Involvement of TRPV1 and TRPA1 in the modulation of pacemaker potentials in the mouse ileum.

BACKGROUND: The roles of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) and subfamily A, member 1 (TRPA1) in mechanisms of gastrointestinal motility are complex. This study aimed to clarify the effects of several TRPV1 and TRPA1 ligands on the electrical potentials generated by pacemaker cells in the mouse-isolated ileum. METHOD: The pacemaker potentials of ileal segments of mice were recorded extracellularly using a 60-channel microelectrode array. The dominant frequencies, average waveform periods and propagation velocities were quantified. The effects of TRPV1 and TRPA1 agonist and antagonist were compared with the baseline recordings. RESULTS: The electrophysiological recordings showed that capsaicin (30 µM to 3 mM), resiniferatoxin (300 µM), capsazepine (100-300 µM), allyl isothiocyanate (300 µM), isovelleral (300 µM), icilin (300 µM), A-967,079 (10 µM), AP18 (20 µM) and HC-030,031 (50 µM) significantly reduced the pacemaker frequency and increased the waveform period relative to the baseline. Conversely, ruthenium red (300 µM) significantly increased the pacemaker frequency and reduced the waveform period. Capsaicin (3 mM) and AP18 (20 µM) also significantly reduced the propagation velocity. However, all tested antagonists failed to inhibit the effects of agonists. AMG9810 (300 µM), but not A-967,079 (300 µM), significantly inhibited the increases in pacemaker frequency caused by increased temperatures. CONCLUSION: Our findings suggest that TRPV1 and TRPA1 play a minor role in regulating pacemaker potentials and that at non-specific actions at other TRP and ion channels most likely contributed to the overall effects on the electrophysiological recordings that we observed.

Intra-gastrointestinal amyloid-ß1-42 oligomers perturb enteric function and induce Alzheimer's disease pathology.

KEY POINTS: Alzheimer's disease (AD) patients and transgenic mice have beta-amyloid (Aß) aggregation in the gastrointestinal (GI) tract. It is possible that Aß from the periphery contributes to the load of Aß in the brain, as Aß has prion-like properties. The present investigations demonstrate that Aß injected into the GI tract of ICR mice is internalised into enteric cholinergic neurons; at 1 month, administration of Aß into the body of the stomach and the proximal colon was observed to partly redistribute to the fundus and jejunum; at 1 year, vagal and cerebral ß-amyloidosis was present, and mice exhibited GI dysfunction and cognitive deficits. These data reveal a previously undiscovered mechanism that potentially contributes to the development of AD. ABSTRACT: Alzheimer's disease (AD) is the most common age-related cause of dementia, characterised by extracellular beta-amyloid (Aß) plaques and intracellular phosphorylated tau tangles in the brain. Aß deposits have also been observed in the gastrointestinal (GI) tract of AD patients and transgenic mice, with overexpression of amyloid precursor protein. In the present studies, we investigate whether intra-GI administration of Aß can potentially induce amyloidosis in the central nervous system (CNS) and AD-related pathology such as dementia. We micro-injected Aß1-42 oligomers (4 µg per site, five sites) or vehicle (saline, 5 µl) into the gastric wall of ICR mice under general anaesthesia. Immunofluorescence staining and in vivo imaging showed that HiLyte Fluor 555-labelled Aß1-42 had migrated within 3 h via the submucosa to nearby areas and was internalised into cholinergic neurons. At 1 month, HiLyte Fluor 555-labelled Aß1-42 in the body of the stomach and proximal colon had partly re-distributed to the fundus and jejunum. At 1 year, the jejunum showed functional alterations in neuromuscular coupling (P < 0.001), and Aß deposits were present in the vagus and brain, with animals exhibiting cognitive impairments in the Y-maze spontaneous alteration test (P < 0.001) and the novel object recognition test (P < 0.001). We found that enteric Aß oligomers induce an alteration in gastric function, amyloidosis in the CNS, and AD-like dementia via vagal mechanisms. Our results suggest that Aß load is likely to occur initially in the GI tract and may translocate to the brain, opening the possibility of new strategies for the early diagnosis and prevention of AD.

