Utility of contrast-enhanced computed tomography in the evaluation of canine insulinoma location

OBJECTIVES: To determine 1) the sensitivity of contrast-enhanced CT (CECT) for detection of primary canine insulinomas and metastases 2) the sensitivity of CECT to locate canine insulinomas within the pancreas and 3) the CECT attenuation pattern of canine insulinomas and post-contrast phase in which insulinomas have the best visibility. METHODS: A retrospective review was performed of the medical records of 27 canine insulinoma patients. Simultaneous occurrence of blood glucose 10 mIU/L (reference interval: 1.4-24.5 mIU/L) were considered diagnostic for insulinoma. The dogs had a mean age of 9.0 ± 1.7 (SD) years and comprised 11 males and 17 females. RESULTS: Using CECT-scans, 26/27 insulinomas were successfully detected. However, CECT-scans predicted the correct location of insulinomas within the pancreas in only 14/27 dogs. In 9/13 inaccurately located insulinoma cases, the location error was major. There was no significant difference between triple, double and single-phase CECT-scans with location accuracies of 54%, 50% and 50%, respectively. Also, there was no specific post-contrast phase in which insulinomas could be visualised best. Detection of lymph node metastases with CECT-scans had a sensitivity of 67% (10/15 lymph node metastases). Detection of liver metastases had a sensitivity of 75% (6/8 liver metastases). This study highlights that major location errors mainly occurred if single- or double-phase CECT-scans were used (6/9 cases). CONCLUSION: It is suggested that triple-phase CECT-scans have superior outcome over single- or double-phase CECT-scans in pre-operative imaging of canine insulinomas

Differential genetic interactions of yeast stress response MAPK pathways.

Genetic interaction screens have been applied with great success in several organisms to study gene function and the genetic architecture of the cell. However, most studies have been performed under optimal growth conditions even though many functional interactions are known to occur under specific cellular conditions. In this study, we have performed a large-scale genetic interaction analysis in Saccharomyces cerevisiae involving approximately 49 × 1,200 double mutants in the presence of five different stress conditions, including osmotic, oxidative and cell wall-altering stresses. This resulted in the generation of a differential E-MAP (or dE-MAP) comprising over 250,000 measurements of conditional interactions. We found an extensive number of conditional genetic interactions that recapitulate known stress-specific functional associations. Furthermore, we have also uncovered previously unrecognized roles involving the phosphatase regulator Bud14, the histone methylation complex COMPASS and membrane trafficking complexes in modulating the cell wall integrity pathway. Finally, the osmotic stress differential genetic interactions showed enrichment for genes coding for proteins with conditional changes in phosphorylation but not for genes with conditional changes in gene expression. This suggests that conditional genetic interactions are a powerful tool to dissect the functional importance of the different response mechanisms of the cell

Chemo-biocatalytic one-pot two-step conversion of cyclic amine to lactam using whole cell monoamine oxidase

BACKGROUND: Most biocatalysts currently involved in one‐pot chemoenzymatic cascades are pure enzymes, while whole cells and crude enzyme extracts remain unexplored. This work aims to develop a chemo‐biocatalytic one‐pot two‐step system involving whole cell monoamine oxidase (MAO, EC 1.4.3.4) coupled with a Cu‐based oxidative system (CuI/H2O2) for the transformation of 1,2,3,4‐tetrahydroisoquinoline (THIQ) to 3,4‐dihydroisoquinolin‐1(2H)‐one (DHIO). RESULTS: MAO‐N variants D9 and D11 were tested as whole cell and crude lysate biocatalysts for biological oxidation. Whole Escherichia coli OverExpress C43(DE3) cells expressing MAO‐N D9 showed the best performance (Vmax = 36.58 mmol L−1 h−1, KM = 8.124 mmol L−1, maximum specific productivity 89.3 μmol min−1 g−1DCW) and were employed in combination with CuI/H2O2 in a sequential one‐pot two‐step process. The biotransformation was scaled‐up to the initial volume of 25 mL and after triple THIQ feeding, 48.2 mmol L−1 of the intermediate 3,4‐dihydroisoquinoline (DHIQ) was obtained with a yield of 71.3%. Afterwards, chemical catalysts (1 mol% CuI and 10 eq. H2O2) were added to the biologically produced DHIQ, which was transformed to ∼30 mmol L−1 DHIO at 69.4% overall yield. CONCLUSION: As MAO‐N variants have wide substrate specificity, this work broadens the portfolio of one‐pot chemoenzymatic processes employing whole cell biocatalysts, representing an alternative to using pure enzymes

Clinical and genetic characterisation of dystrophin-deficient muscular dystrophy in a family of Miniature Poodle dogs

