Altered resting-state amygdala functional connectivity after 36 hours of total sleep deprivation.

Recent neuroimaging studies have identified a potentially critical role of the amygdala in disrupted emotion neurocircuitry in individuals after total sleep deprivation (TSD). However, connectivity between the amygdala and cerebral cortex due to TSD remains to be elucidated. In this study, we used resting-state functional MRI (fMRI) to investigate the functional connectivity changes of the basolateral amygdala (BLA) and centromedial amygdala (CMA) in the brain after 36 h of TSD.Fourteen healthy adult men aged 25.9 ± 2.3 years (range, 18-28 years) were enrolled in a within-subject crossover study. Using the BLA and CMA as separate seed regions, we examined resting-state functional connectivity with fMRI during rested wakefulness (RW) and after 36 h of TSD.TSD resulted in a significant decrease in the functional connectivity between the BLA and several executive control regions (left dorsolateral prefrontal cortex [DLPFC], right dorsal anterior cingulate cortex [ACC], right inferior frontal gyrus [IFG]). Increased functional connectivity was found between the BLA and areas including the left posterior cingulate cortex/precuneus (PCC/PrCu) and right parahippocampal gyrus. With regard to CMA, increased functional connectivity was observed with the rostral anterior cingulate cortex (rACC) and right precentral gyrus.These findings demonstrate that disturbance in amygdala related circuits may contribute to TSD psychophysiology and suggest that functional connectivity studies of the amygdala during the resting state may be used to discern aberrant patterns of coupling within these circuits after TSD

Acetylation of Lysine 201 Inhibits the DNA-Binding Ability of PhoP to Regulate Salmonella Virulence.

The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix-turn-helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms

Brain areas that exhibited altered functional connectivity after 36 h TSD.

<p>The left sides of the images in transverse views represent the right hemisphere.</p

Whole-brain functional connectivity patterns of the BLA and CMA before and after 36-h TSD.

<p>Brain regions with positive correlations are displayed in warm colors, while negative correlations are displayed in cool colors.</p

PhoP can be acetylated and deacetylated <i>in vitro</i> and <i>in vivo</i>.

<p>(A) The acetylation levels of PhoP in the wild type strain and <i>pat</i> deletion mutant. 6×His-tagged PhoP was expressed using pCDSS-<i>phoP</i> in the <i>pat</i> deletion mutant or the wild type <i>S</i>. Typhimurium. Acetylation levels of the purified PhoP proteins were detected with the pan anti-acetyllysine antibody (α-Acetyl), and the anti-PhoP antibody was used as a loading control. Western blots were independently repeated at least three times. (B) CobB deacetylates PhoP <i>in vitro</i>. PhoP (0.2 μg/μl) was purified and incubated with or without CobB (0.1 μg/μl), NAM (10 mM), NAD⁺ (1 mM). The acetylation levels were determined by Western blot. Western blots are representative of at least three independent replicates. (C) Pat acetylates PhoP <i>in vitro</i>. PhoP (0.2 μg/μl) was purified from the wild type strain and incubated with or without Pat (0.2 μg/μl) and Ac-CoA (0.2 mM). The acetylation levels were determined by Western blot, and Western blots are representative of at least three independent replicates.</p

Activation results from single-group, whole-brain, voxel-wise analysis of the basolateral amygdale.

<p>Activation results from single-group, whole-brain, voxel-wise analysis of the basolateral amygdale.</p

Acetylation of PhoP K201 regulates intracellular replication and inflammation response in macrophages.

