GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

GABAB receptors are expressed in human aortic smooth muscle cells and regulate the intracellular Ca2+ concentration

The aim of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter gamma-aminobutyric acid (GABAB), in human aortic smooth muscle cells (HASMCs), and to explore if altering receptor activation modified intracellular Ca(2+) concentration ([Ca(2+)]i) of HASMCs. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABABR1 and GABABR2 in cultured HASMCs. Immunohistochemistry was used to localize the two subunits in human left anterior descending artery (LAD). The effects of the GABAB receptor agonist baclofen on [Ca(2+)]i in cultured HASMCs were demonstrated using fluo-3. Both GABABR1 and GABABR2 mRNA and protein were identified in cultured HASMCs and antibody staining was also localized to smooth muscle cells of human LAD. 100 μM baclofen caused a transient increase of [Ca(2+)]i in cultured HASMCs regardless of whether Ca(2+) was added to the medium, and the effects were inhibited by pre-treatment with CGP46381 (selective GABAB receptor antagonist), pertussis toxin (a Gi/o protein inhibitor), and U73122 (a phospholipase C blocker). GABAB receptors are expressed in HASMCs and regulate the [Ca(2+)]i via a Gi/o-coupled receptor pathway and a phospholipase C activation pathway

GABAB1 and GABAB2 receptor subunits co-expressed in cultured human RPE cells regulate intracellular Ca2+ via Gi/o-protein and phospholipase C pathways

GABAB receptors associate with Gi/o-proteins that regulate voltage-gated Ca(2+) channels and thus the intracellular Ca(2+) concentration ([Ca(2+)]i), there is also reported cross-regulation of phospholipase C. These associations have been studied extensively in the brain and also shown to occur in non-neural cells (e.g. human airway smooth muscle). More recently GABAB receptors have been observed in chick retinal pigment epithelium (RPE). The aims were to investigate whether the GABAB receptor subunits, GABAB1 and GABAB2, are co-expressed in cultured human RPE cells, and then determine if the GABAB receptor similarly regulates the [Ca(2+)]i of RPE cells and if phospholipase C is involved. Human RPE cells were cultured from 5 donor eye cups. Evidence for GABAB1 and GABAB2 mRNAs and proteins in the RPE cell cultures were investigated using real time PCR, western blots and immunofluorescence. The effects of the GABAB receptor agonist baclofen, antagonist CGP46381, a Gi/o-protein inhibitor pertussis toxin, and the phospholipase C inhibitor U73122 on [Ca(2+)]i in cultured human RPE were demonstrated using Fluo-3. Both GABAB1 and GABAB2 mRNA and protein were identified in cell cultures of human RPE; antibody staining was co-localized to the cell membrane and cytoplasm. One-hundred μM baclofen caused a transient increase in the [Ca(2+)]i of RPE cells regardless of whether Ca(2+) was added to the buffer. Baclofen induced increases in the [Ca(2+)]i were attenuated by pre-treatment with CGP46381, pertussis toxin, and U73122. GABAB1 and GABAB2 are co-expressed in cell cultures of human RPE. GABAB receptors in RPE regulate the [Ca(2+)]i via a Gi/o-protein and phospholipase C pathway.

Displaced granulosa cells in peritoneal washings : a rare diagnostic pitfall

Developing follicles and follicular cysts in the ovary are lined by granulosa cells. Approximately the size of histiocytes, non-neoplastic granulosa cells have scant granular to foamy cytoplasm and mildly atypical hyperchromatic nuclei, which may be mitotically active. 1 Displaced granulosa cells, derived from normal follicles and introduced into ovarian vascular channels, ovarian stroma and the fallopian tube, have been reported to cause diagnostic difficulty in histol- ogy, as they may mimic small cell carcinoma or other metastatic carcinomas. 2–4 The cells are thought to be displaced artefactually due to surgical trauma or during sectioning in the laboratory or during ovulation...

