High-throughput mapping of a dynamic signaling network in mammalian cells

Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor-beta (TGFbeta) pathway. Analysis using self-organizing maps and k-means clustering identified links of the TGFbeta pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGFbeta type I receptor localization for efficient TGFbeta-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.     

Graduate students' teaching experiences improve their methodological research skills

Science, technology, engineering, and mathematics (STEM) graduate students are often encouraged to maximize their engagement with supervised research and minimize teaching obligations. However, the process of teaching students engaged in inquiry provides practice in the application of important research skills. Using a performance rubric, we compared the quality of methodological skills demonstrated in written research proposals for two groups of early career graduate students (those with both teaching and research responsibilities and those with only research responsibilities) at the beginning and end of an academic year. After statistically controlling for preexisting differences between groups, students who both taught and conducted research demonstrate significantly greater improvement in their abilities to generate testable hypotheses and design valid experiments. These results indicate that teaching experience can contribute substantially to the improvement of essential research skills.     

Species-specific sensitivity of earthworm coelomocytes to dermal metal (Cd,Cu, Ni,Pb, Zn) exposures: Methodological approach

Earthworm immune-competent cells (coelomocytes) can be adversely affected by soil metal contamination. The aim of the present paper was to perform comparative studies on the coelomocytes of four lumbricid species dermally exposed for 2 days to filter paper soaked with Ni, Cu, Zn, Cd, or Pb chlorides (1 mM). Coelomocytes were expelled by electro-stimulation, counted, and assayed ex vivo by flow cytometry and spectrofluorometry. The coelomocytes of Allolobophora chlorotica and Dendrobaena veneta contain both amoebocytes and riboflavin-storing eleocytes; consequently, they were subjected to assays based on flow cytometric quantification of autofluorescent eleocytes and spectrofluorometric measurement of riboflavin content in coelomocyte lysates. In Al. chlorotica, the number of coelomocytes, the percentage of eleocytes, and the amount of riboflavin were significantly lower in Cu-exposed worms although these cytometric parameters were less affected by Ni, Zn, Cd exposure, and entirely unaffected by Pb exposure. In D. veneta, such cellular effects were also only observed in Cu-exposed worms. The coelomic fluids of Aporrectodea caliginosa and Lumbricus rubellus are constitutionally impoverished of eleocytes; therefore, these species could not be subjected to assays measuring the riboflavin content of these granular cells. Rather, the amoebocytes of Ap. caliginosa and L. rubellus were subjected to flow cytometric measurements of in vitro neutral red uptake (NR). However, the NR uptake assay was technically demanding, requiring a strictly normalized incubation period over all samples to yield useful comparative data. In contrast, the riboflavin content in the coelomocyte lysates of eleocyte-rich species appears to be a robust and convenient immune-function biomarker of environmental stress.     

A study to evaluate buprenorphine at 40 μg kg(-1) compared to 20 μg kg(-1) as a post-operative analgesic in the dog

ObjectiveComparison of the analgesic effect of buprenorphine at 20 or 40 μg kg^?1.Study designAn investigator ‘blinded’, randomised study.AnimalsTwenty six dogs presented for ovariohysterectomy.MethodsDogs were premedicated intramuscularly with acepromazine 0.03 mg kg^?1 and buprenorphine at either 20 (B20, n = 12) or 40 μg kg^?1 (B40, n= 14) followed by anaesthetic induction with propofol and maintenance with isoflurane. During anaesthesia non invasive blood pressure, heart rate, respiratory rate, blood oxygen saturation, inspired and expired volatile agent, end-tidal carbon dioxide and ECG were recorded. Pain and sedation were assessed using interactive VAS scores; mechanical nociceptive thresholds were measured at the wound and hindlimb - all were assessed before and up to 22 hours after administration. Carprofen was used for rescue analgesia.ResultsThere were no significant differences between the two groups for any of the parameters examined. Rescue analgesia was required around 5 hours after administration of buprenorphine in a significant number of animals. Sedation was good preoperatively and scores decreased over time postoperatively. Hock thresholds did not change over time; wound thresholds decreased significantly compared to the baseline value from 3 hours onwards.ConclusionsAdministration of buprenorphine at either 20 or 40 μg kg^?1 IM with acepromazine provided good pre-operative sedation. Cardiovascular and respiratory values remained within clinically acceptable limits during anaesthesia. There was no evidence that increasing dose increased adverse events that may be associated with opioid administration (e.g. bradycardia and respiratory depression).Clinical relevanceIncreasing the dose of buprenorphine from 20 to 40 μg kg^?1 did not provide any benefits with respect to analgesia after ovariohysterectomy as assessed using the VAS scoring system.     

