brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Brain Slice Biotinylation: An <em>Ex Vivo</em> Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

University of Massachusetts Medical School

The detection of nonsmall cell lung cancer (NSCLC) at an early stage presents a daunting challenge due to the lack of a specific noninvasive marker. The discovery of microRNAs (miRNAs), particularly those found in serum, has opened a new avenue for tumor diagnosis. To determine whether the expression profile of serum miRNAs can serve as a NSCLC fingerprint, we performed Taqman probe-based quantitative RT-PCR assay to selected differentially expressed serum miRNAs from a sample set including 400 NSCLC cases and 220 controls, and risk score analysis to evaluate the diagnostic value of the serum miRNA profiling system. After a two-phase selection and validation process, 10 miRNAs were found to have significantly different expression levels in NSCLC serum samples compared with the control serum samples. Risk score analysis showed that this panel of miRNAs was able to distinguish NSCLC cases from controls with high sensitivity and specificity. Under ROC curves, the AUC for tumor identification in training set and validation set were 0.966 and 0.972, respectively. Furthermore, the expression profile of the 10-serum miRNAs was correlated with the stage of NSCLC patients, especially in younger patients and patients with current smoking habits. More importantly, the serum miRNA-based biomarker for early NSCLC detection was supported by a retrospective analysis in which the 10-serum miRNA profile could accurately classify serum samples collected up to 33 months ahead of the clinical NSCLC diagnosis. Taken together, we demonstrate that the profiling of 10-serum miRNAs provides a novel noninvasive biomarker for NSCLC diagnosis.

Identification of ten serum microRNAs from a genome-wide serum microRNA expression profile as novel noninvasive biomarkers for nonsmall cell lung cancer diagnosis.

Acrylamide (AA) is known to be a neurotoxic, genotoxic, and carcinogenic compound. The presence of AA in foods causes public health concerns. In our previous study, we found that allicin can effectively reduce AA content in Maillard model system. However, there has been no report on whether allicin can reduce AA-induced toxicity in vitro and in vivo. In our present study, we evaluated the protective effect of allicin against AA-induced hepatocyte damage in cultured mouse primary hepatocytes and mouse liver. Our date suggested that allicin significantly decreased the levels of maleic dialdehyde (MDA) and 8-hydroxy-desoxyguanosine (8-OHdG) both in vitro and in vivo study. Allicin also markedly increased the activity of total superoxide dismutase (SOD) and level of glutathione (GSH). The in vitro study revealed that 15 μM allicin was the optimum concentration for inhibiting AA-induced hepatocyte damage. The in vivo study revealed that 20mg/kg b.w./day allicin was the optimum dose for inhibiting AA-induced hepatocyte damage. The protective effects of allicin against AA-induced hepatocyte damage may be due to its ability to scavenge free radicals and its effective recovery of the antioxidative defense system, and its ability to block the epoxidation process of AA to GA by inhibiting P450 enzyme.

Protective effect of allicin against acrylamide-induced hepatocyte damage in vitro and in vivo.

Laparoscopic surgery is now widely performed to treat various abdominal diseases. Currently, carbon dioxide is the most frequently used gas for insufflation of the abdominal cavity (pneumoperitoneum). Many other gases have been introduced as alternatives to carbon dioxide for establishing pneumoperitoneum.

Gases for establishing pneumoperitoneum during laparoscopic abdominal surgery.

Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.

Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton.

In this study, a solid phase microextraction-gas chromatography/mass spectrometry (SPMEGC/MS) method was developed to detection the levels of furan in 191 selected food products obtained from the Chinese markets. The performance of the analysis method was evaluated by some quality parameters such as limit of detection (LOD), limit of quantification (LOQ), linearity, recovery, and runto-run (n=6) and day-to-day (n=18) precisions. The LOD and the LOQ of the method were 0.059 ng/g and 0.175 ng/g, respectively. The recovery of the method in fruit juice, canned jam, and infant formula ranged from 93.25% to 104.69%. The results from this study showed that furan was detected in almost all analyzed samples. Furan contents in different food samples varied greatly according to the raw materials and processing conditions. The higher contents of furan were detected in traditional Chinese liquor (61.63 ng/g), coffee (71.36 ng/g), tea (68.28 ng/g) and pickle (85.63 ng/g). The dietary intake of furan was estimated to be 0.093 μg/kg b.w.t/day in adults and 0.333 μg/kg b.w.t/day in infants at 6 months. Further studies are needed to reveal other sources of dietary furan exposure.

Detection of furan levels in select Chinese foods by solid phase microextraction-gas chromatography/mass spectrometry method and dietary exposure estimation of furan in the Chinese population.

Sijia Wu

Program in Neuroscience,
Graduate School of Biomedical Sciences,
Program in Neuroscience, Graduate School of Biomedical Sciences

University of Massachusetts Medical School

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Sijia Wu

Program in Neuroscience,Graduate School of Biomedical Sciences,Program in Neuroscience, Graduate School of Biomedical Sciences

University of Massachusetts Medical School

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Program in Neuroscience,
Graduate School of Biomedical Sciences,
Program in Neuroscience, Graduate School of Biomedical Sciences