H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

BACKGROUND: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS). METHODS: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response. RESULTS: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay. CONCLUSION: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

Youth off the Streets "Our Place, Walgett Youth and Young Families Project" Project Evaluation.

The Project: • YOTS is a major youth specific agency established in 1991. It is a non-denominational, non-discriminatory and not-for-profit organisation, providing a wide range of services and offering a full continuum of care. It seeks to build on the strengths and positive aspects of marginalised young people and communities. • The 'Our Place, Walgett Youth and Young Families Project' further develops an existing YOTS capacity to provide services to Aboriginal young people. • The project adopted an action-research and community development model in which YOTS worked in partnership with the Youth Sub-committee of the Walgett Interagency. • Specific goals/objectives of the program were to: Coordinate youth and young family activities in partnership with local services and the community to build self-esteem, pride, resilience, motivation and skills; Contribute to the prevention and reduction of homelessness, unstable and unsafe housing and disruptive mobility (Walgett/Redfern) in youth and young families; Increase and improve collaborative engagement between youth and family focused services; and, research, adapt and implement Australian and international best-practice homelessness prevention/reduction initiatives to contribute to new models of practice relevant to rural and regional areas. • The project centred around an out-reach model that focused on providing a safe space with relevant structured activities coordinated by YOTS youth and family workers. Through community and service provider consultation, it was proposed that local services could coordinate strategies and activities and run them, where possible, from the centre, providing ease of access in a safe and supportive context. • Specific activities included: Implementing regular meetings with the stakeholders and community representatives; Developing a Terms of Reference for YOTS presence in the Walgett community; Undertaking a community consultation prior to finalising program activities; Implementing a range of recreational activities (sports, music, arts and crafts) early on in the activity; Implementing young family support initiatives; implementing a volunteering program, including volunteer support to young families through intergenerational volunteering; running a series of Culture and Healing Camps in partnership with local Elders and other services; Running a series of Music Camps; Providing alternative education support and referrals in partnership with local schools; Researching, identifying and adapting other best-practice models.

“You could scream the place down”: Five poems on the experience of aged care

This article presents five poems constructed from interviews with older people adjusting to living in residential aged care. They are part of the “Inside Aged Care” project, on-going longitudinal phenomenological research tracking the lived experience of aged care from the perspective of residents, family members and service providers. Poetry, through the process of poetic transcription, provided an engaging, evocative and almost visceral way to help us appreciate what it might be like to be ageing in aged care. To date, despite a growing body of work documenting the importance and impact of research in the form of poetry, applying a literary lens is rare in gerontological research. At a very practical level, therefore, we hope these poems help older people, their families, students and those working in aged care better understand the unique world and perspective of new aged care residents.

Towards Vision-Based Pose- and Condition-Invariant Place Recognition along Routes

Vision-based place recognition involves recognising familiar places despite changes in environmental conditions or camera viewpoint (pose). Existing training-free methods exhibit excellent invariance to either of these challenges, but not both simultaneously. In this paper, we present a technique for condition-invariant place recognition across large lateral platform pose variance for vehicles or robots travelling along routes. Our approach combines sideways facing cameras with a new multi-scale image comparison technique that generates synthetic views for input into the condition-invariant Sequence Matching Across Route Traversals (SMART) algorithm. We evaluate the system’s performance on multi-lane roads in two different environments across day-night cycles. In the extreme case of day-night place recognition across the entire width of a four-lane-plus-median-strip highway, we demonstrate performance of up to 44% recall at 100% precision, where current state-of-the-art fails.