Sulprostone-Induced Gastric Dysrhythmia in the Ferret: Conventional and Advanced Analytical Approaches.

Nausea and emesis resulting from disease or drug treatment may be associated with disrupted gastric myoelectric activity (GMA). Conventional analytical techniques can determine the relative degrees of brady-, normo-, and tachygastric power, but lose information relative to the basic slow wave shape. The aim of the present study was to investigate the application of advanced analytical techniques in the analysis of disrupted GMA recorded after administration of sulprostone, a prostaglandin E3 / 1 agonist, in ferrets. Ferrets were implanted with radiotelemetry devices to record GMA, blood pressure, heart rate (HR) and core body temperature 1 week before the administration of sulprostone (30 µg/kg) or vehicle (saline, 0.5 mL/kg). GMA was initially analyzed using fast Fourier transformations (FFTs) and a conventional power partitioning. Detrended fluctuation analysis (DFA) was also applied to the GMA recordings to reveal information relative to the fluctuation of signals around local trends. Sample entropy (SampEn) analysis was used for examining the regularity of signals. Conventional signal processing techniques revealed that sulprostone increased the dominant frequency (DF) of slow waves, with an increase in the percentage power of the tachygastric range and a decrease in the percentage power of the normogastric range. DFA revealed that sulprostone decreased the fluctuation function, indicative of a loss of the variability of GMA fluctuations around local trends. Sulprostone increased SampEn values, indicating a loss of regularity in the GMA data. Behaviorally, sulprostone induced emesis and caused defecation. It also increased blood pressure and elevated HR, with an associated decrease in HR variability (HRV). Further analysis of HRV revealed a decrease in both low-frequency (LF) and high-frequency (HF) components, with an overall increase in the LF/HF ratio. Sulprostone did not affect core body temperature. In conclusion, DFA and SampEn permit a detailed analysis of GMA, which is necessary to understand the action of sulprostone to modulate gastric function. The action to decrease HRV and increase the LF/HF ratio may be consistent with a shift toward sympathetic nervous system dominance, commonly seen during nausea.

On the analytical formulation of excess noise in avalanche photodiodes with dead space.

Simple, approximate formulas are developed to calculate the mean gain and excess noise factor for avalanche photodiodes using the dead-space multiplication theory in the regime of small multiplication width and high applied electric field. The accuracy of the approximation is investigated by comparing it to the exact numerical method using recursive coupled integral equations and it is found that it works for dead spaces up to 15% of the multiplication width, which is substantial. The approximation is also tested for real materials such as GaAs, InP and Si for various multiplication widths, and the results found are accurate within ∼ 15% of the actual noise, which is a significant improvement over the local-theory noise formula. The results obtained for the mean gain also confirm the recently reported relationship between experimentally determined local ionization coefficients and the enabled non-local ionization coefficients.

Modification of flower colour by suppressing ß-ring carotene hydroxylase genes in Oncidium.

Oncidium 'Gower Ramsey' (Onc. GR) is a popular cut flower, but its colour is limited to bright yellow. The ß-ring carotene hydroxylase (BCH2) gene is involved in carotenoid biogenesis for pigment formation. However, the role of BCH2 in Onc. GR is poorly understood. Here, we investigated the functions of three BCH2 genes, BCH-A2, BCH-B2 and BCH-C2 isolated from Onc. GR, to analyse their roles in flower colour. RT-PCR expression profiling suggested that BCH2 was mainly expressed in flowers. The expression of BCH-B2 remained constant while that of BCH-A2 gradually decreased during flower development. Using Agrobacterium tumefaciens to introduce BCH2 RNA interference (RNAi), we created transgenic Oncidium plants with down-regulated BCH expression. In the transgenic plants, flower colour changed from the bright yellow of the wild type to light and white-yellow. BCH-A2 and BCH-B2 expression levels were significantly reduced in the transgenic flower lips, which make up the major portion of the Oncidium flower. Sectional magnification of the flower lip showed that the amount of pigmentation in the papillate cells of the adaxial epidermis was proportional to the intensity of yellow colouration. HPLC analyses of the carotenoid composition of the transgenic flowers suggested major reductions in neoxanthin and violaxanthin. In conclusion, BCH2 expression regulated the accumulation of yellow pigments in the Oncidium flower, and the down-regulation of BCH-A2 and BCH-B2 changed the flower colour from bright yellow to light and white-yellow.