Four full-sibling intact male Miniature Poodles were evaluated at 4–19 months of age. One was clinically normal and three were affected. All affected dogs were reluctant to exercise and had generalised muscle atrophy, a stiff gait and a markedly elevated serum creatine kinase activity. Two affected dogs also showed poor development, learning difficulties and episodes of abnormal behaviour. In these two dogs, investigations into forebrain structural and metabolic diseases were unremarkable; electromyography demonstrated fibrillation potentials and complex repetitive discharges in the infraspinatus, supraspinatus and epaxial muscles. Histopathological, immunohistochemical and immunoblotting analyses of muscle biopsies were consistent with dystrophin-deficient muscular dystrophy. DNA samples were obtained from all four full-sibling male Poodles, a healthy female littermate and the dam, which was clinically normal. Whole genome sequencing of one affected dog revealed a >5 Mb deletion on the X chromosome, encompassing the entire DMD gene. The exact deletion breakpoints could not be experimentally ascertained, but we confirmed that this region was deleted in all affected males, but not in the unaffected dogs. Quantitative polymerase chain reaction confirmed all three affected males were hemizygous for the mutant X chromosome, while the wildtype chromosome was observed in the unaffected male littermate. The female littermate and the dam were both heterozygous for the mutant chromosome. Forty-four Miniature Poodles from the general population were screened for the mutation and were homozygous for the wildtype chromosome. The finding represents a naturally-occurring mutation causing dystrophin-deficient muscular dystrophy in the dog

Th1 type lymphocyte reactivity to metals in patients with total hip arthroplasty

<p>Abstract</p> <p>Background</p> <p>All prostheses with metallic components release metal debris that can potentially activate the immune system. However, implant-related metal hyper-reactivity has not been well characterized. In this study, we hypothesized that adaptive immunity reaction(s), particularly T-helper type 1 (Th1) responses, will be dominant in any metal-reactivity responses of patients with total joint replacements (TJAs). We tested this hypothesis by evaluating lymphocyte reactivity to metal "ions" in subjects with and without total hip replacements, using proliferation assays and cytokine analysis.</p> <p>Methods</p> <p>Lymphocytes from young healthy individuals without an implant or a history of metal allergy (Group 1: n = 8) were used to assess lymphocyte responses to metal challenge agents. In addition, individuals (Group 2: n = 15) with well functioning total hip arthroplasties (average Harris Hip Score = 91, average time in-situ 158 months) were studied. Age matched controls with no implants were also used for comparison (Group 3, n = 8, 4 male, 4 female average age 70, range 49–80). Group 1 subjects' lymphocyte proliferation response to Aluminum⁺³, Cobalt⁺², Chromium⁺³, Copper⁺², Iron⁺³, Molybdenum⁺⁵, Manganeese⁺², Nickel⁺², Vanadium⁺³and Sodium⁺²chloride solutions at a variety of concentrations (0.0, 0.05, 0.1, 0.5, 1.0 and 10.0 mM) was studied to establish toxicity thresholds. Mononuclear cells from Group 2 and 3 subjects were challenged with 0.1 mM CrCl₃, 0.1 mM NiCl₂, 0.1 mM CoCl₂and approx. 0.001 mM titanium and the reactions measured with proliferation assays and cytokine analysis to determine T-cell subtype prominence.</p> <p>Results</p> <p>Primary lymphocytes from patients with well functioning total hip replacements demonstrated a higher incidence and greater magnitude of reactivity to chromium than young healthy controls (p < 0.03). Of the 15 metal ion-challenged subjects with well functioning total hip arthroplasties, 7 demonstrated a proliferative response to Chromium, Nickel, Cobalt and/or Titanium (as defined by a statistically significant >2 fold stimulation index response, p < 0.05) and were designated as metal-reactive. Metals such as Cobalt, Copper, Manganese, and Vanadium were toxic at concentrations as low as 0.5 mM while other metals, such as Aluminum, Chromium, Iron, Molybdenum, and Nickel, became toxic at much higher concentrations (>10 mM). The differential secretion of signature T-cell subsets' cytokines (Th1 and Th2 lymphocytes releasing IFN-gamma and IL-4, respectively) between those total hip arthroplasty subjects which demonstrated metal-reactivity and those that did not, indicated a Th1 type (IFN-gamma) pro-inflammatory response.</p> <p>Conclusion</p> <p>Elevated proliferation and production of IFN-gamma to metals in hip arthroplasty subjects' lymphocytes indicates that a Th1 (vs. Th2) type response is likely associated with any metal induced reactivity. The involvement of an elevated and specific lymphocyte response suggests an <it>adaptive </it>(macrophage recruiting) immunity response to metallic implant debris rather than an <it>innate </it>(nonspecific) immune response.</p

