Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements. Here, we describe the techniques needed to generate low noise (<0.5 pA) electrodes. The techniques have been modified from published descriptions by previous researchers (1,2). Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber. Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip. The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal. Lastly, the electrodes are placed in an oven to cure the epoxy. Careful handling of the electrodes is critical to ensure that they are made consistently and without damage. This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells

Enhancing students’ motivation to learn software engineering programming techniques: a collaborative and social interaction approach

To motivate students to study advanced programming techniques, including the use of architectural styles such as the model–view–controller pattern, we have con-ducted action research upon a project based-learning approach. In addition to collabo-ration, the approach includes students’ searching and analysis of scientific documents and their involvement in communities of practice outside academia. In this paper, we report the findings of second action research cycle, which took place throughout the fourth semester of a six-semester program. As with the previous cycle during the pre-vious academic year, students did not satisfactorily achieve expected learning out-comes. More groups completed the assigned activities, but results continue to reflect poor engagement in the communities of practice and very low performance in other learning tasks. From the collected data we have identified new approaches and recom-mendations for subsequent research.Fundação para a Ciência e Tecnologia (FCT), Portugal, for Ph.D. Grants SFRH/BD/91309/2012 and SFRH/BD/87815/201

Longitudinal Assessment of Growth in Hypoplastic Left Heart Syndrome: Results From the Single Ventricle Reconstruction Trial

Background: We sought to characterize growth between birth and age 3 years in infants with hypoplastic left heart syndrome who underwent the Norwood procedure. Methods and Results: We performed a secondary analysis using the Single Ventricle Reconstruction Trial database after excluding patients 2 SD below normal). Failure to find consistent risk factors supports the strategy of tailoring nutritional therapies to patient‐ and stage‐specific targets. Clinical Trial Registration URL: http://clinicaltrials.gov/. Unique identifier: NCT00115934

Ages at menarche- and menopause-related genetic variants in relation to terminal duct lobular unit involution in normal breast tissue

PURPOSE: Reduced levels of terminal duct lobular unit (TDLU) involution, as reflected by higher numbers of TDLUs and acini per TDLU, have been associated with higher breast cancer risk. Younger age at menarche and older age at menopause have been previously related to lower levels of TDLU involution. To determine a possible genetic link, we examined whether single nucleotide polymorphisms (SNPs) previously established in genome-wide association studies (GWAS) for ages at menarche and menopause are associated with TDLU involution. METHODS: We conducted a pooled analysis of 862 women from two studies. H&E tissue sections were assessed for numbers of TDLUs and acini/TDLU. Poisson regression models were used to estimate associations of 36 menarche- and 21 menopause-SNPs with TDLU counts, acini counts/TDLU, and the product of these two measures, adjusting for age and study site. RESULTS: Fourteen percent of evaluated SNPs (8 SNPs) were associated with TDLU counts at p<0.05, suggesting an enrichment of associations with TDLU counts. However, only menopause-SNPs had >50% that were either significantly or nonsignficantly associated with TDLU measures in the directions consistent with their relationships shown in GWAS. Among 10 SNPs that were statistically significantly associated with at least one TDLU involution measure (p<0.05), seven SNPs (rs466639: RXRG; rs2243803: SLC14A2; rs2292573: GAB2; rs6438424: 3q13.32; rs7606918: METAP1D; rs11668344: TMEM150B; rs1635501: EXO1) were associated in the consistent directions. CONCLUSIONS: Our data suggest that the loci associated with ages at menarche and menopause may influence TDLU involution, suggesting some shared genetic mechanisms. However, larger studies are needed to confirm the results

Dual Functions of ASCIZ in the DNA Base Damage Response and Pulmonary Organogenesis

Zn2+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion or knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis

Abnormal Intracellular Accumulation and Extracellular Aβ Deposition in Idiopathic and Dup15q11.2-q13 Autism Spectrum Disorders

<div><h3>Background</h3><p>It has been shown that amyloid ß (Aβ), a product of proteolytic cleavage of the amyloid β precursor protein (APP), accumulates in neuronal cytoplasm in non-affected individuals in a cell type–specific amount.</p> <h3>Methodology/Principal Findings</h3><p>In the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15) and autism spectrum disorder (ASD). In spite of interindividual differences within each examined group, levels of intraneuronal Aβ load were significantly greater in the dup(15) autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test). In eight regions, intraneuronal Aβ load differed significantly between idiopathic autism and control groups (p<0.0001). The intraneuronal Aβ was mainly N-terminally truncated. Increased intraneuronal accumulation of Aβ_17–40/42 in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aβ accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aβ_1–40/42 detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aβ and an extracellular deposition of full-length Aβ in nonfibrillar plaques.</p> <h3>Conclusions/Significance</h3><p>The higher prevalence of excessive Aβ accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15) compared to subjects with idiopathic ASD, supports the concept of mechanistic and functional links between autism, epilepsy and alterations of APP processing leading to neuronal and astrocytic Aβ accumulation and diffuse plaque formation.</p> </div