MicroRNA-17-5p post-transcriptionally regulates p21 expression in irradiated betel quid chewing-related oral squamous cell carcinoma cells.

BACKGROUND AND PURPOSE: Betel nut chewing is associated with oral cavity cancer in Taiwan. OC3 is an oral carcinoma cell line that was established from cells collected from a long-term betel nut chewer who does not smoke. After we found that microRNA-17-5p (miR-17-5p) is induced in OC3 cells, we used this cell line to examine the biological role(s) of this microRNA in response to exposure to ionizing radiation. MATERIALS AND METHODS: A combined SYBR green-based real-time PCR and oligonucleotide ligation assay was used to examine the expression of the miR-17 polycistron in irradiated OC3 cells. The roles of miR-17-5p and p21 were evaluated with specific antisense oligonucleotides (ODN) that were designed and used to inhibit their expression. Expression of the p21 protein was evaluated by Western blotting. The clonogenic assay and annexin V staining were used to evaluate cell survival and apoptosis, respectively. Cells in which miR-17-5p was stably knocked down were used to create ectopic xenografts to evaluate in vivo the role of miR-17-5p. RESULTS: A radiation dose of 5 Gy significantly increased miR-17-5p expression in irradiated OC3 cells. Inhibition of miR-17-5p expression enhanced the radiosensitivity of the OC3 cells. We found that miR-17-5p downregulates radiation-induced p21 expression in OC3 cells and, by using a tumor xenograft model, it was found that p21 plays a critical role in increasing the radiosensitivity of OC3 cells in vitro and in vivo. CONCLUSION: miR-17-5p is induced in irradiated OC3 cells and it downregulates p21 protein expression, contributing to the radioresistance of OC3 cells.

Construction of a Der p2-transgenic plant for the alleviation of airway inflammation.

In clinical therapy, the amount of antigen administered to achieve oral tolerance for allergic diseases is large, and the cost is a major consideration. In this study, we used tobacco plants to develop a large-scale protein production system for allergen-specific immunotherapy, and we investigated the mechanisms of oral tolerance induced by a transgenic plant-derived antigen. We used plants (tobacco leaves) transgenic for the Dermatophagoides pteronyssinus 2 (Der p2) antigen to produce Der p2. Mice received total protein extract from Der p2 orally once per day over 6 days (days 0-2 and days 6-8). Mice were also sensitized and challenged with yeast-derived recombinant Der p2 (rDer p2), after which the mice were examined for airway hyper-responsiveness and airway inflammation. After sensitization and challenge with rDer p2, mice that were fed with total protein extracted from transgenic plants showed decreases in serum Der p2-specific IgE and IgG1 titers, decreased IL-5 and eotaxin levels in bronchial alveolar lavage fluid, and eosinophil infiltration in the airway. In addition, hyper-responsiveness was also decreased in mice that were fed with total protein extracted from transgenic plants, and CD4(+)CD25(+)Foxp3(+) regulatory T cells were significantly increased in mediastinal and mesenteric lymph nodes. Furthermore, splenocytes isolated from transgenic plant protein-fed mice exhibited decreased proliferation and increased IL-10 secretion after stimulation with rDer p2. The data here suggest that allergen-expressing transgenic plants could be used for therapeutic purposes for allergic diseases.

Lepidopteran-active variable-region sequence imparts coleopteran activity in eCry3.1Ab, an engineered Bacillus thuringiensis hybrid insecticidal protein.

