Characterising the benthic Phormidium autumnale-dominated biofilm community and anatoxin production throughout biofilm succession

There has been an increase in the prevalence and intensity of Phormidium autumnale-dominated benthic blooms in New Zealand over the last decade. This species produces the potent neurotoxins Anatoxin-a, Homoanatoxin-a and their derivatives, and consumption of P. autumnale biofilms has led to over 70 dog deaths since 2005. The mechanisms regulating the dominance and toxicity of P. autumnale are still unclear, as these blooms can reach high biomass in low nutrient conditions. Benthic biofilms are composed of multiple taxa and usually harbor a complex community of bacteria and other microbes, which can change over time and interact to facilitate biofilm development and metabolic processing. Prior to this thesis, the microbial composition of P. autumnale-dominated biofilms was unknown. This study provides insights into the relationships of this neurotoxic cyanobacterium with microbial components of the biofilm community. Benthic biofilms were sampled every two to four days for 32 days from three sites in the Hutt River (Wellington) following a high flow event. A combination of microscopy and molecular techniques (bacterial ARISA and Illumina™ sequencing) were used to identify the micro-algal and bacterial components of the biofilm throughout its development. Variation in total anatoxin production was measured using LC-MS and changes in toxic P. autumnale cell numbers were quantified using QPCR. A suite of environmental variables (point velocity, depth, flow, conductivity, temperature and nutrients) were also monitored throughout the study period. Three distinct phases of microbial succession were identified (early, mid and late) using non-metric multidimensional cluster analyses. The micro-algal community composition (including P. autumnale) shifted from early to mid-phase ca. 16 days after the flushing flow and from mid to late phase at ca. day 30. The ARISA and Illumina™ sequencing showed the bacterial community shifts occurred ca. 4 and 9 days before the respective micro-algal community shifts. These analyses indicate a close coupling of the micro-algal and bacterial communities and may suggest bacterial driven succession. However, bacteria are likely to depend on micro-algal by-products for nutrition from the mid-phases onward and assessment of the metabolic processes occurring within the biofilms is needed to clarify this. Phormidium autumnale was dominant in the biofilm from an early stage in development and grew exponentially despite an influx of diatoms at day 20. None of the environmental parameters measured could explain the temporal variation in micro-algal and bacterial communities, which suggested that intrinsic rather than extrinsic factors were more important in regulating succession. This further supports the hypothesis that biofilm microbes may facilitate P. autumnale dominance. There was a significant variation in anatoxins per cell over time (p = 0.034). Production of anatoxins was greatest in the mid-phase of succession (208 fg cell⁻¹), coinciding with an increase in diatom biomass, which could implicate anatoxins as allelopathic chemicals that alleviate the effects of competition on P. autumnale. Changes in proportions of the different anatoxin variants produced over time also aligned with the three successional phases in both the micro-algal and bacterial communities, providing further evidence of a relationship between anatoxin production and microbial biofilm components. Bacterial taxa of the Alphaproteobacteria were dominant within the early bacterial community, but were surpassed by the Betaproteobacteria and Flavobacteria in mid and late phases. Bacterial genera involved in exopolysaccharide production, alkaline phosphatase activity and biopolymer degradation were identified. These attributes are important in the formation, maintenance and break-down of biofilms and therefore strengthen the likelihood of linkages between the micro-algal and bacterial community. Further investigations into functional roles of the biofilm components are needed to infer relationships between P. autumnale and the bacterial community. A clear pattern of microbial succession is described here and linkages between the micro-algal and bacterial communities are evident. Future work should focus on the functional attributes of microbes occurring at different stages of succession to further understand how P. autumnale dominates these benthic communities

Screening Historical Sexualities: A Roundtable on Sodomy, South Africa, and Proteus

