Sympathetic nerve-derived ATP regulates renal medullary vasa recta diameter via pericyte cells: a role for regulating medullary blood flow?

Pericyte cells are now known to be a novel locus of blood flow control, being able to regulate capillary diameter via their unique morphology and expression of contractile proteins. We have previously shown that exogenous ATP causes constriction of vasa recta via renal pericytes, acting at a variety of membrane bound P2 receptors on descending vasa recta (DVR), and therefore may be able to regulate medullary blood flow (MBF). Regulation of MBF is essential for appropriate urine concentration and providing essential oxygen and nutrients to this region of high, and variable, metabolic demand. Various sources of endogenous ATP have been proposed, including from epithelial, endothelial, and red blood cells in response to stimuli such as mechanical stimulation, local acidosis, hypoxia, and exposure to various hormones. Extensive sympathetic innervation of the nephron has previously been shown, however the innervation reported has focused around the proximal and distal tubules, and ascending loop of Henle. We hypothesize that sympathetic nerves are an additional source of ATP acting at renal pericytes and therefore regulate MBF. Using a rat live kidney slice model in combination with video imaging and confocal microscopy techniques we firstly show sympathetic nerves in close proximity to vasa recta pericytes in both the outer and inner medulla. Secondly, we demonstrate pharmacological stimulation of sympathetic nerves in situ (by tyramine) evokes pericyte-mediated vasoconstriction of vasa recta capillaries; inhibited by the application of the P2 receptor antagonist suramin. Lastly, tyramine-evoked vasoconstriction of vasa recta by pericytes is significantly less than ATP-evoked vasoconstriction. Sympathetic innervation may provide an additional level of functional regulation in the renal medulla that is highly localized. It now needs to be determined under which physiological/pathophysiological circumstances that sympathetic innervation of renal pericytes is important

Transplantation in HD: Are We Transplanting the Right Cells?

Cell replacement therapy is a viable option for the treatment of Huntington\u27s disease (HD), where the aim is to replace the lost medium spiny projection neurons of the striatum. The intra‐striatal engraftment of developing striatal precursors harvested from the foetal brain has provided proof of concept in both rodent models and human patients that these primary foetal tissue grafts can bring about a degree of functional recovery in a HD‐degenerated brain. With the advent of pluripotent stem cell technologies, novel, potential alternative donor cell sources have become available. Ongoing studies are assessing the capacity of these cells to differentiate towards striatal precursors for transplantation in HD. Here, we review the characteristics of potential donor cells for HD with respect to available cell markers, functional properties and maturity of cells upon transplantation. We consider the optimal composition of the donor cell population, that is, whether a heterogeneous population containing all cell types from the developing striatum (the whole ganglionic eminence) is preferable to a more homogeneous population of striatal projection neurons, as directed by differentiation protocols applied to pluripotent stem cells. Furthermore, we consider what might be required to improve transplant efficacy and success, with respect to striatal differentiation of transplanted cells and functional improvement

Co-production of 11α-hydroxyprogesterone and ethanol using recombinant yeast expressing fungal steroid hydroxylases

Background Bioethanol production from sustainable sources of biomass that limit effect on food production are needed and in a biorefinery approach co-products are desirable, obtained from both the plant material and from the microbial biomass. Fungal biotransformation of steroids was among the first industrial biotransformations allowing corticosteroid production. In this work, the potential of yeast to produce intermediates needed in corticosteroid production is demonstrated at laboratory scale following bioethanol production from perennial ryegrass juice. Results Genes encoding the 11?-steroid hydroxylase enzymes from Aspergillus ochraceus (11?-SHAoch) and Rhizopus oryzae (CYP509C12) transformed into Saccharomyces cerevisiae for heterologous constitutive expression in p425TEF. Both recombinant yeasts (AH22:p11?-SHAoch and AH22:p509C12) exhibited efficient progesterone bioconversion (on glucose minimal medial containing 300 ?M progesterone) producing either 11?-hydroxyprogesterone as the sole metabolite (AH22:p11?-SHAoch) or a 7:1 mixture of 11?-hydroxyprogesterone and 6?-hydroxyprogesterone (AH22:p509C12). Ethanol yields for AH22:p11?-SHAoch and AH22:p509C12 were comparable resulting in ?75% conversion of glucose to alcohol. Co-production of bioethanol together with efficient production of the 11-OH intermediate for corticosteroid manufacture was then demonstrated using perennial ryegrass juice. Integration of the 11?-SHAoch gene into the yeast genome (AH22:11?-SHAoch+K) resulted in a 36% reduction in yield of 11?-hydroxyprogesterone to 174 ?mol/L using 300 ?M progesterone. However, increasing progesterone concentration to 955 ?M and optimizing growth conditions increased 11?-hydroxyprogesterone production to 592 ?mol/L product formed. Conclusions The progesterone 11?-steroid hydroxylases from A. ochraceus and R. oryzae, both monooxygenase enzymes of the cytochrome P450 superfamily, have been functionally expressed in S. cerevisiae. It appears that these activities in fungi are not associated with a conserved family of cytochromes P450. The activity of the A. ochraceous enzyme was important as the specificity of the biotransformation yielded just the 11-OH product needed for corticosteroid production. The data presented demonstrate how recombinant yeast could find application in rural biorefinery processes where co-production of value-added products (11?-hydroxyprogesterone and ethanol) from novel feedstocks is an emergent and attractive possibility.publishersversionPeer reviewe