A unique, coleopteran-active protein, termed eCry3.1Ab, was generated following variable-region exchange of a Bacillus thuringiensis lepidopteran-active protein, Cry1Ab, with a Cry3A region. Our results support the hypothesis that this variable-region exchange is responsible for imparting strong bioactivity against the larvae of western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte), a pest species which is not susceptible to either parent protein sequence. This study demonstrates the potential of successfully engineering a portion(s) of a lepidopteran-active B. thuringiensis sequence so that it has activity against coleopterans. Further elucidation of the eCry3.1Ab activity indicated the importance of variable regions 4 to 6 that were derived from Cry1Ab instead of Cry1Ac. There was some flexibility in making domain III of engineered hybrid insecticidal proteins even more Cry1Ab-like and retaining activity, while there was less flexibility in making domain III more Cry3A-like and retaining activity. In vitro binding studies with brush border membrane vesicles demonstrated that there was specific binding of chymotrypsin-processed modified Cry3A (mCry3A), which was not diminished by addition of a 100-fold molar excess of chymotrypsin-processed eCry3.1Ab or unprocessed eCry3.1Ab. In addition, in the converse experiment, specific binding of chymotrypsin-processed eCry3.1Ab was not diminished by the presence of a 75-fold molar excess of chymotrypsin-processed mCry3A. These data support the hypothesis that eCry3.1Ab can interact with different binding sites than the activated form of mCry3A in the WCR brush border and may provide a different mode of action from the standpoint of resistance management.

Trait stacking in transgenic crops: challenges and opportunities.

In recent years, there has been a rapid increase in the planting of transgenic crops with stacked traits. Most of these products have been formed by conventional breeding, i.e. the crossing of transgenic plant (event) containing individual transgenes with other event(s) containing single or double transgenic traits. Many biotech companies are developing stacked trait products with increasing numbers of insect and herbicide tolerance genes for controlling a broad range of insect pests and weeds. There has also been an increase in development of technologies for molecular stacking of multiple traits in a single transgene locus. In this review we look at the status of stacked trait products, crop trait stacking technologies and the technical challenges we are facing. We also review recent progress in developing technology for assembling large transgene arrays in vitro (molecular stacks), their delivery to crop plants and issues they pose for transgene expression.

Normative apparent diffusion coefficient values in the developing fetal brain.

BACKGROUND AND PURPOSE: Previous studies of diffusion-weighted imaging (DWI) in fetuses are limited. Because of the need for normative data for comparison with young fetuses and preterm neonates with suspected brain abnormalities, we studied apparent diffusion coefficient (ADC) values in a population of singleton, nonsedated, healthy fetuses. MATERIALS AND METHODS: DWI was performed in 28 singleton nonsedated fetuses with normal or questionably abnormal results on sonography and normal fetal MR imaging results; 10 fetuses also had a second fetal MR imaging, which included DWI. ADC values in the periatrial white matter (WM), frontal WM, thalamus, basal ganglia, cerebellum, and pons were plotted against gestational age and analyzed with linear regression. We compared mean ADC in different regions using the Tukey Honestly Significant Difference test. We also compared rates of decline in ADC with increasing gestational age across different areas by using the t test with multiple comparisons correction. Neurodevelopmental outcome was assessed. RESULTS: Median gestational age was 24.28 weeks (range, 21-33.43 weeks). Results of all fetal MR imaging examinations were normal, including 1 fetus with a normal variant of a cavum velum interpositum. ADC values were highest in the frontal and periatrial WM and lowest in the thalamus and pons. ADC declined with increasing gestational age in periatrial WM (P = .0003), thalamus (P < .0001), basal ganglia (P = .0035), cerebellum (P < .0001), and pons (P = .024). Frontal WM ADC did not significantly change with gestational age. ADC declined fastest in the cerebellum, followed by the thalamus. CONCLUSIONS: Regional differences in nonsedated fetal ADC values and their evolution with gestational age likely reflect differences in brain maturation and are similar to published data in premature neonates.

From enzyme to zymogen: engineering Vip2, an ADP-ribosyltransferase from Bacillus cereus, for conditional toxicity.

