Knocking down Wnt9a mRNA levels increases cellular proliferation

Wnts are secreted lipid-modified signaling proteins. Activation of Wnt signalling in many tissues has also been associated with cancer. In many eukaryotes, expression of nuclear-encoded mRNA can be strongly inhibited by the presence of a small double-stranded RNA corresponding to exon sequences in the mRNA. In this study we used pAVU6+27 vectors, which have SalI and XbaI clone sites, to construct the siRNA expression vectors for human Wnt9a. Two kinds of small interfering RNA inserts were designed, synthesized and visually tested for efficacy by in situ hybridization, the results demonstrated that in the cells, transfected with U6+27 cassettes with anti-Wnt9a hairpin siRNA inserts, dramatically reduced Wnt9a signals were observed as compared to the untransfected cells. The results of flow cytometry analysis showed that the cell proliferation was promoted after lowering expression of the human Wnt9a in MCF-7 cells by RNAi, but was inhibited after over-expression of human Wnt9a. These results suggests the expression level of human Wnt9a in MCF-7 that breast cancer may play a role in adjusting the rate of cellular proliferation.     

C3G regulates cortical neuron migration, preplate splitting and radial glial cell attachment

Neuronal migration is integral to the development of the cerebral cortex and higher brain function. Cortical neuron migration defects lead to mental disorders such as lissencephaly and epilepsy. Interaction of neurons with their extracellular environment regulates cortical neuron migration through cell surface receptors. However, it is unclear how the signals from extracellular matrix proteins are transduced intracellularly. We report here that mouse embryos lacking the Ras family guanine nucleotide exchange factor, C3G (Rapgef1, Grf2), exhibit a cortical neuron migration defect resulting in a failure to split the preplate into marginal zone and subplate and a failure to form a cortical plate. C3G-deficient cortical neurons fail to migrate. Instead, they arrest in a multipolar state and accumulate below the preplate. The basement membrane is disrupted and radial glial processes are disorganised and lack attachment in C3G-deficient brains. C3G is activated in response to reelin in cortical neurons, which, in turn, leads to activation of the small GTPase Rap1. In C3G-deficient cells, Rap1 GTP loading in response to reelin stimulation is reduced. In conclusion, the Ras family regulator C3G is essential for two aspects of cortex development, namely radial glial attachment and neuronal migration.     

Optical protein sensor for detecting cancer markers in saliva

A surface immobilized optical protein sensor has been utilized to detect Interleukin-8 (IL-8) protein, an oral cancer marker, and can reach limit of detection (LOD) at 1.1pM in buffer without using enzymatic amplification. Only after applying enzymatic amplification to increase the signal level by a few orders of magnitude, ELISA can reach the LOD of 1pM level. We then develop the confocal optics based sensor for further reducing the optical noise and can extend the LOD of the surface immobilized optical protein sensor two orders in magnitude. These improvements have allowed us to detect IL-8 protein at 4.0fM in buffer. In addition, these sensitive LODs were achieved without the use of enzymatic signal amplification, such that the simplified protocol can further facilitate the development of point-of-care devices. The ultra sensitive optical protein sensor presented in this paper has a wide number of applications in disease diagnoses. Measurements for detecting biomarkers in clinical sample are much more challenging than the measurements in buffer, due to high background noise contributed by large collections of non-target molecules. We used clinical saliva samples to validate the functionality of the optical protein sensor. Clinical detection of disease-specific biomarkers in saliva offers a non-invasive, alternative approach to using blood or urine. Currently, the main challenge of using saliva as a diagnostic fluid is its inherently low concentration of biomarkers. We compare the measurements of 40 saliva samples; half from oral cancer patients and half from a control group. The data measured by the optical protein sensor is compared with the traditional Enzyme-Linked Immunosorbant Assay (ELISA) values to validate the accuracy of our system. These positive results enable us to proceed to using confocal optical protein sensor to detect other biomarkers, which have much lower concentrations.     