Neural stem cells for cell replacement therapy in Huntington's disease

The research reported in this thesis focused on the potential of neural precursor cells to provide a suitable source of neurones which can be used in cell replacement strategies for Huntington's disease. Specifically, the parameters affecting the differentiation of these cells into neuronal phenotypes were addressed and increasing the survival of proliferating and differentiating neurones was attempted. In vivo characteristics and the fibre projections of primary and 10 day expanded ENPs was also assessed. The limitations of xenografts in this thesis led to the search for an alternative model system for such experiments. Chapter Three involved an extensive study investigating the effects of a range of concentrations of FGF-2 and EGF on the proliferation and more importantly the neuronal differentiation of murine ENPs over 6 passages in culture, and it was found that the concentration had an effect on the neuronal proportion as well as the neuronal yield of these cultures. Chapter 4 examined the turnover of neuronal precursors in the ENP population cultured in the presence of FGF2 and EGF, using BrdU. The ongoing proliferation of neuronal precursors within ENP cultures was observed and the addition of the growth factors: CNTF, BDNF, HGF and NGF, to enhance the survival of these neurons on differentiation had no effect. Chapter 5 examined the potential of 10 day expanded human striatal ENPs to maintain a striatal like phenotype both in vitro and in vivo in comparison to primary foetal tissue. In vitro after 10 days expansion ENPs differentiated into DARPP-32 positive neurons and this characteristic was maintained in vivo, in a lesion model of HD, albeit to a much lesser extent. This study was limited by the need for ongoing immunosuppression which reduced the life span of the host animal. Chapter 6 investigates further the potential of ENPs. The ability for these cells to send long projections in the host brain and therefore repairing the circuitry lost or damaged as a result of the disease. A four way analysis was carried out examining both alio- and xenograft environments with both primary and 10 day expanded ENPs. Mouse grafts were used to address the allograft environment given that such an experiment is not possible with human tissue and both human and mouse tissue addressed the xenograft environment. To overcome the issues associated with labelling the grafted tissue in the host brain, several techniques were employed, including the use of the GFP transgenic mouse, lentiviral labelling of the cells with the LacZ gene and iontophoretic labelling of the graft with anterograde tracers. ENP grafts were shown to send out longer projections than that of primary tissue although this may be due to migration of the grafted cells. Chapter 7 addresses the issue of immunosuppression of xenografted animals. An alternative model system was explored with the hypothesis being that it would be possible to tolerise the animal in the neonatal period to the xenograft tissue that would subsequently be used for intrastriatal grafting in the adult. Indeed, tolerising the animal resulted in healthy surviving grafts in the adult without the need for daily immunosuppression. Work presented in this thesis contributes some understanding to the biology of neural stem cells and neural xenografts that may ultimately be used for neural transplantation therapies in HD

Charcoal reflectance reveals early Holocene boreal deciduous forests burned at high intensities

Published onlineJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.Wildfire size, frequency, and severity are increasing in the Alaskan boreal forest in response to climate warming. One of the potential impacts of this changing fire regime is the alteration of successional trajectories, from black spruce to mixed stands dominated by aspen, a vegetation composition not experienced since the early Holocene. Such changes in vegetation composition may consequently alter the intensity of fires, influencing fire feedbacks to the ecosystem. Paleorecords document past wildfire-vegetation dynamics and as such, are imperative for our understanding of how these ecosystems will respond to future climate warming. For the first time, we have used reflectance measurements of macroscopic charcoal particles (>180μm) from an Alaskan lake-sediment record to estimate ancient charring temperatures (termed pyrolysis intensity). We demonstrate that pyrolysis intensity increased markedly from an interval of birch tundra 11 ky ago (mean 1.52%Ro; 485°C), to the expansion of trees on the landscape ~10.5 ky ago, remaining high to the present (mean 3.54%Ro; 640°C) irrespective of stand composition. Despite differing flammabilities and adaptations to fire, the highest pyrolysis intensities derive from two intervals with distinct vegetation compositions. 1) the expansion of mixed aspen and spruce woodland at 10 cal. kyr BP, and 2) the establishment of black spruce, and the modern boreal forest at 4 cal. kyr BP. Based on our analysis, we infer that predicted expansion of deciduous trees into the boreal forest in the future could lead to high intensity, but low severity fires, potentially moderating future climate-fire feedbacks