The adenosine diphosphate (ADP)-ribosyltransferase, Vip2 (vegetative insecticidal protein), from Bacillus cereus in combination with another protein from the same organism, Vip1, has insecticidal activity against western corn rootworm larvae. The Vip2 protein exerts its intracellular poisoning effect by modifying actin and preventing actin polymerization. Due to the nature of this toxin, expression of Vip2 in planta is lethal. In this work, we attempted to build an enzyme precursor (proenzyme, zymogen) that would silently reside in one biological system (e.g. plants or yeast) and be activated in the other (insect larvae). Our approach involved engineering a random propeptide library at the C-terminal end of Vip2 and selecting for malfunctional enzyme variants in yeast. A selected proenzyme (proVip2) possesses reduced enzymatic activity as compared with the wild-type Vip2 protein, but remains a potent toxin toward rootworm larvae. In addition, upon analysis of the digestive fate of the engineered enzyme precursor in rootworm larvae, we demonstrated that 'zymogenized' Vip2 can be proteolytically activated by rootworm digestive enzyme machinery. This report represents an example of applying a protein engineering strategy for the creation of a plant-tolerated, zymogen-like form of an otherwise toxic protein. This approach may outline a novel path to address challenges associated with utilizing toxic proteins in certain biotechnological applications.

Fabrication of high-aspect-ratio Fresnel zone plates by e-beam lithography and electroplating.

The fabrication of gold Fresnel zone plates, by a combination of e-beam lithography and electrodeposition, with a 30 nm outermost zone width and a 450 nm-thick structure is described. The e-beam lithography process was implemented with a careful evaluation of applied dosage, tests of different bake-out temperatures and durations for the photoresist, and the use of a developer without methylisobutylketone. Electrodeposition with a pulsed current mode and with a specially designed apparatus produced the desired high-aspect-ratio nanostructures. The fabricated zone plates were examined by electron microscopy and their performances were assessed using a transmission X-ray microscope. The results specifically demonstrated an image resolution of 40 nm.

An engineered chymotrypsin/cathepsin G site in domain I renders Bacillus thuringiensis Cry3A active against Western corn rootworm larvae.

The western corn rootworm remains one of the most important pests of corn in the United States despite the use of many pest management tools. Cry3A, the first coleopteran-active Bacillus thuringiensis toxin isolated, has not been useful for control of the corn rootworm pest complex. Modification of Cry3A so that it contained a chymotrypsin/cathepsin G protease recognition site in the loop between alpha-helix 3 and alpha-helix 4 of domain I, however, resulted in consistent activity of the toxin ("mCry3A") against neonate western corn rootworm. In vitro chymotrypsin digests showed that there was a substantial difference between the enzyme sensitivity of mCry3A and the enzyme sensitivity of Cry3A, with mCry3A rapidly converted from a 67-kDa form to a approximately 55-kDa form. The introduced protease site was also recognized in vivo, where the approximately 55-kDa form of mCry3A toxin was rapidly generated and associated with the membrane fraction. After a point mutation in mcry3A that resulted in the elimination of the native domain I chymotrypsin site (C terminal to the introduced chymotrypsin/cathepsin G protease site of mCry3A), the in vitro and in vivo digestion patterns remained the same, demonstrating that the introduced site was required for the enhanced activity. Also, 55-kDa mCry3A generated by cleavage with chymotrypsin exhibited specific binding to western corn rootworm brush border membrane, whereas untreated 67-kDa mCry3A did not. These data indicate that the mCry3A toxicity for corn rootworm larvae was due to the introduction of a chymotrypsin/cathepsin G site, which enhanced cleavage and subsequent binding of the activated toxin to midgut cells.

Ablation of advanced tongue or base of tongue cancer and reconstruction with free flap: functional outcomes.

AIM: To evaluate the functional outcomes of patients who underwent total or nearly total glossectomy for advanced tongue or base of tongue cancer. MATERIAL AND METHODS: We used the radial forearm free flap (RFFF), anterior lateral thigh flap (ALTF) or fibular osteocutaneous flap (FOCF) to reconstruct the oral defect after radical resection in 39 patients undergoing total or nearly total glossectomy with laryngeal preservation. RESULTS: Good functional outcomes, measured by independent feeding, speech and swallowing were achieved in 35, 36 and 35 patients, respectively. The cumulative 4-year survival rates were 63.8% for tongue cancer and 42.9% for base of tongue cancer. CONCLUSION: Reconstruction with free flaps is a feasible method to restore the functional outcomes in speech and deglutition among patients who undergo total or nearly total glossectomy with laryngeal preservation.