Solid-phase microextraction for determining twelve orange flavour compounds in a model beverage emulsion

Solid-phase microextraction (SPME) coupled to gas chromatography has been applied for the headspace analysis (HS) of 12 target flavour compounds in a model orange beverage emulsion. The main volatile flavour compounds studied were: acetaldehyde, ethyl acetate, alpha-pinene, ethyl butyrate, beta-pinene, myrcene, limonene, gamma-terpinene, octanal, decanal, linalool and citral (neral plus geranial). After screening the fibre type, the effect of other HS-SPME variables such as adsorption temperature (25-55 degrees C), extraction time (10-40 min), sample concentration (1-100% w/w), sample amount (5-10 g) and salt amount (0-30% w/w) were determined using a two-level fractional factorial design (2(5-2)) that was expanded further to a central composite design. It was found that an extraction process using a carboxen-polydimethylsiloxane fibre coating at 15 masculineC for 50 min with 5 g of diluted emulsion 1% (w/w) and 30% (w/w) of sodium chloride under stirring mode resulted in the highest HS extraction efficiency. For all volatile flavour compounds, the linearity values were accurate in the concentration ranges studied (r(2) > 0.97). Average recoveries that ranged from 90.3 to 124.8% showed a good accuracy for the optimised method. The relative standard deviation for six replicates of all volatile flavour compounds was found to be less than 15%. For all volatile flavour compounds, the limit of detection ranged from 0.20 to 1.69 mg/L.     

ROC-based utility function maximization for feature selection and classification with applications to high-dimensional protease data

SUMMARY: In medical diagnosis, the diseased and nondiseased classes are usually unbalanced and one class may be more important than the other depending on the diagnosis purpose. Most standard classification methods, however, are designed to maximize the overall accuracy and cannot incorporate different costs to different classes explicitly. In this article, we propose a novel nonparametric method to directly maximize the weighted specificity and sensitivity of the receiver operating characteristic curve. Combining advances in machine learning, optimization theory, and statistics, the proposed method has excellent generalization property and assigns different error costs to different classes explicitly. We present experiments that compare the proposed algorithms with support vector machines and regularized logistic regression using data from a study on HIV-1 protease as well as six public available datasets. Our main conclusion is that the performance of proposed algorithm is significantly better in most cases than the other classifiers tested. Software package in MATLAB is available upon request.     

LRRK2 R1628P increases risk of Parkinson’s disease: replication evidence

We showed that the frequency of a LRRK2 variant (c.4883G > C, R1628P) was higher in Parkinson’s disease (PD) compared to controls (8.4 vs. 3.4%, P = 0.046, OR 2.5, 95% CI 1.1–5.6). In the multivariate logistic regression (with adjustments made for the effect of age, age of onset, and gender), the heterozygous R1628P genotype was associated with an increased risk of PD compared to controls (OR 3.3, 95% CI 1.4– 7.9, P = 0.007). We provided an independent confirmation that the R1628P variant increases the risk of PD among Chinese.     

Altered Dopamine Receptor and Dopamine Transporter Binding and Tyrosine Hydroxylase mRNA Expression Following Perinatal NMDA Receptor Blockade

This study examined how perinatal phencyclidine (PCP) treatment would affect dopamine D2 receptor and dopamine transporter (DAT) binding at different stages after treatment cessation. Female rat pups received injections of PCP (10 mg/kg, s.c.) or saline on postnatal day (PN)7, 9 and 11. D2 receptor and transporter binding was examined at four time-points (PN12, 18, 32 and 96) following injections. PCP treatment altered D2 receptor binding throughout development, with a final end-point of 22-33% decreased binding at adulthood in the nucleus accumbens and caudate putamen (P < 0.01), accompanied by a small but significant increase in DAT binding in the caudate putamen. Tyrosine hydroxylase mRNA expression was also significantly increased by 25% (P < 0.05) in the ventral tegmental area of adult rats, suggesting that this model may produce a long-term increase in dopamine output. This study demonstrates that early insult to the brain from NMDA receptor hypofunction alters the dopaminergic system at different stages of development.     

Trace amine-associated receptor 1 (TAAR1) is activated by amiodarone metabolites

Amiodarone (Cordarone, Wyeth-Ayerst Pharmaceuticals) is a clinically available drug used to treat a wide variety of cardiac arrhythmias. We report here the synthesis and characterization of a panel of potential amiodarone metabolites that have significant structural similarity to thyroid hormone and its metabolites the iodothyronamines. Several of these amiodarone derivatives act as specific agonists of the G protein-coupled receptor (GPCR) trace amine-associated receptor 1 (TAAR(1)). This result demonstrates a novel molecular target for amiodarone derivatives with potential clinical significance.