<p>(A) The replication of bacteria in RAW264.7 cells. Net growth between 2 h and 24 h was calculated from the fold change in c.f.u. /ml recovered at these time points. Error bars represented SD from at least three independent experiments. (B) The replication of bacteria in primary peritoneal macrophages. Net growth between 2 h and 24 h was calculated from the fold change in c.f.u. /ml recovered at these time points. Error bars represented SD from at least three independent experiments. (C) Effect of the mimic acetylated or non-acetylated K201 on its DNA-binding ability in macrophage. RAW264.7 cells infected by chromosome PhoP-Flag mutants were collected at 24 h post-infection. DNA fragments bound to PhoP were enriched by immunoprecipitation. The relative levels of candidate genes’ promoters before (Input) and after (Flag-ChIP) IP were determined by qPCR, yielding the ChIP/Input ratio (expressed as %). Error bars represented SD from at least three independent experiments. (D) The transcription levels of candidate genes in chromosome <i>phoP</i> mutants. Bacteria were grown to OD₆₀₀~0.4, and harvested to isolate total RNA. The transcriptional level was determined by qPCR with the methods of 2^−ΔΔCt. The relative expression of tested gene was normalized to that of 16S rRNA. (E) Acetylation level of PhoP K201 in macrophages. After 24 h treatment, the intracellular bacteria were harvested for IP assay. Flag-fused PhoP proteins were immunoprecipitated by the anti-Flag antibody. The acetylation level of K201 was determined by the anti-PhoP K201Ac specific antibody (α-K201Ac), while the acetylation level of PhoP was determined by the pan anti-acetyllysine antibody (α-Acetyl). Western blots are representative of at least three independent biological replicates. (F) Comparison of the transcription levels of candidate genes between the wild type PhoP strain and PhoP K201R mutant on the <i>pat</i> deletion background. Bacteria were grown to OD₆₀₀~0.4, and harvested to isolate total RNA. The transcriptional levels of candidate genes were determined by qPCR with the methods of 2^−ΔΔCt. (G) Cytokines detection by ELISA. RAW264.7 cells were infected with chromosome PhoP mutants (MOI = 10) for 24 h. Cell culture supernatants were collected to detect the amounts of several cytokines after infection. Error bars represented SD from at least three independent experiments. *, <i>P</i><0.05; **, <i>P</i><0.01, ANOVA analysis.</p

Inflammatory pathology in the ceca of PhoP eWT, eK201Q, eK201R, and eK201A mutants infected mice.

<p>Inflammatory pathology in the ceca of PhoP eWT, eK201Q, eK201R, and eK201A mutants infected mice.</p

Acetylation of K201 impairs the binding ability of PhoP to its promoter.

<p>(A) Acetylation levels of PhoP in different strains. Bacteria were grown to log-phase (OD₆₀₀~0.4) and harvested for IP assay. Flag-fused PhoP proteins were immunoprecipitated by the anti-Flag antibody. The acetylation level of PhoP K201 was determined by the anti-PhoP K201Ac specific antibody, while the acetylation level of PhoP was determined by the pan anti-acetyllysine antibody. Western blots are representative of at least three independent replicates. (B) The acetylation level of PhoP K201Ac. The wild type PhoP and PhoP K201Ac were purified as described in “Materials and Methods” section. The acetylation levels were determined by Western blot using the pan anti-acetyllysine antibody and the anti-PhoP K201Ac specific antibody, respectively. Western blots are representative of two independent replicates. (C) CobB deacetylates PhoP K201Ac <i>in vitro</i>. PhoP K201Ac (0.2 μg/μl) was incubated with or without CobB (0.1 μg/μl), NAM (10 mM) and NAD⁺ (1 mM). The acetylation levels were determined by Western blot. Western blots are representative of two independent replicates. (D) DNA-binding abilities of PhoP and PhoP K201Ac. EMSA was used to test the binding of PhoP at indicated concentrations (lanes 2 to 5 and lanes 7 to 10) to 6’-FAM-labeled <i>phoP</i> promoter. Lane 1 and lane 6 represent the labeled DNA alone. Left panel: The wild type PhoP was incubated with probes. Right panel: PhoP K201Ac was incubated with probes. EMSA is representative of at least three independent replicates. (E) Binding of PhoP or PhoP K201Ac to the <i>phoP</i> promoter by SPR. PhoP was diluted to 1 μM, 0.6 μM, 0.3 μM (up to down) in SPR buffer and injected for 180 s (association phase). This was followed by injection of SPR buffer alone (dissociation phase). Left panel: PhoP, Right panel: PhoP K201Ac. (F) Effect of enzymatic acetylation on <i>phoP</i> DNA-binding ability in macrophage. RAW264.7 cells infected by the wild type <i>S</i>. Typhimurium strain or <i>pat</i> deletion mutant were collected at 24 h post-infection. DNA fragments bound to PhoP were enriched by immunoprecipitation. The relative levels of candidate gene promoters before (Input) and after (Flag-ChIP) IP were determined by qPCR, yielding the ChIP/Input ratio (expressed as %). Error bars represented SD from at least three independent experiments. (G)Acetylation of PhoP K201 does not affect phosphorylation of PhoP. The wild type PhoP (positive control), PhoP K201Ac and PhoP D52A (negative control) were incubated with [³²P]-AcP, and detected by exposing overnight on a phosphor screen.</p