Distally based sural island flap for the reconstruction of a large soft tissue defect in an open tibial fracture with occluded anterior and posterior tibial arteries-a case report.

To repair a 10 x 16 cm soft tissue defect of right lower leg that accompanied with occluded anterior and posterior tibial arteries in a 30-year-old man who sustained comminuted type III(B) Gustilo open tibial and fibular fracture, we present a successful reconstruction by using a large distally based sural island flap perfused by the lowermost perforator of the peroneal artery. This flap is a useful alternative for distal extremity reconstruction when anterior and posterior tibial arteries are occluded, as large flaps can be elevated safely based on only one peroneal perforator.

A pilot bridging data integration and analytics: BioMediator and R?

Biological research today involves aggregating and analyzing large amounts of data from disparate sources. Tools such as the University of Washington's BioMediator system integrate heterogeneous data. Analytic packages such as the R environment have a rich set of tools to analyze biomedical research data. Our pilot project bridged data integration and analytics in a general way by successfully incorporating the BioMediator system into the R platform for specific analyses on neurophysiologic research data.

Percutaneous puncture and pre-operative cyanoacrylate obliteration of a traumatic false aneurysm of an angular artery branch.

We report a case of traumatic false aneurysm developed in the right glabella in a 5-year-old boy 3 weeks after an innocuous fall. Ultrasound, CT and facial arteriography did not reveal the feeding artery. After direct puncture of the glabellar bulge and rapid aspiration of blood, percutaneous contrast agent infusion revealed that the false aneurysm was supplied by the contralateral angular artery. Intralesional obliteration with cyanoacrylate was subsequently performed smoothly. Succeeding excision was easy and the cosmetic outcome was excellent.

Development of a method for the determination of pyrimethamine concentrations in feeds by ion-pairing high-performance liquid chromatography.

An ion-pairing reversed-phase high-performance liquid chromatography (HPLC) method with diode array detection at 280 nm was developed to determine pyrimethamine concentrations in feed for laying hens. Pyrimethamine was extracted with a mixture of 5% isobutanol and 95% benzene, and the extract was cleaned up on an alumina column. The drug was eluted from an Intersil ODS-3V column (250 by 4.6 mm) with a mixture of 25% acetonitrile and 75% water (vol/vol) containing 0.01 M tetramethylammonium chloride as an ion-pairing agent and adjusted with acetic acid to pH 3.5. The flow rate was 1.0 ml/min. Mean recovery of pyrimethamine from supplemented feeds at concentrations of 2, 4, and 5 microg/g of feed were 100.5, 103.5, and 100.8%, respectively. Precision within a day ranged from 4.3 to 7.0% for the three concentrations, and day-to-day precision was 5.3% for feed supplemented at a concentration of 4 microg/g. No chromatographic interference was detected from other 2,4-diaminopyrimidine compounds or other major drugs used in poultry.

High-performance liquid chromatographic determination of carbadox, olaquindox, furazolidone, nitrofurazone, and nitrovin in feed.

A high-performance liquid chromatography with gradient programming method was developed to determine the amount of carbadox (CBX), olaquindox (OLQ), furazolidone (FZ), nitrofurazone (NF), and nitrovin (NTV) in feed simultaneously. Complete separation of the drugs was obtained using a C8 silica gel column with gradients of acetonitrile as mobile phase. The mobile phase used an acetonitrile gradient with an initial hold time of 1 min at 0% acetonitrile, followed by an increase to 50% acetonitrile over 10 min. The correlation coefficients (r) for calibration curves of the five feed additives were greater than 0.999. The relative standard deviations (RSDs) of peak areas from four injections for these drugs at three concentrations were less than 3.0%, although the RSD for NTV at 5 ppm was somewhat large (6.7%). The medicated feeds were extracted by pretreating with water, extracted with 95% dimethylformamide overnight at room temperature, and cleaned up on a column of alumina oxide. Recoveries of CBX, OLQ, FZ, NF, and NTV from low level spiked feed were 102.0, 94.6, 97.4, 110.6, and 66.0%, respectively, and from high level spiked feed, they were 114.09, 99.1, 97.3, 109.9, and 62.7%, respectively.