(Excerpt) Proteus (2003; 100 min., Canada and South Africa) is a low-budget feature film, directed by John Greyson (Toronto) and Jack Lewis (Cape Town), that made the international rounds of “art cinema” and queer festivals in 2003 and 2004, with limited theatrical release in New York, Toronto, and other cities. The film advances Greyson’s and Lewis’s experiments with political essay-narrative forms both in their respective documentary, experimental, and dramatic videos dating back to the early 1980s (including Lewis’s Apostles of Civilized Vice [1999]) and in Greyson’s theatrical feature films beginning with Urinal in 1988. Based on an early-eighteenth-century court record, Proteus narrates the meeting, sexual relationship, and eventual trial and execution for sodomy of two prisoners in the Dutch Cape Colony, the Dutchman Rijkhaart Jacobsz and the Khoi Claas Blank. Subsidiary narratives focus on the Scottish botanist Virgil Niven, who observed the prisoners, and on the contemporaneous crackdown on sodomites in Amsterdam. GLQ initiated the following “virtual conversation” among the two directors, Israeli queer legal theorist Noa Ben-Asher, American film scholar R. Bruce Brasell, American film critic Daniel Garrett, and South African historian Susan Newton-King. Though it will “spoil” the plot for readers who have not seen the movie, we offer it as a lively debate about one of the more interesting entries in the new “new queer cinema.” The debate explores the precarious and artful interrelationship of histories, nations, narratives, and the law; cinematic intent and spectatorial interpretation; same-sexuality, conjugality, and difference; and even, as one participant dares to put it, love

Peer review manual : instructions and checklists

https://egrove.olemiss.edu/aicpa_guides/1546/thumbnail.jp

Assessment of nursing care and teaching: A qualitative approach

Background: The reform of the Spanish higher education to adapt to the European Higher Education Area involves, among other issues, the students’ participation in the curriculum assessment. The aim is to understand the insights of both nursing professionals and current undergraduate students of nursing on the connection between the knowledge acquired throughout the degree and the professional healthcare practice. Methods: An exploratory, descriptive qualitative study was carried out at a Spanish University. By convenience sampling, twelve nursing professionals and twelve 4th-year students of the Degree in Nursing were included. In two phases, twelve semi-structured interviews and two focus groups were conducted in order to triangulate data. A thematic analysis of data was carried out, later to be coded by two researchers. Results: Two main themes were identified: Evidence-based nursing vs. experience-based nursing, and a theory-practice gap. The topics that were specifically highlighted were the coexistence between professionals who work according to evidence and those whose work is based on experience, and the lack of connection between the training received during the degree and actual healthcare practice. Conclusion: Nursing care work varies in terms of the implementation of evidence-based care. Nursing training is perceived as being affected by a theory-practice gap. To achieve the linking between nursing theory and practice, a great effort on stakeholders would be needed

Identification of genes regulating the plant-specific expression of the ItmM gene in Epichloe festucae : this thesis is presented as a partial fulfillment of the requirements for the degree of Master of Science (Msc) in Genetics at Massey University, Palmerston North, New Zealand

The fungal endophyte Epichloë festucae forms a largely mutualistic association with the ryegrass species Lolium perenne. E. festucae produces a range of bio-protective alkaloids that protect the host grass from herbivory by both mammals and insects. One such alkaloid, Lolitrem B, is a potent mycotoxin and the causative agent of ryegrass staggers in livestock. Ten genes required for biosynthesis of lolitrem B are encoded in the ltm gene cluster. The ltm genes are expressed in a plant-specific manner, with high levels of expression in planta and very low levels of expression in culture. The mechanism regulating ltm gene expression is unknown but it is predicted to involve signalling from the host plant. The ltmM gene was chosen for use in the investigation of ltm gene regulation because the flanking regions do not contain retrotransposon sequence, which surrounds much of the ltm gene cluster. To identify fungal genes involved in the plant-induced expression of ltmM, a mutagenesis and screening system was developed using a PltmM-gusA ‘knock-in’ construct to detect expression from the ltmM promoter. Agrobacterium tumefaciens-mediated T-DNA mutagenesis was used to create a set of mutants with random insertions in the genome. Mutants were then screened for altered PltmM-gusA expression, both in culture and in planta. Three mutants were identified with increased PltmM-gusA expression in culture, however, no mutants were identified with loss of PltmM-gusA expression in planta. This indicates that a mechanism of repression is involved in the plant-induced expression of ltmM, either directly or indirectly. TM mutants of interest were also observed for altered symbiosis phenotypes. Mutants were identified with reduced colonisation rates and altered hyphal growth in planta. Integration sites were identified for two colonisation mutants and the disrupted genes are predicted to be the CTP:cholinephosphate cytidylyltransferase (CCT) gene PCT1 and the mitogen-activated protein kinsase kinase (MAPKK) gene mkk2