PhoP K201 acetylation is involved in magnesium and acid tolerance response.

<p>(A) Acetylation levels of PhoP under different concentrations of magnesium. Bacteria were grown to log-phase in LB supplemented with different amount of magnesium. Flag-fused PhoP proteins were immunoprecipitated by the anti-Flag antibody. The acetylation level of PhoP was determined by the pan anti-acetyllysine antibody (α-Acetyl). Western blots are representative of at least three independent replicates. (B) Acetylation proportion of K201 under different concentrations of magnesium. Flag-fused PhoP proteins were immunoprecipitated by the anti-Flag antibody, whose acetylation fraction of K201 was determined by the anti-PhoP K201Ac specific antibody (α-K201Ac) and the anti-PhoP antibody (α-PhoP) by 2-fold serial dilution of PhoP K201Ac. Set the α-PhoP gray value to “y1”, the α-K201Ac gray value to “y2” and the protein content to “x” to draw the two scatter diagrams. The standard curves were shown as indicated. The gray values of three samples were mapped to the standard curves, so as to get the content of total PhoP (3.37 ng, 3.23 ng and 2.94 ng) and K201Ac (0.55 ng,1.22 ng and 2.03 ng). Finally, the acetylation fraction of each sample was K201Ac/ total PhoP (16%, 38% and 69%). (C) The survival rates of chromosome <i>phoP</i> mutants in log-phase ATR. Log-phase <i>S</i>. Typhimurium cells were adapted in EG medium at pH 5.8 for 1 h to induce ATR, and then cells were harvested and resuspended in the same volume of EG medium at pH 3.3. After 1 h or 2 h incubation, viable counts were recorded. *, <i>P</i><0.05, ANOVA analysis. (D) Different DNA-binding abilities of K201 mutations in log-phase ATR. Chromosome Flag-tag sequence fused <i>phoP</i> mutants were treated by EG medium at pH 5.0 for 1 h, and cells were collected and the DNA fragments bound to PhoP were immunoprecipitated according to the ChIP assay presented in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005458#sec014" target="_blank">Materials and Methods</a> section. The relative amounts of candidate gene promoters before (Input) and after (Flag-ChIP) IP were determined by qPCR, yielding the ChIP/Input ratio (expressed as %). Error bars represented SD from at least three independent experiments. *, <i>P</i><0.05; **, <i>P</i><0.01, ANOVA analysis. (E) Acetylation level of PhoPK201 after acid stimulation. After treatment with EG medium at pH 5.0or pH 7.7 for 1 h, cells were harvested for IP assay. Flag-fused PhoP proteins were immunoprecipitated by the anti-Flag antibody. The acetylation level of PhoP K201 was determined by the anti-PhoP K201Ac specific antibody (α-K201Ac), while the acetylation level of PhoP was determined by the pan anti-acetyl lysine antibody (α-Acetyl). Western blots are representative of at least three independent biological replicates.</p