Young Adults, Social Networks, and Addiction Recovery: Post Treatment Changes in Social Ties and Their Role as a Mediator of 12-Step Participation

Background: Social factors play a key role in addiction recovery. Research with adults indicates individuals with substance use disorder (SUD) benefit from mutual-help organizations (MHOs), such as Alcoholics Anonymous, via their ability to facilitate adaptive network changes. Given the lower prevalence of sobriety-conducive, and sobriety-supportive, social contexts in the general population during the life-stage of young adulthood, however, 12-step MHOs may play an even more crucial recovery-supportive social role for young adults, but have not been investigated. Greater knowledge could enhance understanding of recovery-related change and inform young adults’ continuing care recommendations. Methods: Emerging adults (N = 302; 18–24 yrs; 26% female; 95% White) enrolled in a study of residential treatment effectiveness were assessed at intake, 1, 3, 6, and 12 months on 12-step attendance, peer network variables (“high [relapse] risk” and “low [relapse] risk” friends), and treatment outcomes (Percent Days Abstinent; Percent Days Heavy Drinking). Hierarchical linear models tested for change in social risk over time and lagged mediational analyses tested whether 12-step attendance conferred recovery benefits via change in social risk. Results: High-risk friends were common at treatment entry, but decreased during follow-up; low-risk friends increased. Contrary to predictions, while substantial recovery-supportive friend network changes were observed, this was unrelated to 12-step participation and, thus, not found to mediate its positive influence on outcome. Conclusions: Young adult 12-step participation confers recovery benefit; yet, while encouraging social network change, 12-step MHOs may be less able to provide social network change directly for young adults, perhaps because similar-aged peers are less common in MHOs. Findings highlight the importance of both social networks and 12-step MHOs and raise further questions as to how young adults benefit from 12-step MHOs

First aid guidelines for psychosis in Asian countries: A Delphi consensus study

BACKGROUND: Guidelines for how a member of the public should give first aid to a person who is becoming psychotic have been developed for English-speaking countries. However, these guidelines may not be appropriate for use in other cultures. A study was therefore carried out to examine whether it was possible to achieve consensus on guidelines that could apply in a range of Asian countries. METHODS: A Delphi consensus study was carried out with a panel of 28 Asian mental health clinicians drawn from Cambodia, China, Hong Kong, Indonesia, Japan, Malaysia, Mongolia, South Korea, Sri Lanka, Taiwan, Thailand and Vietnam. The panel was given a 211 item questionnaire about possible first aid actions and asked to rate whether they thought these should be included in guidelines. Panel members were invited to propose additional items. RESULTS: After three Delphi rounds, there were 128 items that were rated as "essential" or "important" by 80% or more of the panel members. These items covered: recognition of psychosis, encouraging and assisting the person to seek help, how to interact with the person, responding to acute psychosis, responding to aggression, and what to do if the person refuses to get professional help. CONCLUSION: Despite the diversity of the countries involved, there was consensus on a core set of first aid items that were considered as suitable for assisting a psychotic person. Future work is needed to develop guidelines for specific countries

Can manipulation of differentiation conditions eliminate proliferative cells from a population of ES cell-derived forebrain cells?

There is preliminary evidence that implantation of primary fetal striatal cells provides functional benefit in patients with Huntington’s disease, a neurodegenerative condition resulting in loss of medium-sized spiny neurons (MSN) of the striatum. Scarcity of primary fetal tissue means it is important to identify a renewable source of cells from which to derive donor MSNs. Embryonic stem (ES) cells, which predominantly default to telencephalic-like precursors in chemically defined medium (CDM), offer a potentially inexhaustible supply of cells capable of generating the desired neurons. Using an ES cell line, with the forebrain marker FoxG1 tagged to the LacZ reporter, we assessed effects of known developmental factors on the yield of forebrain-like precursor cells in CDM suspension culture. Addition of FGF2, but not DKK1, increased the proportion of FoxG1- expressing cells at day 8 of neural induction. Oct4 was expressed at day 8, but was undetectable by day 16. Differentiation of day 16 precursors generated GABA-expressing neurons, with few DARPP32 positive MSNs. Transplantation of day 8 precursor cells into quinolinic acid-lesioned striata resulted in generation of teratomas. However, transplantation of day 16 precursors yielded grafts expressing neuronal markers including NeuN, calbindin and parvalbumin, but no DARPP32 6 weeks post-transplantation. Manipulation of fate of ES cells requires optimization of both concentration and timing of addition of factors to culture systems to generate the desired phenotypes. Furthermore, we highlight the value of increasing the precursor phase of ES cell suspension culture when directing differentiation toward forebrain fate, so as to dramatically reduce the risk of teratoma formation