Nutrient cycling in a Pinus patula plantation in the Mpumalanga Province, South Africa

The accumulation of litter on the forest floor was identified as a potential problem in managed plantations of Pinus patula (Schlechtd. et Cham.) in the Mpumalanga Province of South Africa in the late 1980s. Litter accumulation in pine plantations is regarded as a threat to site productivity as organic acids are released, moisture penetration is altered and nutrients are immobilised within the litter. This study examines the cycling of nutrients in a 42-year-old P. patula stand in which litter has accumulated. Samples of the vegetation, litter and soil components were collected and chemically analysed for total nitrogen (N), phosphorus (P) and the major cations potassium (K⁺), calcium (Ca²⁺) and magnesium (Mg²⁺). Complete nutrient budgets for N and P, and the cation pool sizes were determined. It was evident from these studies that large reserves of N (1442 kg ha⁻¹) and P (103 kg ha⁻¹) are stored in the litter layers, with levels of cations being low. The presence of large nutrient reserves within the litter and the predominance of fine feeder roots distributed within this layer indicated that a tightly closed plant–litter–plant nutrient cycle was in operation for the cycling of N and P. This may not be true for the major cations. Management of the litter should ensure retention of as many nutrients as possible in the system. This could be achieved through controlled burning to reduce nutrient loss through volatilisation; increasing forest floor temperatures by altering the planting density and application of dolomitic lime to replace cations and to alleviate the acidic conditions making the litter more favourable for decomposing organisms.     

Cut-flower life of dry transported carnations as influenced by different silver form pre-treatments

Twenty-four hours of pre-treatment with AgNO₃ increased the vase-life of flowers from 3 to 4 days for carnations kept in water or in preservative solutions after simulated transport conditions. A mixture of AgNO₃ with sodium thiosulphate or with EDTA was considerably better than AgNO₃, while such complexing agents as CDTA, EDDHA and EDPA were less effective. Determination of radioactive silver (Ag) in dissected carnation parts treated with different Ag complexes showed a positive correlation between vase-life and the amount of tracer Ag in the plants.     

Investigation of changes in the middle latency auditory evoked potential during anesthesia with sevoflurane in dogs

OBJECTIVE: To investigate the middle latency auditory evoked potential (MLAEP) in awake dogs and dogs anesthetized with 2 concentrations of sevoflurane. ANIMALS: 10 adult Beagles. PROCEDURE: The MLAEP was recorded while dogs were awake and anesthetized with sevoflurane (end-tidal concentration, 2.7% or 3.5%).Three needle electrodes were inserted SC, and click stimuli were delivered biaurally. Signal acquisition, averaging, and analysis were performed by use of computer software developed in-house. Signals were recorded for 128 milliseconds, and the responses to 1,024 stimuli were averaged. Waveforms from 10 recordings in each dog were averaged, and latencies of peaks were measured. Data acquired for awake dogs and dogs anesthetized with high and low sevoflurane concentrations were compared statistically. RESULTS: Sevoflurane anesthesia attenuated the MLAEP so that only peaks P0, Na, and Pa could be identified. The MLAEP changes were maximal at the lower concentration of sevoflurane evaluated. The latencies of these peaks were significantly shorter in awake dogs, compared with values in anesthetized dogs. No difference in the peak latency was detected between the sevoflurane concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: In terms of CNS responsiveness, the effects of anesthesia with sevoflurane are similar to those of anesthesia with isoflurane. Data suggest that sevoflurane is not the inhalant agent of choice in a research setting where electroencephalographic measurements are to be recorded during anesthesia. The depression of the MLAEP waveform by sevoflurane also suggests that the MLAEP is not a suitable tool with which to monitor anesthetic depth during sevoflurane anesthesia in dogs.     