Increasing leucine concentration stimulates mechanistic target of rapamycin signaling and cell growth in C2C12 skeletal muscle cells

Leucine is a key amino acid for initiating translation in muscle cells, but the dose-dependent effects of leucine on intracellular signaling are poorly characterized. This study examined the effect that increasing doses of leucine would have on changes in mechanistic target of rapamycin (mTOR)–mediated signaling, rates of protein synthesis, and cell size in C2C12 cells. We hypothesized that a leucine “threshold” exists, which represents the minimum stimulus required to initiate mTOR signaling in muscle cells. Acute exposure to 1.5, 3.2, 5.0, and 16.1 mM leucine increased phosphorylation of mTORSer2448 (~1.4-fold; P < .04), 4E-BP1 Thr37/46 (~1.9-fold; P < .001), and rpS6Ser235/6 (~2.3-fold; P < .001). However, only p70S6kThr389 exhibited a dose-dependent response to leucine with all treatments higher than control (~4-fold; P < .001) and at least 5 mM higher than the 1.5-mM concentration (1.2-fold; P < .02). Rates of protein synthesis were not altered by any treatment. Seven days of exposure to 0.5, 1.5, 5.0, and 16.5 mM leucine resulted in an increase in cell size in at least 5 mM treatments (~1.6-fold, P < .001 vs control). Our findings indicate that even at low leucine concentrations, phosphorylation of proteins regulating translation initiation signaling is enhanced. The phosphorylation of p70S6kThr389 follows a leucine dose-response relationship, although this was not reflected by the acute protein synthetic response. Nevertheless, under the conditions of the present study, it appears that leucine concentrations of at least 5 mM are necessary to enhance cell growth.

A non-fullerene electron acceptor based on fluorene and diketopyrrolopyrrole building blocks for solution-processable organic solar cells with an impressive open-circuit voltage

A novel solution-processable non-fullerene electron acceptor 6,6′-(5,5′-(9,9-dioctyl-9H-fluorene-2,7-diyl)bis(thiophene-5,2-diyl))bis(2,5-bis(2-ethylhexyl)-3-(thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione) (DPP1) based on fluorene and diketopyrrolopyrrole conjugated moieties was designed, synthesized and fully characterized. DPP1 exhibited excellent solubility and high thermal stability which are essential for easy processing. Upon using DPP1 as an acceptor with the classical electron donor poly(3-hexylthiophene), solution processable bulk-heterojunction solar cells afforded a power conversion efficiency of 1.2% with a high open-circuit voltage (1.1 V). As per our knowledge, this value of open circuit voltage is one of the highest values reported so far for a bulk-heterojunction device using DPP1 as a non-fullerene acceptor.

Visual place recognition for persistent robot navigation in changing environments

This thesis demonstrates that robots can learn about how the world changes, and can use this information to recognise where they are, even when the appearance of the environment has changed a great deal. The ability to localise in highly dynamic environments using vision only is a key tool for achieving long-term, autonomous navigation in unstructured outdoor environments. The proposed learning algorithms are designed to be unsupervised, and can be generated by the robot online in response to its observations of the world, without requiring information from a human operator or other external source.

"Nursing is no place for men" ― A thematic analysis of male nursing students experiences of undergraduate nursing education

Background: Surprisingly, opinion about whether men are suitable within the profession continues to be a divided issue. Men enter the profession for a multitude of reasons, yet barriers whether emotional, verbal or sexual are still present. Aim: The aim of this study was to examine the experience of men “training” to be registered nurses within a regional New Zealand context. Design: A Narrative Analysis approach was used. Participants: Five New Zealand men currently undertaking their bachelor of nursing degree at a regional tertiary institute were interviewed as to their experiences of what it meant to be a man in “training”. Method: A thematic analysis was undertaken and guided by an understanding of the way personal narratives informs the human sciences especially within the context of nursing praxis. Four key themes were identified. Results: Four key themes were identified: A career with flexibility and promise; perceived gender inequality in providing care; developing professional boundaries with female colleagues and being unique has its advantages. Conclusion: The men in this study were attracted to the profession by career stability and advancement; the opportunities for travel also figured highly. At times they felt excluded and marginalised because of their minority status within their group and the feminine nature of the curriculum. The men attempted to dispel the myth around male nurse sexual stereotypes. Some of the students behaved in a manner to exert their heterosexualness. The students in this study sensed their vulnerability in choosing nursing as a career. However, all the participants saw nursing as viable and portable career in terms of advancement and travel.

