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Phosphatases, together with kinases and transcription factors, are key components in cellular signalling networks. Here, we present a systematic functional analysis of the phosphatases in Cryptococcus neoformans, a fungal pathogen that causes life-threatening fungal meningoencephalitis. We analyse 230 signature-tagged mutant strains for 114 putative phosphatases under 30 distinct in vitro growth conditions, revealing at least one function for 60 of these proteins. Large-scale virulence and infectivity assays using insect and mouse models indicate roles in pathogenicity for 31 phosphatases involved in various processes such as thermotolerance, melanin and capsule production, stress responses, O-mannosylation, or retromer function. Notably, phosphatases Xpp1, Ssu72, Siw14, and Sit4 promote blood-brain barrier adhesion and crossing by C. neoformans. Together with our previous systematic studies of transcription factors and kinases, our results provide comprehensive insight into the pathobiological signalling circuitry of C. neoformans., Phosphatases are key components in cellular signalling networks. Here, the authors present a systematic functional analysis of phosphatases of the fungal pathogen Cryptococcus neoformans, revealing roles in virulence, stress responses, O-mannosylation, retromer function and other processes.

Sleep abnormality often accompanies the impairment of cognitive function. Both rapid eye movement (REM) and non-REM (NREM) sleep have associated with improved memory performance. However, the role of composition in NREM sleep, consisting of light and deep NREM, for memory formation is not fully understood. We investigated how the dynamics of NREM sleep states influence memory consolidation. Thalamocortical (TC) neuron-specific phospholipase C β4 (PLCβ4) knockout (KO) increased the total duration of NREM sleep, consisting of destabilized light NREM and stabilized deep NREM. Surprisingly, the longer NREM sleep did not improve memory consolidation but rather impaired it in TC-specific PLCβ4 KO mice. Memory function was positively correlated with the stability of light NREM and spindle activity occurring in maintained light NREM period. Our study suggests that a single molecule, PLCβ4, in TC neurons is critical for tuning the NREM sleep states and thus affects sleep-dependent memory formation.

Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, we study the roles played by fungal kinases and transcription factors (TFs) in blood-brain barrier (BBB) crossing and brain infection in mice. We use a brain infectivity assay to screen signature-tagged mutagenesis (STM)-based libraries of mutants defective in kinases and TFs, generated in the C. neoformans H99 strain. We also monitor in vivo transcription profiles of kinases and TFs during host infection using NanoString technology. These analyses identify signalling components involved in BBB adhesion and crossing, or survival in the brain parenchyma. The TFs Pdr802, Hob1, and Sre1 are required for infection under all the conditions tested here. Hob1 controls the expression of several factors involved in brain infection, including inositol transporters, a metalloprotease, PDR802, and SRE1. However, Hob1 is dispensable for most cellular functions in Cryptococcus deuterogattii R265, a strain that does not target the brain during infection. Our results indicate that Hob1 is a master regulator of brain infectivity in C. neoformans., Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, the authors identify fungal kinases and transcription factors involved in blood-brain barrier crossing and brain infection in mice.

Large DNA molecules have been utilized as a model system to investigate polymer physics. However, DNA visualization via intercalating dyes has generated equivocal results due to dye-induced structural deformation, particularly unwanted unwinding of the double helix. Thus, the contour length increases and the persistence length changes so unpredictably that there has been a controversy. In this paper, we used TAMRA-polypyrrole to stain single DNA molecules. Since this staining did not change the contour length of B-form DNA, we utilized TAMRA-polypyrrole stained DNA as a tool to measure the persistence length by changing the ionic strength. Then, we investigated DNA stretching in nanochannels by varying the ionic strength from 0.06 mM to 47 mM to evaluate several polymer physics theories proposed by Odijk, de Gennes and recent papers to deal with these regimes.

Reactions of 1-adamantylmethyl chloroformate (, 1) and 1-adamantylmethyl fluoroformate (, 2) in hydroxylic solvents have been studied. Application of the extended Grunwald-Winstein (G-W) equation to solvolyses of 1 in a variety of pure and binary solvents indicates an addition-elimination pathway in the majority of the solvents except an ionization pathway in the solvents of relatively low nucleophilcity and high ionizing power. The solvolyses of 2 show an addition-elimination pathway in all of the mixed solvents. The leaving group effects (), the kinetic solvent isotope effects (KSIEs, ), and the enthalpy and entropy of activation for the solvolyses of 1 and 2 were also calculated. The selectivity values (S) for each solvent composition are reported and discussed. These observations are compared with those previously reported for other alkyl haloformate esters.

The "parent" tertiary alkyl chloroformate, tert-butyl chloroformate, is unstable, but the tert-butyl chlorothioformate (1) is of increased stability and a kinetic investigation of the solvolyses is presented. Analyses in terms of the simple and extended Grunwald-Winstein equations are carried out. The original one-term equation satisfactorily correlates the data with a sensitivity towards changes in solvent ionizing power of 0.73 ±0.03. When the two-term equation is applied, the sensitivity towards changes in solvent nucleophilicity of 0.13 ± 0.09 is associated with a high (0.17) probability that the term that it governs is not statistically significant.

The specific rates of solvolysis of isobutyl fluoroformate (1) have been measured at 40.0 °C in 22 pure and binary solvents. These results correlated well with the extended Grunwald-Winstein (G-W) equation, which incorporated the N(T) solvent nucleophilicity scale and the Y(Cl) solvent ionizing power scale. The sensitivities (l and m-values) to changes in solvent nucleophilicity and solvent ionizing power, and the k(F)/k(Cl) values are very similar to those observed previously for solvolyses of n-octyl fluoroformate, consistent with the additional step of an addition-elimination pathway being rate-determining. The solvent deuterium isotope effect value (k(MeOH)/k(MeOD)) for methanolysis of 1 was determined, and for solvolyses in ethanol, methanol, 80% ethanol, and 70% TFE, the values of the enthalpy and the entropy of activation for the solvolysis of 1 were also determined. The results are compared with those reported earlier for isobutyl chloroformate (2) and other alkyl haloformate esters and mechanistic conclusions are drawn.

The “parent” tertiary alkyl chloroformate, tert-butyl chloroformate, is unstable, but the tert-butyl chlorothioformate (1) is of increased stability and a kinetic investigation of the solvolyses is presented. Analyses in terms of the simple and extended Grunwald-Winstein equations are carried out. The original one-term equation satisfactorily correlates the data with a sensitivity towards changes in solvent ionizing power of 0.73 ±0.03. When the two-term equation is applied, the sensitivity towards changes in solvent nucleophilicity of 0.13 ± 0.09 is associated with a high (0.17) probability that the term that it governs is not statistically significant.