Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve

Schwann cells (SCs) are the supporting cells of the peripheral nervous system and originate from the neural crest. They play a unique role in the regeneration of injured peripheral nerves and have themselves a highly unstable phenotype as demonstrated by their unexpectedly broad differentiation potential. Thus, SCs can be considered as dormant, multipotent neural crest-derived progenitors or stem cells. Upon injury they de-differentiate via cellular reprogramming, re-enter the cell cycle and participate in the regeneration of the nerve. Here we describe a protocol for efficient generation of neurospheres from intact adult rat and murine sciatic nerve without the need of experimental in vivo pre-degeneration of the nerve prior to Schwann cell isolation. After isolation and removal of the connective tissue, the nerves are initially plated on poly-D-lysine coated cell culture plates followed by migration of the cells up to 80% confluence and a subsequent switch to serum-free medium leading to formation of multipotent neurospheres. In this context, migration of SCs from the isolated nerve, followed by serum-free cultivation of isolated SCs as neurospheres mimics the injury and reprograms fully differentiated SCs into a multipotent, neural crest-derived stem cell phenotype. This protocol allows reproducible generation of multipotent Schwann cell-derived neurospheres from sciatic nerve through cellular reprogramming by culture, potentially marking a starting point for future detailed investigations of the de-differentiation process.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage.

Dynamic game soundtrack generation in response to a continously varying emotional trajectory

Dynamic soundtracking presents various practical and aesthetic challenges to composers working with games. This paper presents an implementation of a system addressing some of these challenges with an affectively-driven music generation algorithm based on a second order Markov-model. The system can respond in real-time to emotional trajectories derived from 2-dimensions of affect on the circumplex model (arousal and valence), which are mapped to five musical parameters. A transition matrix is employed to vary the generated output in continuous response to the affective state intended by the gameplay.

Challenges in quantifying wind generation's contribution to securing peak demand

Wind generation's contribution to supporting peak electricity demand is one of the key questions in wind integration studies. Differently from conventional units, the available outputs of different wind farms cannot be approximated as being statistically independent, and hence near-zero wind output is possible across an entire power system. This paper will review the risk model structures currently used to assess wind's capacity value, along with discussion of the resulting data requirements. A central theme is the benefits from performing statistical estimation of the joint distribution for demand and available wind capacity, focusing attention on uncertainties due to limited histories of wind and demand data; examination of Great Britain data from the last 25 years shows that the data requirements are greater than generally thought. A discussion is therefore presented into how analysis of the types of weather system which have historically driven extreme electricity demands can help to deliver robust insights into wind's contribution to supporting demand, even in the face of such data limitations. The role of the form of the probability distribution for available conventional capacity in driving wind capacity credit results is also discussed.

My generation(s): cycles, branding and renewal in E4’s 'Skins'

In an era of fragmenting audience and diversified viewing platforms, youth television needs to move fast and make a lot of noise in order to capture and maintain the attention of the teenage viewer. British ensemble youth drama Skins (E4, 2007-2013) calls attention to itself with its high doses of drugs, chaotic parties and casual attitudes towards sexuality. It also moves quickly, shedding its cast every two seasons as they graduate from school, then renewing itself with a fresh generation of 16 year old characters - three cycles in total. This essay will explore the challenges of maintaining audience connections whilst resetting the narrative clock with each cycle. I suggest that the development of the Skins brand was key to the programme’s success. Branding is particularly important for an audience demographic who increasingly consume their television outside of broadcast flow and essential for a programme which renews its cast every two years. The Skins brand operate as a framework, as the central audience draw, have the strength to maintain audience connections when it ‘graduates’ those characters they identify with at the close of each cycle and starts again from scratch. This essay will explore how the Skins brand constructs a cohesive identity across its multiple generations, yet also consider how the cyclic form poses challenges for the programme’s representations and narratives. This cyclic form allows Skins to repeatedly reach out to a new audience who comes of age alongside each new generation and to reflect shifts in British youth culture. Thus Skins remains ever-youthful, seeking to maintain an at times painfully hip identity. Yet the programme has a somewhat schizophrenic identity, torn between its roots in British realist drama and surrealist comedy and an escapist aspirational glamour that shows the influence of US Teen TV. This combination results in a tendency towards a heightened melodrama at odds with Skins claims for authenticity - its much vaunted teenage advisors and young writers - with the cyclic structure serving to amplify the programme’s excessive tendencies. Each cycle wrestles with a need for continuity and familiarity - partly maintained through brand, aesthetic and setting - yet a desire for freshness and originality, to assert difference from what has gone before. I suggest that the inevitable need for each cycle to ‘top’ what has gone before results in a move away from character-based intimacy and the everyday to high-stakes drama and violence which sits uncomfortably within British youth television.