Triangle Momentum

This research investigates the breakout of security prices from periods of sideways drift known as Triangles. Contributions are made to the existing literature by considering returns conditionally based on Triangles in particular terms of how momentum traders time positions, and by then using alternative statistical methods to more clearly show results. Returns are constructed by scanning for Triangle events, and determining simulated trader returns from predetermined price levels. These are compared with a Naive model consisting of randomly sampled events of comparable measure. Modelling of momentum results is achieved using a marked point Poisson process based approach, used to compare arrival times and profit/losses. These results are confirmed using a set of 10 day return heuristics using bootstrapping to define confidence intervals. Using these methods applied to CRSP US equity data inclusive from years 1960 to 2017, US equities show a consistent but weak predictable return contribution after Triangle events occur; however, the effect has decreased over time, presumably as the market becomes more efficient. While these observed short term momentum changes in price have likely been compensated to a degree by risk, they do show that such patterns have contained forecastable information about US equities. This shows that prices have likely weakly been affected by past prices, but that currently the effect has reduced to the point that it is of negligible size as of 2017

Triangle Momentum

This research investigates the breakout of security prices from periods of sideways drift known as Triangles. Contributions are made to the existing literature by considering returns conditionally based on Triangles in particular terms of how momentum traders time positions, and by then using alternative statistical methods to more clearly show results. Returns are constructed by scanning for Triangle events, and determining simulated trader returns from predetermined price levels. These are compared with a Naive model consisting of randomly sampled events of comparable measure. Modelling of momentum results is achieved using a marked point Poisson process based approach, used to compare arrival times and profit/losses. These results are confirmed using a set of 10 day return heuristics using bootstrapping to define confidence intervals.  Using these methods applied to CRSP US equity data inclusive from years 1960 to 2017, US equities show a consistent but weak predictable return contribution after Triangle events occur; however, the effect has decreased over time, presumably as the market becomes more efficient. While these observed short term momentum changes in price have likely been compensated to a degree by risk, they do show that such patterns have contained forecastable information about US equities. This shows that prices have likely weakly been affected by past prices, but that currently the effect has reduced to the point that it is of negligible size as of 2017.</p

