10 research outputs found
FTIR difference and resonance raman spectroscopy of rhodopsins with applications to optogenetics
Thesis (Ph. D.)--Boston UniversityThe major aim of this thesis is to investigate the molecular basis for the function
of several types of rhodopsins with special emphasis on their application to the new
field of optogenetics. Rhodopsins are transmembrane biophotonic proteins with 7
a-helices and a retinal chromophore. Studies included Archaerhodopsin 3 (AR3), a
light driven proton pump similar to the extensively studied bacteriorhodopsin (BR);
channelrhodopsins 1 and 2, light-activated ion channels; sensory rhodopsin II (SRII),
a light-sensing protein that modulates phototaxis used in archaebacteria; and squid
rhodopsins (sRho), the major photopigment in squid vision and a model for human
melanopsin, which controls circadian rythms.
The primary techniques used in these studies were FTIR difference spectroscopy
and resonance Raman spectroscopy. These techniques, in combination with site directed
mutagenesis and other biochemical methodologies produced new knowledge
regarding the structural changes of the retinal chromophore, the location and function
of internal water molecules as well as specific amino acids and peptide backbone.
Specialized techniques were developed that allowed rhodopsins to be studied in intact
membrane environments and in some cases in vivo measurements were made on
rhodopsin heterologously expressed in E. coli thus allowing the effects of interacting
proteins and membrane potential to be investigated.
Evidence was found that the local environment of one or more internal water
molecules in SRII is altered by interaction with its cognate transducer, Htrii, and is
also affected by the local lipid environment. In the case of AR3, many of the broad
IR continuum absorption changes below 3000 cm-1, assigned to networks of water
molecules involved in proton transport through cytoplasmic and extracellular portions
in BR, were found to be very similar to BR. Bands assigned to water molecules
near the Schiff base postulated to be involved in proton transport were, however,
shifted or absent. Structural changes of internal water molecules and possible bands
associated with the interaction with ,8-arrestins were also detected in photoactivated
squid rhodopsin when transformed to the acid Meta intermediate. Near-IR confocal
resonance Raman measurements were performed both on AR3 reconstituted into E.
coli polar lipids and in vivo in E. coli expressing AR3 in the absence and presence of
a negative transmembrane potential. On the basis of these measurements, a model
is proposed which provides a possible explanation for the observed fluorescence dependence of AR3 and other microbial rhodopsins on transmembrane potential
Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19
IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19.
Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19.
DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022).
INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days.
MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes.
RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively).
CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes.
TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570
Near-IR Resonance Raman Spectroscopy of Archaerhodopsin 3: Effects of Transmembrane Potential
Archaerhodopsin 3 (AR3) is a light driven proton pump
from <i>Halorubrum sodomense</i> that has been used as a
genetically
targetable neuronal silencer and an effective fluorescent sensor of
transmembrane potential. Unlike the more extensively studied bacteriorhodopsin
(BR) from <i>Halobacterium salinarum</i>, AR3 readily incorporates
into the plasma membrane of both <i>E. coli</i> and mammalian
cells. Here, we used near-IR resonance Raman confocal microscopy to
study the effects of pH and membrane potential on the AR3 retinal
chromophore structure. Measurements were performed both on AR3 reconstituted
into <i>E. coli</i> polar lipids and <i>in vivo</i> in <i>E. coli</i> expressing AR3 in the absence and presence
of a negative transmembrane potential. The retinal chromophore structure
of AR3 is in an all-trans configuration almost identical to BR over
the entire pH range from 3 to 11. Small changes are detected in the
retinal ethylenic stretching frequency and Schiff Base (SB) hydrogen
bonding strength relative to BR which may be related to a different
water structure near the SB. In the case of the AR3 mutant D95N, at
neutral pH an all-trans retinal O-like species (O<sup>all‑trans</sup>) is found. At higher pH a second 13-cis retinal N-like species (N<sup>13‑cis</sup>) is detected which is attributed to a slowly
decaying intermediate in the red-light photocycle of D95N. However,
the amount of N<sup>13‑cis</sup> detected is less in <i>E. coli</i> cells but is restored upon addition of carbonyl
cyanide <i>m</i>-chlorophenyl hydrazone (CCCP) or sonication,
both of which dissipate the normal negative membrane potential. We
postulate that these changes are due to the effect of membrane potential
on the N<sup>13‑cis</sup> to M<sup>13‑cis</sup> levels
accumulated in the D95N red-light photocycle and on a molecular level
by the effects of the electric field on the protonation/deprotonation
of the cytoplasmic accessible SB. This mechanism also provides a possible
explanation for the observed fluorescence dependence of AR3 and other
microbial rhodopsins on transmembrane potential