characterization. This methodology opens the possibility to study other multiheme cytochromes from Gram-positive bacteria, allowing the extracellular electron transfer mechanisms of this class of organisms to be unraveled.

Diagnostic immunohistochemistry of canine and feline intracalvarial tumors in the age of brain biopsies.

The focus of immunohistochemistry as applied to nervous system tumors is in identifying the neoplasm present and evaluating margins between normal and neoplastic tissue. Although not always utilized by specialists in neuropathology, immunohistochemistry remains useful to resolve concerns about the differentiation and rate of tumor growth. The aims of this review are to discuss the utility of immunohistochemical reagents currently used in diagnosis of canine and feline intracalvarial tumors, to indicate the applicability of some tests currently used in human nervous system tumors for domestic species, and to evaluate a few less commonly used reagents. A panel of biomarkers is usually needed to confirm a diagnosis, with groups of reagents for leptomeningeal, intraparenchymal, and ventricular neoplasms. In the future, signature genetic alterations found among feline and canine brain tumors--as correlated prospectively with diagnosis, rate of enlargement, or response to treatment--may result in new immunohistochemical reagents to simplify the task of diagnosis. Prospective studies determining the type and proportion of stem cell marker expression on patient longevity are likely to be fruitful and suggest new therapies. Due to increased frequency of biopsy or partial resection of tumors from the living patient, biomarkers are needed to serve as accurate prognostic indicators and assist in determining the efficacy of developing therapeutic options in nervous system tumors of dogs and cats.

The effects of conformation and intermolecular hydrogen bonding on the structure and IR spectra of flutamide; a study based on the matrix isolation technique, ab initio and DFT calculations.

In this study, stable conformers of flutamide referred to as an anticancer drug were searched through a relaxed potential energy surface scan carried out at the B3LYP/6-31G(d) level of theory. This was followed by geometry optimization and thermochemistry calculations performed with the HF-SCF, MP2, B3LYP methods and the 6-31G(d), 6-311++G(d,p), aug-cc-pvTZ basis sets for each of the determined minimum energy conformers. The results revealed that flutamide has at least five stable conformers and two of them provide the major contribution to the observed matrix isolation infrared (IR) spectra of the molecule. The effects of conformational variety and intermolecular hydrogen bonding interactions on the observed IR spectra of flutamide were interpreted in the light of the vibrational spectral data obtained for the most stable monomer and dimer forms of the molecule at the same levels of theory. Pulay's "Scaled Quantum Mechanical-Force Field (SQM-FF)" method was used in the refinement of the calculated harmonic wavenumbers, IR intensities and potential energy distributions. This scaling method which proved its superiority to both anharmonic frequency calculations and other scaling methods helped us to correctly interpret the remarkable differences between the matrix IR spectra of flutamide in argon and the condensed phase IR spectra of the molecule in solvents such as KBr, H2O, D2O, ethanol and methanol.

Evidence for direct electron transfer by a gram-positive bacterium isolated from a microbial fuel cell.

Despite their importance in iron redox cycles and bioenergy production, the underlying physiological, genetic, and biochemical mechanisms of extracellular electron transfer by Gram-positive bacteria remain insufficiently understood. In this work, we investigated respiration by Thermincola potens strain JR, a Gram-positive isolate obtained from the anode surface of a microbial fuel cell, using insoluble electron acceptors. We found no evidence that soluble redox-active components were secreted into the surrounding medium on the basis of physiological experiments and cyclic voltammetry measurements. Confocal microscopy revealed highly stratified biofilms in which cells contacting the electrode surface were disproportionately viable relative to the rest of the biofilm. Furthermore, there was no correlation between biofilm thickness and power production, suggesting that cells in contact with the electrode were primarily responsible for current generation. These data, along with cryo-electron microscopy experiments, support contact-dependent electron transfer by T. potens strain JR from the cell membrane across the 37-nm cell envelope to the cell surface. Furthermore, we present physiological and genomic evidence that c-type cytochromes play a role in charge transfer across the Gram-positive bacterial cell envelope during metal reduction.

Endogenous steroids and financial risk taking on a London trading floor.

Little is known about the role of the endocrine system in financial risk taking. Here, we report the findings of a study in which we sampled, under real working conditions, endogenous steroids from a group of male traders in the City of London. We found that a trader's morning testosterone level predicts his day's profitability. We also found that a trader's cortisol rises with both the variance of his trading results and the volatility of the market. Our results suggest that higher testosterone may contribute to economic return, whereas cortisol is increased by risk. Our results point to a further possibility: testosterone and cortisol are known to have cognitive and behavioral effects, so if the acutely elevated steroids we observed were to persist or increase as volatility rises, they may shift risk preferences and even affect a trader's ability to engage in rational choice.

X-ray spectromicroscopy in soil and environmental sciences.