Nonsense suppressor therapy of cystinosis

Cystinosis is a rare, recessive lysosomal storage disorder caused by mutations in the cystinosin (CTNS) gene. CTNS encodes a H+-driven transmembrane transporter responsible for efflux of cystine from the lysosome. Loss-of-function mutations result in pathologic cystine accumulation in the lysosomes of all tissues of the body, leading to the formation of cystine crystals and, eventually, cell death. The kidney is the first organ affected. Proximal tubule dysfunction is evident by 6 months due to the huge lysosomal burden of proteins endocytosed from tubular fluid and progressive dysfunction of glomerular cells then leads to end-stage renal failure by 10 years. Current therapy involves the drug cysteamine, which allows chemical depletion of intralysosomal cystine. However, while this strategy slows organ deterioration somewhat, it does not prevent renal failure or progressive myopathy, which causes death by about 30 years. The clinical limitations of cysteamine therapy suggest there may be additional functions of CTNS protein beyond its role as a cystine channel. Indeed, recent observations indicate that CTNS has a role both in endocytic trafficking and autophagy. Thus, an effective cure for cystinosis will require restoration of all functions of CTNS protein in the cell.Interestingly, due to a founder effect, many cystinosis patients in Quebec harbor a nonsense mutation (W138X). We hypothesized that it might be possible to restore all functions of the CTNS protein in these patients by tapping the ability of aminoglycosides to overcome premature termination codons (PTCs). These drugs bind to the ribosome and induce wobble during translation, allowing insertion of a near-cognate tRNA and thus, translational readthrough. The recent development of a non-toxic aminoglycoside (ELX-02) offers the prospect of direct clinical application. Using CTNSW138X/W138X patient fibroblasts, we show that ELX-02 can normalize CTNS mRNA levels and reduce pathologic cystine accumulation by 73%. This effect is comparable to the effect of cysteamine used at peak levels achieved in patients. Furthermore, ELX-02 had a similar effect on a CTNSW138XX/57kbDel compound heterozygote where only one allele is amenable to nonsense suppression. We have also shown in our CtnsY226X/Y226X mouse model, that ELX-02 is concentrated and retained in kidney tissue for >24 hours after a relatively low dose of 10mg/kg. At this dose, ELX-02 reduces pathologic cystine accumulation in kidney tissue to about 52% of untreated levels.Our results demonstrate that ELX-02 induces sufficient translational readthrough of the French Canadian W138X mutation to produce a clinically relevant reduction in pathologic cystine accumulation. The concentration and longevity of ELX-02 in kidney suggests that it may also be useful in other hereditary renal diseases, particularly those with a high percentage of nonsense mutations, such as polycystic kidney disease.La cystinose est un défaut héréditaire rare et récessif causé par des mutations dans le gène de la cystinosine (CTNS). Le produit de ce gène est un transporteur de protons transmembranaire qui est responsable de l'efflux de la cystine du lysosome. Les mutations résultent en l'accumulation de la cystine dans les lysosomes de tous les tissues, ce qui induit la formation de cristaux de cystine et éventuellement la mort cellulaire. Le rein en est particulièrement affecté. Le dysfonctionnement du tubule proximal est évident à l'âge de 6 mois et le dysfonctionnement progressif des cellules glomérulaires mène à une insuffisance rénale terminale à l'âge de 10 ans. Le traitement actuel est la cysteamine, une drogue qui permet la déplétion chimique de la cystine intra-lysosomale et permet de ralentir la détérioration de l'organe. Cependant, elle ne prévient pas l'insuffisance rénale terminale ou la myopathie progressive, ce qui cause la mort du patient à environ 30 ans. Les limites cliniques de cette thérapie suggèrent que la protéine CTNS possède d'autres fonctions mis à part son rôle comme transporteur de cystine. En effet, des études récentes indiquent que CTNS joue un rôle dans les processus d'endocytose et l'autophagie. Conséquemment, un traitement efficace devrait maintenir toutes les fonctions de CTNS dans la cellule. En outre, en raison d'un effet fondateur, une grande majorité des patients atteints de cystinose au Québec possèdent une mutation non-sens (W138X). Notre hypothèse est qu'il serait possible de rétablir toutes les fonctions de la protéine CTNS dans ces patients en utilisant la capacité des aminoglycosides de transcender les codons de terminaison prématurés (CTPs). Ces antibiotiques se lient au ribosome et induisent un appariement non canonique lors de la traduction, permettant ainsi sa continuité. Le développement récent d'un aminoglycoside non-toxique (ELX-02) offre des possibilités d'applications cliniques. En utilisant les fibroblastes CTNSW138X/W138X d'un patient, nous avons démontré que ELX-02 peut rectifier les niveaux d'ARN messager de CTNS et réduire l'accumulation pathologique de cystine par 73%. Ceci est comparable à l'effet de la dose maximale de cysteamine utilisée par les patients. Par surcroît, ELX-02 a un effet similaire sur un hétérozygote CTNSW138XX/57kbDel dont une allèle est propice à la suppression des mutations non-sens. Nous avons aussi démontré, en utilisant notre modèle de souris CtnsY226X/Y226X, que ELX-02 est concentré et retenu dans le rein pour plus de 24 heures après une dose relativement faible de 10 mg/kg. A cette dose, ELX-02 réduit l'accumulation pathologique de cystine dans le rein à environ 52% des niveaux non-traités. Nos résultats suggèrent que ELX-02 corrige partiellement l'effet de la mutation franco-canadienne W138X, ce qui permet une diminution de l'accumulation pathologique de cystine. La rétention et la concentration de ELX-02 dans le rein suggèrent que cette drogue serait utile pour traiter d'autres maladies héréditaires du rein, particulièrement celles avec un grand pourcentage de mutations non-sens, telle que la polykystose rénale