The Evolutionary Pathway to Obligate Scavenging in Gyps Vultures

The evolutionary pathway to obligate scavenging in Gyps vultures remains unclear. We propose that communal roosting plays a central role in setting up the information transfer network critical for obligate scavengers in ephemeral environments and that the formation of a flotilla-like foraging group is a likely strategy for foraging Gyps vultures. Using a spatial, individual-based, optimisation model we find that the communal roost is critical for establishing the information network that enables information transfer owing to the spatial-concentration of foragers close to the roost. There is also strong selection pressure for grouping behaviour owing to the importance of maintaining network integrity and hence information transfer during foraging. We present a simple mechanism for grouping, common in many animal species, which has the added implication that it negates the requirement for roost-centric information transfer. The formation of a flotilla-like foraging group also improves foraging efficiency through the reduction of overlapping search paths. Finally, we highlight the importance of consideration of information transfer mechanisms in order to maximise the success of vulture reintroduction programmes

Plasma levels of phosphorylated tau 181 are associated with cerebral metabolic dysfunction in cognitively impaired and amyloid-positive individuals

Alzheimer’s disease biomarkers are primarily evaluated through MRI, PET and CSF methods in order to diagnose and monitor disease. Recently, advances in the assessment of blood-based biomarkers have shown promise for simple, inexpensive, accessible and minimally invasive tools with diagnostic and prognostic value for Alzheimer’s disease. Most recently, plasma phosphorylated tau181 has shown excellent performance. The relationship between plasma phosphorylated tau181 and cerebral metabolic dysfunction assessed by [18F]fluorodeoxyglucose PET in Alzheimer’s disease is still unknown. This study was performed on 892 older individuals (297 cognitively unimpaired; 595 cognitively impaired) from the Alzheimer’s Disease Neuroimaging Initiative cohort. Plasma phosphorylated tau181 was assessed using single molecular array technology and metabolic dysfunction was indexed by [18F]fluorodeoxyglucose PET. Cross-sectional associations between plasma and CSF phosphorylated tau181 and [18F]fluorodeoxyglucose were assessed using voxelwise linear regression models, with individuals stratified by diagnostic group and by β-amyloid status. Associations between baseline plasma phosphorylated tau181 and longitudinal (24 months) rate of brain metabolic decline were also assessed in 389 individuals with available data using correlations and voxelwise regression models. Plasma phosphorylated tau181 was elevated in β-amyloid positive and cognitively impaired individuals as well as in apolipoprotein E ε4 carriers and was significantly associated with age, worse cognitive performance and CSF phosphorylated tau181. Cross-sectional analyses showed strong associations between plasma phosphorylated tau181 and [18F]fluorodeoxyglucose PET in cognitively impaired and β-amyloid positive individuals. Voxelwise longitudinal analyses showed that baseline plasma phosphorylated tau181 concentrations were significantly associated with annual rates of metabolic decline in cognitively impaired individuals, bilaterally in the medial and lateral temporal lobes. The associations between plasma phosphorylated tau181 and reduced brain metabolism, primarily in cognitively impaired and in β-amyloid positive individuals, supports the use of plasma phosphorylated tau181 as a simple, low-cost, minimally invasive and accessible tool to both assess current and predict future metabolic dysfunction associated with Alzheimer’s disease, comparatively to PET, MRI and CSF methods

Molecular Binding Mechanism of TtgR Repressor to Antibiotics and Antimicrobials

A disturbing phenomenon in contemporary medicine is the prevalence of multidrug-resistant pathogenic bacteria. Efflux pumps contribute strongly to this antimicrobial drug resistance, which leads to the subsequent failure of clinical treatments. The TtgR protein of Pseudomonas putida is a HTH-type transcriptional repressor that controls expression of the TtgABC efflux pump, which is the main contributor to resistance against several antimicrobials and toxic compounds in this microbe. One of the main strategies to modulate the bacterial resistance is the rational modification of the ligand binding target site. We report the design and characterization of four mutants-TtgRS77A, TtgRE78A, TtgRN110A and TtgRH114A - at the active ligand binding site. The biophysical characterization of the mutants, in the presence and in the absence of different antimicrobials, revealed that TtgRN110A is the variant with highest thermal stability, under any of the experimental conditions tested. EMSA experiments also showed a different dissociation pattern from the operator for TtgRN110A, in the presence of several antimicrobials, making it a key residue in the TtgR protein repression mechanism of the TtgABC efflux pump. We found that TtgRE78A stability is the most affected upon effector binding. We also probe that one mutation at the C-terminal half of helix-α4, TtgRS77A, provokes a severe protein structure distortion, demonstrating the important role of this residue in the overall protein structure and on the ligand binding site. The data provide new information and deepen the understanding of the TtgR-effector binding mechanism and consequently the TtgABC efflux pump regulation mechanism in Pseudomonas putida.This work was supported by Spanish Ministry of Economy and Competitiveness, National programme for Recruitment and Incorporation of Human Resources, Subprogramme: Ramon y Cajal RYC-2009-04570 and grant P11-CVI-7391 from Junta de Andalucía and EFDR (European Regional Development Fund)