X-ray microscopy is capable of imaging particles in the nanometer size range directly with sub-micrometer spatial resolution and can be combined with high spectral resolution for spectromicroscopy studies. Two types of microscopes are common in X-ray microscopy: the transmission X-ray microscope and the scanning transmission X-ray microscope; their set-ups are explained in this paper. While the former takes high-resolution images from an object with exposure times of seconds or faster, the latter is very well suited as an analytical instrument for spectromicroscopy. The morphology of clusters or particles from soil and sediment samples has been visualized using a transmission X-ray microscope. Images are shown from a cryo-tomography experiment based on X-ray microscopy images to obtain information about the three-dimensional structure of clusters of humic substances. The analysis of a stack of images taken with a scanning transmission X-ray microscope to combine morphology and chemistry within a soil sample is shown. X-ray fluorescence is a method ideally applicable to the study of elemental distributions and binding states of elements even on a trace level using X-ray energies above 1 keV.

Characterization and mode of inheritance of a paroxysmal dyskinesia in Chinook dogs.

BACKGROUND: Paroxysmal dyskinesias are episodes of abnormal, involuntary movement or muscle tone, distinguished from seizures by the character of the episode and lack of seizure activity on ictal EEG. HYPOTHESIS: Paroxysmal dyskinesia is an inherited, autosomal recessive disorder in Chinook dogs. ANIMALS: Families of Chinook dogs with paroxysmal dyskinesia. METHODS: Pedigrees and medical histories were reviewed for 299 Chinook dogs. A family of 51 dogs was used for analysis. Episodes were classified as seizures, paroxysmal dyskinesia, or unknown, and segregation analysis was performed. RESULTS: Paroxysmal dyskinesia was identified in 16 of 51 dogs and characterized by an inability to stand or ambulate, head tremors, and involuntary flexion of 1 or multiple limbs, without autonomic signs or loss of consciousness. Episode duration varied from minutes to an hour. Inter-ictal EEGs recorded on 2 dogs with dyskinesia were normal. Three dogs with dyskinesia also had generalized tonic-clonic seizures. One of 51 dogs had episodes of undetermined type. Phenotype was unknown for 6 of 51 dogs, and 28 dogs were unaffected. Segregation was consistent with an autosomal recessive trait. CONCLUSIONS AND CLINICAL IMPORTANCE: This movement disorder is prevalent in the Chinook breed, and consistent with a partially penetrant autosomal recessive or polygenic trait. Insufficient evidence exists for definitive localization; episodes may be of basal nuclear origin, but atypical seizures and muscle membrane disorders remain possible etiologies. The generalized seizures may be a variant phenotype of the same mutation that results in dyskinesia, or the 2 syndromes may be independent.

Branching out in new directions: the control of root architecture by lateral root formation.

Plant roots are required for the acquisition of water and nutrients, for responses to abiotic and biotic signals in the soil, and to anchor the plant in the ground. Controlling plant root architecture is a fundamental part of plant development and evolution, enabling a plant to respond to changing environmental conditions and allowing plants to survive in different ecological niches. Variations in the size, shape and surface area of plant root systems are brought about largely by variations in root branching. Much is known about how root branching is controlled both by intracellular signalling pathays and by environmental signals. Here, we will review this knowledge, with particular emphasis on recent advances in the field that open new and exciting areas of research.

The effect of the apolipoprotein E gene polymorphisms and haplotypes on behavioural and psychological symptoms in probable Alzheimer's disease.

BACKGROUND: Patients with Alzheimer's disease and dementia commonly suffer from behavioural and psychological symptoms of dementia (BPSD). A genetic component to BPSD development in Alzheimer's disease has been demonstrated. Several studies have investigated whether the exon 4 epsilon2/epsilon3/epsilon4 haplotype of the apolipoprotein E (APOE) gene is associated with BPSD, with variable results. OBJECTIVE: We investigated the exon 4 polymorphisms and extended this study to include promoter polymorphisms and the resultant haplotypes across the gene. METHODS: Our large independent cohort of 388 patients with longitudinal measures of BPSD assessed by the Neuropsychiatric Inventory was used to analyse whether any of these variants were associated with the presence of BPSD. RESULTS: We revealed several significant relationships before correction for multiple testing. The exon 4 haplotype was associated with hallucinations and anxiety, A-491T with irritability, T-427C with agitation/aggression and appetite disturbances, and T-219C with depression. Haplotype analyses of all variants did not reveal any statistically significant findings. CONCLUSIONS: Our data and a review of previous studies showed a diversity of relationships, suggesting that these findings might be due to chance and so collectively do not support a role for the APOE gene in BPSD.

Cerebrospinal Fluid Levels of Phosphorylated Neurofilament Heavy as a Diagnostic Marker of Canine Degenerative Myelopathy.