Ultrastructural morphologic evaluation of the phenotype of valvular interstitial cells in dogs with myxomatous degeneration of the mitral valve

OBJECTIVE: To evaluate morphologic changes in valvular interstitial cells of dogs and to find evidence for disease-associated phenotypic changes in these cells. ANIMALS: 5 clinically normal dogs and 5 dogs with severe mitral valve endocardiosis. PROCEDURE: Mitral valve leaflets were evaluated by use of transmission electron microscopy. Differences in cell type and cell location were identified. RESULTS: A change in cell type toward a myofibroblast or smooth muscle cell phenotype was detected, with the smooth muscle cell type being most common. These cells had long amorphous cytoplasmic extensions, fibrillar cytoplasm, incomplete basal lamina, few mitochondria, and eccentrically placed nuclei but lacked smooth endoplasmic reticulum or Golgi complexes. Remaining valvular interstitial cells had heterochromatic nuclei and produced only minimal quantities of collagen. Compared with normal valves, myxomatous valves ha many interstitial-like cells located adjacent to the endothelium. Deeper within the abnormal valves, cells with a heterogenous phenotype formed groupings that appeared to be anchored to adjacent collagen. CONCLUSIONS AND CLINICAL RELEVANCE: Myxomatous degeneration of the mitral valve in dogs is associated with phenotypic alteration, changing from an interstitial to a mixed myofibroblast or smooth muscle cell phenotype. A closer association between interstitial cells and the endothelium is evident in diseased valves. In response to the disease process, valvular interstitial cells of dogs appear to change toward a smooth muscle phenotype, possibly in an attempt to maintain valve tone and mechanical function.     

Transport and distribution of lindane and simazine in a riverine environment: measurements in bed sediments and modelling

Aquatic sediments often remove hydrophobic contaminants from fresh waters. The subsequent distribution and concentration of contaminants in bed sediments determines their effect on benthic organisms and the risk of re-entry into the water and/or leaching to groundwater. This study examines the transport of simazine and lindane in aquatic bed sediments with the aim of understanding the processes that determine their depth distribution. Experiments in flume channels (water flow of 10 cm s(-1)) determined the persistence of the compounds in the absence of sediment with (a) de-ionised water and (b) a solution that had been in contact with river sediment. In further experiments with river bed sediments in light and dark conditions, measurements were made of the concentration of the compounds in the overlying water and the development of bacterial/algal biofilms and bioturbation activity. At the end of the experiments, concentrations in sediments and associated pore waters were determined in sections of the sediment at 1 mm resolution down to 5 mm and then at 10 mm resolution to 50 mm depth and these distributions analysed using a sorption-diffusion-degradation model. The fine resolution in the depth profile permitted the detection of a maximum in the concentration of the compounds in the pore water near the surface, whereas concentrations in the sediment increased to a maximum at the surface itself. Experimental distribution coefficients determined from the pore water and sediment concentrations indicated a gradient with depth that was partly explained by an increase in organic matter content and specific surface area of the solids near the interface. The modelling showed that degradation of lindane within the sediment was necessary to explain the concentration profiles, with the optimum agreement between the measured and theoretical profiles obtained with differential degradation in the oxic and anoxic zones. The compounds penetrated to a depth of 40-50 mm over a period of 42 days.