| 1. |
- Aref, Mohaddeseh, et al.
(författare)
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Potentiometric pH Nanosensor for Intracellular Measurements: Real-Time and Continuous Assessment of Local Gradients
- 2021
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:47, s. 15744-15751
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Tidskriftsartikel (refereegranskat)abstract
- We present a pH nanosensor conceived for single intracellular measurements. The sensing architecture consisted of a two-electrode system evaluated in the potentiometric mode. We used solid-contact carbon nanopipette electrodes tailored to produce both the indicator (pH nanosensor) and reference electrodes. The indicator electrode was a membrane-based ion-selective electrode containing a receptor for hydrogen ions that provided a favorable selectivity for intracellular measurements. The analytical features of the pH nanosensor revealed a Nernstian response (slope of -59.5 mV/pH unit) with appropriate repeatability and reproducibility (variation coefficients of <2% for the calibration parameters), a fast response time (<5 s), adequate medium-term drift (0.7 mV h(-)(1)), and a linear range of response including physiological and abnormal cell pH levels (6.0-8.5). In addition, the position and configuration of the reference electrode were investigated in cell-based experiments to provide unbiased pH measurements, in which both the indicator and reference electrodes were located inside the same cell, each of them inside two neighboring cells, or the indicator electrode inside the cell and the reference electrode outside of (but nearby) the studied cell. Finally, the pH nanosensor was applied to two cases: (i) the tracing of the pH gradient from extra-to intracellular media over insertion into a single PC12 cell and (ii) the monitoring of variations in intracellular pH in response to exogenous administration of pharmaceuticals. It is anticipated that the developed pH nanosensor, which is a label-free analytical tool, has high potential to aid in the investigation of pathological states that manifest in cell pH misregulation, with no restriction in the type of targeted cells.
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| 2. |
- Fei, Keke, et al.
(författare)
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CpxR regulates the Rcs phosphorelay system in controlling the Ysc-Yop type III secretion system in Yersinia pseudotuberculosis
- 2021
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Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 167:1
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Tidskriftsartikel (refereegranskat)abstract
- The CpxRA two-component regulatory system and the Rcs phosphorelay system are both employed by the Enterobacteriaceae family to preserve bacterial envelope integrity and function when growing under stress. Although both systems regulate several overlapping physiological processes, evidence demonstrating a molecular connection between Cpx and Rcs signalling outputs is scarce. Here, we show that CpxR negatively regulates the transcription of the rcsB gene in the Rcs phosphorelay system in Yersinia pseudotuberculosis. Interestingly, transcription of rcsB is under the control of three promoters, which were all repressed by CpxR. Critically, synthetic activation of Cpx signalling through mislocalization of the NlpE lipoprotein to the inner membrane resulted in an active form of CpxR that repressed activity of rcsB promoters. On the other hand, a site-directed mutation of the phosphorylation site at residue 51 in CpxR generated an inactive non-phosphorylated variant that was unable to regulate output from these rcsB promoters. Importantly, CpxR-mediated inhibition of rcsB transcription in turn restricted activation of the Ysc-Yop type III secretion system (T3SS). Moreover, active CpxR blocks zinc-mediated activation of Rcs signalling and the subsequent activation of lcrF transcription. Our results demonstrate a novel regulatory cascade linking CpxR-RcsB-LcrF to control production of the Ysc-Yop T3SS.
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| 3. |
- Fei, Keke, et al.
(författare)
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LcrQ coordinates with the YopD-LcrH complex to repress lcrF expression and control type III secretion by Yersinia pseudotuberculosis
- 2021
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Ingår i: mBio. - : American Society for Microbiology (ASM). - 2161-2129 .- 2150-7511. ; 12:3
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Tidskriftsartikel (refereegranskat)abstract
- Human-pathogenic Yersinia species employ a plasmid-encoded type III secretion system (T3SS) to negate immune cell function during infection. A critical element in this process is the coordinated regulation of T3SS gene expression, which involves both transcriptional and posttranscriptional mechanisms. LcrQ is one of the earliest identified negative regulators of Yersinia T3SS, but its regulatory mechanism is still unclear. In a previous study, we showed that LcrQ antagonizes the activation role played by the master transcriptional regulator LcrF. In this study, we confirm that LcrQ directly interacts with LcrH, the chaperone of YopD, to facilitate the negative regulatory role of the YopD-LcrH complex in repressing lcrF expression at the posttranscriptional level. Negative regulation is strictly dependent on the YopD-LcrH complex, more so than on LcrQ. The YopD-LcrH complex helps to retain cytoplasmic levels of LcrQ to facilitate the negative regulatory effect. Interestingly, RNase E and its associated protein RhlB participate in this negative regulatory loop through a direct interaction with LcrH and LcrQ. Hence, we present a negative regulatory loop that physically connects LcrQ to the posttranscriptional regulation of LcrF, and this mechanism incorporates RNase E involved in mRNA decay.
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| 4. |
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| 5. |
- Hu, Keke, et al.
(författare)
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Electrochemical Measurements Reveal Reactive Oxygen Species in Stress Granules**
- 2021
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Ingår i: Angewandte Chemie - International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 60:28, s. 15302-15306
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Tidskriftsartikel (refereegranskat)abstract
- Stress granules (SGs) are membrane-less organelles that assemble in the cytoplasm to organize cellular contents and promote rapid adaptation during stress. To understand how SGs contribute to physiological functions, we used electrochemical measurements to detect electroactive species in SGs. With amperometry, we discovered that reactive oxygen species (ROS) are encapsulated inside arsenite-induced SGs, and H2O2 is the main species. The release kinetics of H2O2 from single SGs and the number of H2O2 molecules were quantified. The discovery that SGs contain ROS implicates them as communicators of the cellular stresses rather than a simple endpoint. This may explain how SGs regulate cellular metabolism and stress responses. This may also help better understand their cytoprotective functions in pathological conditions associated with SGs such as neurodegenerative diseases (NDs), cancers and viral infections. © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.
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