BACKGROUND: No definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF-H) is a promising biomarker for nervous system diseases. HYPOTHESIS/OBJECTIVE: Cerebrospinal fluid (CSF) and serum pNF-H is a detectable biological marker for diagnosis of canine DM. ANIMALS: Fifty-three DM-affected, 27 neurologically normal, 7 asymptomatic at-risk, and 12 DM mimic dogs. METHODS: Archived CSF and serum pNF-H concentrations were determined by a commercially available ELISA. A receiver-operating characteristic (ROC) curve was generated with CSF values. RESULTS: Compared with old control dogs, median CSF pNF-H concentration was increased in all stages of DM; old dogs 5.1 ng/mL (interquartile range [IQR] 1.4-9.3) versus DM stage 1 23.9 ng/mL (IQR 20.8-29.6; P < .05) versus DM stage 2 36.8 ng/mL (IQR 22.9-51.2; P < .0001) versus DM stage 3 25.2 ng/mL (IQR 20.2-61.8; P < .001) versus DM stage 4 38.0 ng/mL (IQR 11.6-59.9; P < .01). Degenerative myelopathy stage 1 dogs had increased median CSF pNF-H concentrations compared with asymptomatic, at-risk dogs (3.4 ng/mL [IQR 1.5-10.9; P < .01]) and DM mimics (6.6 ng/mL [IQR 3.0-12.3; P < .01]). CSF pNF-H concentration >20.25 ng/mL was 80.4% sensitive (confidence interval [CI] 66.09-90.64%) and 93.6% specific (CI 78.58-99.21%) for DM. Area under the ROC curve was 0.9467 (CI 0.92-0.9974). No differences in serum pNF-H concentration were found between control and DM-affected dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: pNF-H concentration in CSF is a sensitive biomarker for diagnosis of DM. Although there was high specificity for DM in this cohort, further study should focus on a larger cohort of DM mimics, particularly other central and peripheral axonopathies.

Standardization of a Videofluoroscopic Swallow Study Protocol to Investigate Dysphagia in Dogs.

BACKGROUND: Videofluoroscopic swallow study (VFSS) is the gold standard for diagnosis of dysphagia in veterinary medicine but lacks standardized protocols that emulate physiologic feeding practices. Age impacts swallow function in humans but has not been evaluated by VFSS in dogs. HYPOTHESIS/OBJECTIVES: To develop a protocol with custom kennels designed to allow free-feeding of 3 optimized formulations of contrast media and diets that address limitations of current VFSS protocols. We hypothesized that dogs evaluated by a free-feeding VFSS protocol would show differences in objective swallow metrics based on age. ANIMALS: Healthy juvenile, adult, and geriatric dogs (n = 24). METHODS: Prospective, experimental study. Custom kennels were developed to maintain natural feeding behaviors during VFSS. Three food consistencies (thin liquid, pureed food, and dry kibble) were formulated with either iohexol or barium to maximize palatability and voluntary prehension. Dogs were evaluated by 16 swallow metrics and compared across age groups. RESULTS: Development of a standardized VFSS protocol resulted in successful collection of swallow data in healthy dogs. No significant differences in swallow metrics were observed among age groups. Substantial variability was observed in healthy dogs when evaluated under these physiologic conditions. Features typically attributed to pathologic states, such as gastric reflux, were seen in healthy dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Development of a VFSS protocol that reflects natural feeding practices may allow emulation of physiology resulting in clinical signs of dysphagia. Age did not result in significant changes in swallow metrics, but additional studies are needed, particularly in light of substantial normal variation.

Novel forms of anaerobic respiration of environmental relevance.

Novel forms of anaerobic respiration continue to be discovered. Many of these are environmentally significant as they have important impacts on the fate of organic carbon and the cycling of many inorganic compounds. Furthermore, anaerobic respiration is becoming increasing recognized as a strategy for the remediation of organic and metal contaminants in the subsurface.

Radioiodinated antibody to carcinoembryonic antigen: binding to normal and cancerous human colon in vitro.

Samples of tumor and normal mucosa from 32 patients with adenocarcinoma of the colorectum were examined for their capacity to bind radioiodinated antibody to carcinoembryonic antigen (anti-CEA) lgG. Twenty-three (72%) of the tumors bound significantly more antibody than the respective normal mucosa. The results indicate that radiolabeled anti-CEA may be useful in the in vivo localization of CEA-producing tumors and metastases in man, and may have application in vitro as a diagnostic marker of precancerous change in colorectal biopsies from patients at risk of developing colorectal cancer.

How to reduce the stress of general dental practice: the need for research into the effectiveness of multifaceted interventions.

While the practice of dentistry has been demonstrated to be significantly stressful, there have been few published studies describing interventions to reduce the stress of dental practitioners. This article describes research into the prevention and alleviation of stress amongst a variety of healthcare professionals, including dental practitioners, and describes the findings from a small scale study of an intervention aimed at general dental practitioners who reported high levels of work related stress. It is argued that to be effective, interventions should be tailored to the individual needs of the practitioner, within a structured intervention framework. Further research into the effectiveness and cost-effectiveness of stress management for dental practitioners is required.