Characterising the benthic Phormidium autumnale-dominated biofilm community and anatoxin production throughout biofilm succession

There has been an increase in the prevalence and intensity of Phormidium autumnale-dominated benthic blooms in New Zealand over the last decade. This species produces the potent neurotoxins Anatoxin-a, Homoanatoxin-a and their derivatives, and consumption of P. autumnale biofilms has led to over 70 dog deaths since 2005. The mechanisms regulating the dominance and toxicity of P. autumnale are still unclear, as these blooms can reach high biomass in low nutrient conditions. Benthic biofilms are composed of multiple taxa and usually harbor a complex community of bacteria and other microbes, which can change over time and interact to facilitate biofilm development and metabolic processing. Prior to this thesis, the microbial composition of P. autumnale-dominated biofilms was unknown. This study provides insights into the relationships of this neurotoxic cyanobacterium with microbial components of the biofilm community. Benthic biofilms were sampled every two to four days for 32 days from three sites in the Hutt River (Wellington) following a high flow event. A combination of microscopy and molecular techniques (bacterial ARISA and Illumina™ sequencing) were used to identify the micro-algal and bacterial components of the biofilm throughout its development. Variation in total anatoxin production was measured using LC-MS and changes in toxic P. autumnale cell numbers were quantified using QPCR. A suite of environmental variables (point velocity, depth, flow, conductivity, temperature and nutrients) were also monitored throughout the study period. Three distinct phases of microbial succession were identified (early, mid and late) using non-metric multidimensional cluster analyses. The micro-algal community composition (including P. autumnale) shifted from early to mid-phase ca. 16 days after the flushing flow and from mid to late phase at ca. day 30. The ARISA and Illumina™ sequencing showed the bacterial community shifts occurred ca. 4 and 9 days before the respective micro-algal community shifts. These analyses indicate a close coupling of the micro-algal and bacterial communities and may suggest bacterial driven succession. However, bacteria are likely to depend on micro-algal by-products for nutrition from the mid-phases onward and assessment of the metabolic processes occurring within the biofilms is needed to clarify this. Phormidium autumnale was dominant in the biofilm from an early stage in development and grew exponentially despite an influx of diatoms at day 20. None of the environmental parameters measured could explain the temporal variation in micro-algal and bacterial communities, which suggested that intrinsic rather than extrinsic factors were more important in regulating succession. This further supports the hypothesis that biofilm microbes may facilitate P. autumnale dominance. There was a significant variation in anatoxins per cell over time (p = 0.034). Production of anatoxins was greatest in the mid-phase of succession (208 fg cell⁻¹), coinciding with an increase in diatom biomass, which could implicate anatoxins as allelopathic chemicals that alleviate the effects of competition on P. autumnale. Changes in proportions of the different anatoxin variants produced over time also aligned with the three successional phases in both the micro-algal and bacterial communities, providing further evidence of a relationship between anatoxin production and microbial biofilm components. Bacterial taxa of the Alphaproteobacteria were dominant within the early bacterial community, but were surpassed by the Betaproteobacteria and Flavobacteria in mid and late phases. Bacterial genera involved in exopolysaccharide production, alkaline phosphatase activity and biopolymer degradation were identified. These attributes are important in the formation, maintenance and break-down of biofilms and therefore strengthen the likelihood of linkages between the micro-algal and bacterial community. Further investigations into functional roles of the biofilm components are needed to infer relationships between P. autumnale and the bacterial community. A clear pattern of microbial succession is described here and linkages between the micro-algal and bacterial communities are evident. Future work should focus on the functional attributes of microbes occurring at different stages of succession to further understand how P. autumnale dominates these benthic communities