CRISPR-based tools for targeted genetic manipulation in pathogenic Sporothrix species

is an emerging fungal pathogen frequently associated with zoonotic transmission of sporotrichosis by contaminated cats. Within 25 years, the disease has spread not only throughout Brazil but now to neighboring countries in Latin America. Thermo-dimorphism, melanin, glycans, adhesins, and secreted vesicles have been associated with the ability of species to cause disease in the mammalian host. Although certain virulence factors have been proposed as potential determinants for sporotrichosis, the scarcity of molecular tools for performing reverse genetics in has significantly impeded the dissection of mechanisms underlying the disease. Here, we demonstrate that PEG-mediated protoplast transformation is a powerful method for heterologous gene expression in and . Combined with CRISPR/Cas9 gene editing, this transformation protocol enabled the deletion of the putative DHN-melanin synthase gene , which is a proposed virulence factor of species. To improve integration of deletion constructs, we deleted the KU homolog that is critical for non-homologous end-joining DNA repair. The use of Δ strains from enhanced homologous-directed repair during transformation resulting in increased targeted gene deletion in combination with CRISPR/Cas9. In conclusion, our CRISPR/Cas9-based transformation protocol provides an efficient tool for targeted gene manipulation in species. IMPORTANCE Sporotrichosis caused by is a disease that requires long periods of treatment and is rapidly spreading across Latin America. The virulence of this fungus and the surge of atypical and more severe presentations of the disease raise the need for an understanding of the molecular mechanisms underlying sporotrichosis, as well as the development of better diagnostics and antifungal therapies. By developing molecular tools for accurate genetic manipulation in this study addresses the paucity of reliable and reproducible tools for stable genetic engineering of species, which has represented a major obstacle for studying the virulence determinants and their roles in the establishment of sporotrichosis

Investigating paradoxical hysteresis effects in the mouse neocortical slice model

Clinically, anesthetic drugs show hysteresis in the plasma drug concentrations at induction versus emergence from anesthesia induced unconsciousness. This is assumed to be the result of pharmacokinetic lag between the plasma and brain effect-site and vice versa. However, recent mathematical and experimental studies demonstrate that anesthetic hysteresis might be due in part to lag in the brain physiology, independent of drug transport delay — so-called “neural inertia”. The aim of this study was to investigate neural inertia in the reduced neocortical mouse slice model. Seizure-like event (SLE) activity was generated by exposing cortical slices to no-magnesium artificial cerebrospinal fluid (aCSF). Concentration–effect loops were generated by manipulating SLE frequency, using the general anesthetic drug etomidate and by altering the aCSF magnesium concentration. The etomidate (24 μM) concentration–effect relationship showed a clear hysteresis, consistent with the slow diffusion of etomidate into slice tissue. Manipulation of tissue excitability, using either carbachol (50 μM) or elevated potassium (5 mM vs 2.5 mM) did not significantly alter the size of etomidate hysteresis loops. Hysteresis in the magnesium concentration–effect relationship was evident, but only when the starting condition was magnesium-containing “normal” aCSF. The in vitro cortical slice manifests pathway-dependent “neural inertia” and may be a valuable model for future investigations into the mechanisms of neural inertia in the cerebral cortex

The Utility of Zebrafish to Study the Mechanisms by which Ethanol Affects Social Behavior and Anxiety During Early Brain Development 2 3

Abstract 27 Exposure to moderate levels of ethanol during brain development has a number of 28 effects on social behavior but the molecular mechanisms that mediate this are not well 32 Embryos were exposed to 20mM ethanol for seven days (48hpf -9dpf) and tested as 33 adults for individual social behavior and shoaling. We also tested their basal anxiety 34 with the novel tank diving test. We found that the ethanol--exposed fish displayed 35 reductions in social approach and shoaling, and an increase in anxiety in the novel tank 36 test. These behavioral differences corresponded to differences in hrt1aa, slc6a4 and oxtr 37 expression. Namely, acute ethanol caused a spike in oxtr and ht1aa mRNA expression

Trophic level asynchrony in rates of phenological change for marine, freshwater and terrestrial environments

Recent changes in the seasonal timing (phenology) of familiar biological events have been one of the most conspicuous signs of climate change. However, the lack of a standardised approach to analysing change has hampered assessment of consistency in such changes among different taxa and trophic levels and across freshwater, terrestrial and marine environments. We present a standardised assessment of 25,532 rates of phenological change for 726 UK terrestrial, freshwater and marine taxa. The majority of spring and summer events have advanced, and more rapidly than previously documented. Such consistency is indicative of shared large-scale drivers. Furthermore, average rates of change have accelerated in a way that is consistent with observed warming trends. Less coherent patterns in some groups of organisms point to the agency of more local scale processes and multiple drivers. For the first time we show a broad scale signal of differential phenological change among trophic levels; across environments advances in timing were slowest for secondary consumers, thus heightening the potential risk of temporal mismatch in key trophic interactions. If current patterns and rates of phenological change are indicative of future trends, future climate warming may exacerbate trophic mismatching, further disrupting the functioning, persistence and resilience of many ecosystems and having a major impact on ecosystem services

Developmental role of acetylcholinesterase in impulse control in zebrafish

Cellular and molecular processes that mediate individual variability in impulsivity, a key behavioural component of many neuropsychiatric disorders, are poorly understood. Zebrafish heterozygous for a nonsense mutation in Ache (achesb55/+) showed lower levels of impulsivity in a 5-choice serial reaction time task (5-CSRTT) than wild type and ache+/+. Assessment of expression of cholinergic (nAChR), serotonergic (5-HT) and dopamine (DR) receptor mRNA in both adult and larval (9dpf) achesb55/+ revealed significant downregulation of Chrna2, Chrna5 and Drd2 mRNA in achesb55/+ larvae, but no differences in adults. Acute exposure to cholinergic agonist/antagonists had no effect on impulsivity, supporting the hypothesis that behavioural effects observed in adults were due to lasting impact of developmental alterations in cholinergic and dopaminergic signalling. This shows the cross-species role of cholinergic signalling during brain development in impulsivity, and suggests zebrafish may be a useful model for the role of cholinergic pathways as a target for therapeutic advances in addiction medicine