Publications by authors named "Paolo Diana"

6 Publications

  • Page 1 of 1

The switching mechanism of the bacterial rotary motor combines tight regulation with inherent flexibility.

EMBO J 2021 Mar 23;40(6):e104683. Epub 2021 Feb 23.

Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, Israel.

Regulatory switches are wide spread in many biological systems. Uniquely among them, the switch of the bacterial flagellar motor is not an on/off switch but rather controls the motor's direction of rotation in response to binding of the signaling protein CheY. Despite its extensive study, the molecular mechanism underlying this switch has remained largely unclear. Here, we resolved the functions of each of the three CheY-binding sites at the switch in E. coli, as well as their different dependencies on phosphorylation and acetylation of CheY. Based on this, we propose that CheY motor switching activity is potentiated upon binding to the first site. Binding of potentiated CheY to the second site produces unstable switching and at the same time enables CheY binding to the third site, an event that stabilizes the switched state. Thereby, this mechanism exemplifies a unique combination of tight motor regulation with inherent switching flexibility.
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http://dx.doi.org/10.15252/embj.2020104683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7957414PMC
March 2021

Early exposure to general anesthesia impairs social and emotional development in rats.

Mol Neurobiol 2020 Jan 7;57(1):41-50. Epub 2019 Sep 7.

Department of Anesthesiology, University of Colorado School of Medicine, 12801 E. 17th Avenue, Rm L18-4100, Aurora, CO, USA.

Several animal and emerging human studies suggest an association between an early exposure to general anesthesia (GA) and long-lasting problems with complex social and emotional behaviors such as inattentiveness, impulsivity, anxiogenic tendencies, as well as difficulties engaging in proper social intercourse, with significant increase in attention deficit and hyperactivity-type behaviors. To further investigate these behaviors, and to examine the potential of presently available rodent behavioral models to guide future assessments of long-term socio-emotional impairments in humans, we examined the long-term effects of GA on anxiety/fear and social behaviors. We exposed male and female Sprague-Dawley infant rats at the peak of their synaptogenesis to either GA containing midazolam (9 mg/kg, i.p.), 70% nitrous oxide (NO) and 0.75% isoflurane (Iso) administered in 29-30% oxygen (experimental), or air (with 30% oxygen) plus the vehicle, 0.1% dimethyl sulfoxide (Sham) for 6 h. Behavioral experiments were conducted at adolescence (the open-field test) and young adulthood (the open-field test, the elevated plus-maze and the social novelty test). We report that an early exposure to GA during critical stages of brain development results in long-lasting increase in risk-taking tendencies and significant changes in the anxiety-related behaviors when tested in young adult rats. In addition, we noted novelty-seeking tendencies/less guarded behavior with changes in social discrimination. We conclude that early exposure to anesthesia may have lasting influences on emotional and social development. Importantly, our results show that currently used rodent behavioral models could be a good correlate to assess long-term socio-emotional GA-induced impairments observed in humans.
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http://dx.doi.org/10.1007/s12035-019-01755-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6980478PMC
January 2020

Lung ultrasound as a monitoring tool in lung transplantation in rodents: a feasibility study.

J Thorac Dis 2018 Jul;10(7):4274-4282

Department of Emergency, Azienda Ospedaliera di Padova, Padova, Italy.

Background: Orthotopic lung transplantation in rats has been developed as a model to study organ dysfunction, but available tools for monitoring the graft function are limited. In this study, lung ultrasound (LUS) is proposed as a new non-invasive monitoring tool in awake rodents.

Methods: LUS was applied to native and graft lung of six rats after left orthotopic transplantation. Rats were monitored with LUS while awake, patterns identified, images evaluated with a scoring system, intra- and inter-rater agreement was assessed and examination times analyzed.

Results: A total of 78 clips were recorded. The median quality score of LUS was 3.66/4 for left hemithorax and 3.71/4 for native right side. The intra-rater agreement was 0.53 and 0.65 and the inter-rater agreement was 0.61 (P<0.01). Median time to complete the examination was 233.0 seconds (IQR 142) for both lungs, lowered from 254.0 seconds (IQR 129.5) (first trimester of study) to 205.5 seconds (IQR 88.5) (second trimester of the study). Significant findings on LUS were confirmed on pathological examination.

Conclusions: LUS in awake rodents without shaving has been shown to be both feasible and safe and the images collected were of good quality and comparable to those obtained in anesthetized rats without bristles.
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http://dx.doi.org/10.21037/jtd.2018.06.52DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6106042PMC
July 2018

Imaging of Single Dye-Labeled Chemotaxis Proteins in Live Bacteria Using Electroporation.

Methods Mol Biol 2018 ;1729:233-246

Berry Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, UK.

For the last 2 decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signaling in E. coli, including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical. For example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart. FPs may interfere with the native interactions of the protein, and their chromophores have low brightness and photostability, and fast photobleaching rates. Electroporation allows for internalization of purified CheY proteins labeled with organic dyes into E. coli cells in controllable concentrations. Using fluorescence video microscopy, it is possible to observe single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.
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http://dx.doi.org/10.1007/978-1-4939-7577-8_19DOI Listing
December 2018

Effective and Safe Use of Glucocorticosteroids for Rescue of Late ARDS.

Case Rep Crit Care 2017 26;2017:6740532. Epub 2017 Feb 26.

Anesthesiology, University of Virginia Health System, Charlottesville, VA, USA.

We describe a case of severe refractory hypoxemia requiring prolonged extra corporeal membrane oxygenation (ECMO) support in a case of postpartum acute respiratory distress syndrome (ARDS). The clinical course was marked by persistently poor lung compliance and several complications of ECMO, that is, significant hemolysis, hemothorax, and intracranial bleeding. We report marked improvement of lung mechanics and respiratory function, leading to accelerated separation from ECMO, following rescue administration of low dose methylprednisolone 24 days after the onset of ARDS. Corticosteroid treatment was safe and well tolerated. In contrast with the conclusions of the 2006 ARDS Network trial, our report establishes a case in support of the use of low dose methylprednisolone as a safe and effective rescue treatment option in selected subsets of patients with nonresolving ARDS.
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http://dx.doi.org/10.1155/2017/6740532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5346401PMC
February 2017

Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli.

Philos Trans R Soc Lond B Biol Sci 2016 11;371(1707)

Biological Physics Research Group, Clarendon Laboratory, Department of Physics, University of Oxford, Oxford OX1 3PU, UK

For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time.This article is part of the themed issue 'The new bacteriology'.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052738PMC
http://dx.doi.org/10.1098/rstb.2015.0492DOI Listing
November 2016