Generation and Evolution of Natal Pulses: Solitary Meanders in the Agulhas Current

Solitar y meanders of the Agulhas Current, so-called Natal pulses, may play an important role in the overall dynamics of this current system. Several hypotheses concer ning the triggering of these pulses are tested using sea sur face height and temperature data from satellites. The data show the for mation of pulses in the Natal Bight area at irregular inter vals ranging from 50 to 240 days. Moving downstream at speeds between 10 and 20 km day 2 1 they sometimes reach sizes of up to 300 km. They seem to play a role in the shedding of Agulhas rings that penetrate the South Atlantic. The inter mittent for mation of these solitar y meanders is argued to be most probably related to barotropic instability of the strongly baroclinic Agulhas Current in the Natal Bight. The vorticity structure of the obser ved basic flow is argued to be stable anywhere along its path. However , a proper perturbation of the jet in the Natal Bight area will allow barotropic instability , because the bottom slope there is considerably less steep than elsewhere along the South African east coast. Using satellite altimetr y these perturbations seem to be related to the inter mittent presence of offshore anticyclonic anomalies, both upstream and eastward of the Natal Bight.

Exploiting existing ground-based remote sensing networks to improve high-resolution weather forecasts

A new generation of high-resolution (1 km) forecast models promises to revolutionize the prediction of hazardous weather such as windstorms, flash floods, and poor air quality. To realize this promise, a dense observing network, focusing on the lower few kilometers of the atmosphere, is required to verify these new forecast models with the ultimate goal of assimilating the data. At present there are insufficient systematic observations of the vertical profiles of water vapor, temperature, wind, and aerosols; a major constraint is the absence of funding to install new networks. A recent research program financed by the European Union, tasked with addressing this lack of observations, demonstrated that the assimilation of observations from an existing wind profiler network reduces forecast errors, provided that the individual instruments are strategically located and properly maintained. Additionally, it identified three further existing European networks of instruments that are currently underexploited, but with minimal expense they could deliver quality-controlled data to national weather services in near–real time, so the data could be assimilated into forecast models. Specifically, 1) several hundred automatic lidars and ceilometers can provide backscatter profiles associated with aerosol and cloud properties and structures with 30-m vertical resolution every minute; 2) more than 20 Doppler lidars, a fairly new technology, can measure vertical and horizontal winds in the lower atmosphere with a vertical resolution of 30 m every 5 min; and 3) about 30 microwave profilers can estimate profiles of temperature and humidity in the lower few kilometers every 10 min. Examples of potential benefits from these instruments are presented.

Caffeic and chlorogenic acids in Ilex paraguariensis extracts are the main inhibitors of AGE generation by methylglyoxal in model proteins

The present study concentrates on the evaluation of the anti-glycation effect of some bioactive substances present in yerba mate (Ilex paraguariensis): 5-caffeoylquinic acid, caffeic acid and a sapogenin (oleanolic acid). Bovine serum albumin and histones were incubated in the presence of methylglyoxal with or without the addition of 5-caffeoylquinic acid, caffeic acid and oleanolic acid. After the incubation period, advanced glycation end product (AGE) fluorescence spectra were performed and protein structural changes were evaluated by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. Chlorogenic acid, caffeic acid are the main substances responsible for the anti-glycation effect of mate tea. (C) 2009 Elsevier B.V. All rights reserved.

Next Generation Sequencing of Pooled Samples Reveals New SNRNP200 Mutations Associated with Retinitis Pigmentosa

The gene SNRNP200 is composed of 45 exons and encodes a protein essential for pre-mRNA splicing, the 200 kDa helicase hBrr2. Two mutations in SNRNP200 have recently been associated with autosomal dominant retinitis pigmentosa (adRP), a retinal degenerative disease, in two families from China. In this work we analyzed the entire 35-Kb SNRNP200 genomic region in a cohort of 96 unrelated North American patients with adRP. To complete this large-scale sequencing project, we performed ultra high-throughput sequencing of pooled, untagged PCR products. We then validated the detected DNA changes by Sanger sequencing of individual samples from this cohort and from an additional one of 95 patients. One of the two previously known mutations (p.S1087L) was identified in 3 patients, while 4 new missense changes (p.R681C, p.R681H, p.V683L, p.Y689C) affecting highly conserved codons were identified in 6 unrelated individuals, indicating that the prevalence of SNRNP200-associated adRP is relatively high. We also took advantage of this research to evaluate the pool-and-sequence method, especially with respect to the generation of false positive and negative results. We conclude that, although this strategy can be adopted for rapid discovery of new disease-associated variants, it still requires extensive validation to be used in routine DNA screenings. (C) 2011 Wiley-Liss, Inc.

Angiotensin II induces superoxide generation via NAD(P)H oxidase activation in isolated rat pancreatic islets

Angiotensin II (Ang II) controls blood pressure, electrolyte balance, cell growth and vascular remodeling. Ang II activates NAD(P)H oxidase in several tissues with important function in the control of insulin secretion. Considering the concomitant occurrence of hypertension, insulin resistance and pancreatic B cell secretion impairment in the development of type II diabetes the aim of the present study was to evaluate the effect of ANG II on NAD(P)H oxidase activation in isolated pancreatic islets. We found that ANGII-induced superoxide generation via NAD(P)H oxidase activation and increased protein and mRNA levels of NAD(P)H oxidase subunits (p47(PHOX) and gp91(PHOX)). (C) 2008 Elsevier B.V. All rights reserved.