Robust and stable transcriptional repression in Giardia using CRISPRi.

Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems

Aquatic fungi from peat swamp palms: Phruensis brunneispora gen. et sp. nov. and its hyphomycete anamorph

Phruensis brunneispora is a new genus and species occurring on decaying trunks of the palm Licuala longecalycata in Sirindhorn Peat Swamp Forest, Thailand. We compare the genus with other aquatic ascomycetes with falcate septate ascospores: Pseudohalonectria and Ophioceras. Ascospores differ from species in these genera in being brown with lighter end cells. Also, the ascus pore is subapical, with a channel leading to the apex. Lollipopaia minuta differs from Phruensis brunneispora in that the ascomata are borne in a stroma, asci have an apical pore and the ascospores are hyaline. No genus was found to accommodate the new species. Molecular analysis of rDNA ribosomal 18S confirmed the exclusion of the new species from Pseudohalonectria, and Ophioceras and Lollipopaia minuta formed a sister group with it. Phruensis brunneispora and Lollipopaia minuta grouped in the Diaporthales with 100% bootstrap support. Therefore, both morphological and molecular evidence supports erecting a new genus to accommodate this taxon. A hyaline Phialophora-like anamorph was formed when single ascospores were plated out on agar. The taxon is described and illustrated with light micrographs.published_or_final_versio

Tirisporella gen. nov., an ascomycete from the mangrove palm Nypa fruticans

Tirisporella beccariana comb.nov. is redescribed from decomposing leaf petiole (or rachis) bases of Nypa fruticans recently collected in Malaysia and the Philippiines. The superficial ascomata bear bitunicate asci with (3-)5(-7)-septate ascospores that are brown and verrucose, except for the prominent hyaline basal cell, and furnished with a distinctive apical appendage that arises from the spore wall. Te ultrastructure of the fungus is contrasted with that of species of Corollospora and Corallicola, with particular reference to the mode of ascospore appendage formation. The species was originally described from a Sarawak collection as Sphaeria becariana and later transferred to Melanomma and given the new name Melanomma cesatianum. Gibberidea nipae is a synonym. The recent collections were compared with type specimens. The fungus is not properly placed in Melanomma or Gibberidea or other known genera and a new genus Tiriporella is described.published_or_final_versio

Vertexicola caudatus gen. et sp. nov., and a new species of Rivulicola from submerged wood in freshwater habitats

Vertexicola caudatus gen. et sp. nov., is described and illustrated from sibmerged wood collected from a creek in the Philippines and a river in Hong Kong. Vertexicola differs from other genera in the Annulatascaceae (Sordariales) in having thickwalled distoseptate ascospores and asci with a tail-like structure. The placement of Vertexicolawithin the Annulatascaceae is discussed and it is compared with other genera in this family. Rivulicola aquatica sp. nov. is also described and illustrated.published_or_final_versio

A study of the combustion chemistry of petroleum and bio-fuel oil asphaltenes

The combustion of heavy fuel oils such as Bunker C and vacuum residual oil (VRO) are widely used for industrial applications such as furnaces, power generation and for large marine engines. There is also the possible use of bio-oils derived from biomass. Combustion of these oils generates carbonaceous particulate emissions and polynuclear aromatic hydrocarbons (PAH) that are both health hazards and have an adverse effect on the climate. This paper explores the mechanism of the formation of fine particulate soot and cenospheres. The chemical structure of petroleum asphaltene have been investigated via pyrolysis techniques. The results are consistent with a structure made up of linked small aromatic and naphthenic clusters with substituent alkyl groups, some in the long chains, with the building blocks held together by bridging groups. Other functional groups also play a role. The corresponding bio-asphaltene is made up of similar aromatic and oxygenated species and behave in an analogous way