Unsupervised online learning of condition-invariant images for place recognition

This paper presents an online, unsupervised training algorithm enabling vision-based place recognition across a wide range of changing environmental conditions such as those caused by weather, seasons, and day-night cycles. The technique applies principal component analysis to distinguish between aspects of a location’s appearance that are condition-dependent and those that are condition-invariant. Removing the dimensions associated with environmental conditions produces condition-invariant images that can be used by appearance-based place recognition methods. This approach has a unique benefit – it requires training images from only one type of environmental condition, unlike existing data-driven methods that require training images with labelled frame correspondences from two or more environmental conditions. The method is applied to two benchmark variable condition datasets. Performance is equivalent or superior to the current state of the art despite the lesser training requirements, and is demonstrated to generalise to previously unseen locations.

Isolation of microvascular endothelial cells from cadaveric corneal limbus

Limbal microvascular endothelial cells (L-MVEC) contribute to formation of the corneal-limbal stem cell niche and to neovascularization of diseased and injuries corneas. Nevertheless, despite these important roles in corneal health and disease, few attempts have been made to isolate L-MVEC with the view to studying their biology in vitro. We therefore explored the feasibility of generating primary cultures of L-MVEC from cadaveric human tissue. We commenced our study by evaluating growth conditions (MesenCult-XF system) that have been previously found to be associated with expression of the endothelial cell surface marker thrombomodulin/CD141, in crude cultures established from collagenase-digests of limbal stroma. The potential presence of L-MVEC in these cultures was examined by flow cytometry using a more specific marker for vascular endothelial cells, CD31/PECAM-1. These studies demonstrated that the presence of CD141 in crude cultures established using the MesenCult-XF system is unrelated to L-MVEC. Thus we subsequently explored the use of magnetic assisted cell sorting (MACS) for CD31 as a tool for generating cultures of L-MVEC, in conjunction with more traditional endothelial cell growth conditions. These conditions consisted of gelatin-coated tissue culture plastic and MCDB-131 medium supplemented with fetal bovine serum (10% v/v), D-glucose (10 mg/mL), epidermal growth factor (10 ng/mL), heparin (50 μg/mL), hydrocortisone (1 μg/mL) and basic fibroblast growth factor (10 ng/mL). Our studies revealed that use of endothelial growth conditions are insufficient to generate significant numbers of L-MVEC in primary cultures established from cadaveric corneal stroma. Nevertheless, through use of positive-MACS selection for CD31 we were able to routinely observe L-MVEC in cultures derived from collagenase-digests of limbal stroma. The presence of L-MVEC in these cultures was confirmed by immunostaining for von Willebrand factor (vWF) and by ingestion of acetylated low-density lipoprotein. Moreover, the vWF+ cells formed aligned cell-to-cell ‘trains’ when grown on Geltrex™. The purity of L-MVEC cultures was found to be unrelated to tissue donor age (32 to 80 years) or duration in eye bank corneal preservation medium prior to use (3 to 10 days in Optisol) (using multiple regression test). Optimal purity of L-MVEC cultures was achieved through use of two rounds of positive-MACS selection for CD31 (mean ± s.e.m, 65.0 ± 20.8%; p<0.05). We propose that human L-MVEC cultures generated through these techniques, in conjunction with other cell types, will provide a useful tool for exploring the mechanisms of blood vessel cell growth in vitro.

Convolutional Neural Network-based place recognition

Recently Convolutional Neural Networks (CNNs) have been shown to achieve state-of-the-art performance on various classification tasks. In this paper, we present for the first time a place recognition technique based on CNN models, by combining the powerful features learnt by CNNs with a spatial and sequential filter. Applying the system to a 70 km benchmark place recognition dataset we achieve a 75% increase in recall at 100% precision, significantly outperforming all previous state of the art techniques. We also conduct a comprehensive performance comparison of the utility of features from all 21 layers for place recognition, both for the benchmark dataset and for a second dataset with more significant viewpoint changes.