Disrupted iron regulation in the brain and periphery in cocaine addiction

Stimulant drugs acutely increase dopamine neurotransmission in the brain, and chronic use leads to neuroadaptive changes in the mesolimbic dopamine system and morphological changes in basal ganglia structures. Little is known about the mechanisms underlying these changes but preclinical evidence suggests that iron, a coenzyme in dopamine synthesis and storage, may be a candidate mediator. Iron is present in high concentrations in the basal ganglia and stimulant drugs may interfere with iron homeostasis. We hypothesised that morphological brain changes in cocaine addiction relate to abnormal iron regulation in the brain and periphery. We determined iron concentration in the brain, using quantitative susceptibility mapping, and in the periphery, using iron markers in circulating blood, in 44 patients with cocaine addiction and 44 healthy controls. Cocaine-addicted individuals showed excess iron accumulation in the globus pallidus, which strongly correlated with duration of cocaine use, and mild iron deficiency in the periphery, which was associated with low iron levels in the red nucleus. Our findings show that iron dysregulation occurs in cocaine addiction and suggest that it arises consequent to chronic cocaine use. Putamen enlargement in these individuals was unrelated to iron concentrations, suggesting that these are co-occurring morphological changes that may respectively reflect predisposition to, and consequences of cocaine addiction. Understanding the mechanisms by which cocaine affects iron metabolism may reveal novel therapeutic targets, and determine the value of iron levels in the brain and periphery as biomarkers of vulnerability to, as well as progression and response to treatment of cocaine addiction.This work was supported by the NIHR Cambridge Biomedical Research Centre and the Behavioural and Clinical Neuroscience Institute (which was supported by a joint award from the Medical Research Council and the Wellcome Trust). The Food Frequency Questionnaire and related analysis software were used in the study. These instruments were initially developed as part of the EPIC-Norfolk Study, which was supported by Cancer Research UK programme grant (C864/A8257)

In the face of threat: neural and endocrine correlates of impaired facial emotion recognition in cocaine dependence.

The ability to recognize facial expressions of emotion in others is a cornerstone of human interaction. Selective impairments in the recognition of facial expressions of fear have frequently been reported in chronic cocaine users, but the nature of these impairments remains poorly understood. We used the multivariate method of partial least squares and structural magnetic resonance imaging to identify gray matter brain networks that underlie facial affect processing in both cocaine-dependent (n = 29) and healthy male volunteers (n = 29). We hypothesized that disruptions in neuroendocrine function in cocaine-dependent individuals would explain their impairments in fear recognition by modulating the relationship with the underlying gray matter networks. We found that cocaine-dependent individuals not only exhibited significant impairments in the recognition of fear, but also for facial expressions of anger. Although recognition accuracy of threatening expressions co-varied in all participants with distinctive gray matter networks implicated in fear and anger processing, in cocaine users it was less well predicted by these networks than in controls. The weaker brain-behavior relationships for threat processing were also mediated by distinctly different factors. Fear recognition impairments were influenced by variations in intelligence levels, whereas anger recognition impairments were associated with comorbid opiate dependence and related reduction in testosterone levels. We also observed an inverse relationship between testosterone levels and the duration of crack and opiate use. Our data provide novel insight into the neurobiological basis of abnormal threat processing in cocaine dependence, which may shed light on new opportunities facilitating the psychosocial integration of these patients.This work was funded by a research grant from the Medical Research Council (G0701497) and supported by the infrastructure of the Behavioural and Clinical Neuroscience Institute (which is supported by a joint award from the Medical Research Council and the Wellcome Trust). This study was jointly sponsored by the Cambridge University Hospitals NHS Foundation Trust and the University of Cambridge. KD Ersche, CC Hagan, and PS Jones are supported by the Medical Research Council, and DG Smith by the Cambridge Overseas Trust.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/tp.2015.5

Take it or leave it: prefrontal control in recreational cocaine users.

Though stimulant drugs such as cocaine are considered highly addictive, some individuals report recreational use over long periods without developing dependence. Difficulties in response inhibition have been hypothesized to contribute to dependence, but previous studies investigating response inhibition in recreational cocaine users have reported conflicting results. Performance on a stop-signal task was examined in 24 recreational cocaine users and 32 healthy non-drug using control participants matched for age, gender and verbal intelligence during functional magnetic resonance imaging scanning. The two groups were further matched on traumatic childhood histories and the absence of family histories of addiction. Results revealed that recreational cocaine users did not significantly differ from controls on any index of task performance, including response execution and stop-signal reaction time, with the latter averaging 198 ms in both groups. Functional magnetic resonance imaging analyses indicated that, compared with controls, stopping in the recreational users was associated with increased activation in the pre-supplementary motor area but not the right inferior frontal cortex. Thus, findings imply intact response inhibition abilities in recreational cocaine users, though the distinct pattern of accompanying activation suggests increased recruitment of brain areas implicated in response inhibition. This increased recruitment could be attributed to compensatory mechanisms that enable preserved cognitive control in this group, possibly relating to their hypothetical resilience to stimulant drug dependence. Such overactivation, alternatively, may be attributable to prolonged cocaine use leading to neuroplastic adaptations.This work was funded by a Medical Research Council (MRC) research grant to KDE, ETB and TWR (G0701497) and was conducted within the Behavioural and Clinical Neuroscience Institute, University of Cambridge, UK, which is supported by a joint award from the MRC and the Wellcome Trust; Both KDE and PSJ were supported by the MRC, SM was supported by a Wellcome Trust grant (089589/Z/09/Z) awarded to TW Robbins.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/tp.2015.8