6 research outputs found
Single-Cell Growth Rates in Photoautotrophic Populations Measured by Stable Isotope Probing and Resonance Raman Microspectrometry
A newmethod tomeasure growth rates of individual photoautotrophic cells by combining stable isotope probing (SIP) and single-cell resonance Raman microspectrometry is introduced. This report explores optimal experimental design and the theoretical underpinnings for quantitative responses of Raman spectra to cellular isotopic composition. Resonance Raman spectra of isogenic cultures of the cyanobacterium, Synechococcus sp., grown in 13C-bicarbonate revealed linear covariance between wavenumber (cm−1) shifts in dominant carotenoid Raman peaks and a broad range of cellular 13C fractional isotopic abundance. Single-cell growth rates were calculated from spectra-derived isotopic content and empirical relationships. Growth rates among any 25 cells in a sample varied considerably;mean coefficient of variation, CV, was 29±3%(s/x), of which only ∼2% was propagated analytical error. Instantaneous population growth rates measured independently by in vivo fluorescence also varied daily (CV ≈ 53%) and were statistically indistinguishable from single-cell growth rates at all but the lowest levels of cell labeling. SCRR censuses of mixtures prepared from Synechococcus sp. and T. pseudonana (a diatom) populations with varying 13C-content and growth rates closely approximated predicted spectral responses and fractional labeling of cells added to the sample. This approach enables direct microspectrometric interrogation of isotopically- and phylogenetically-labeled cells and detects as little as 3% changes in cellular fractional labeling. This is the first description of a non-destructive technique to measure single-cell photoautotrophic growth rates based on Raman spectroscopy and well-constrained assumptions, while requiring few ancillary measurements
Single-Cell Growth Rates in Photoautotrophic Populations Measured by Stable Isotope Probing and Resonance Raman Microspectrometry
A new method to measure growth rates of individual photoautotrophic cells by combining stable isotope probing (SIP) and single-cell resonance Raman microspectrometry is introduced. This report explores optimal experimental design and the theoretical underpinnings for quantitative responses of Raman spectra to cellular isotopic composition. Resonance Raman spectra of isogenic cultures of the cyanobacterium, Synechococcus sp., grown in 13C-bicarbonate revealed linear covariance between wavenumber (cm−1) shifts in dominant carotenoid Raman peaks and a broad range of cellular 13C fractional isotopic abundance. Single-cell growth rates were calculated from spectra-derived isotopic content and empirical relationships. Growth rates among any 25 cells in a sample varied considerably; mean coefficient of variation, CV, was 29 ± 3% (σ/x¯), of which only ~2% was propagated analytical error. Instantaneous population growth rates measured independently by in vivo fluorescence also varied daily (CV ≈ 53%) and were statistically indistinguishable from single-cell growth rates at all but the lowest levels of cell labeling. SCRR censuses of mixtures prepared from Synechococcus sp. and T. pseudonana (a diatom) populations with varying 13C-content and growth rates closely approximated predicted spectral responses and fractional labeling of cells added to the sample. This approach enables direct microspectrometric interrogation of isotopically- and phylogenetically-labeled cells and detects as little as 3% changes in cellular fractional labeling. This is the first description of a non-destructive technique to measure single-cell photoautotrophic growth rates based on Raman spectroscopy and well-constrained assumptions, while requiring few ancillary measurements
Solution Structure of Duplex DNA Containing a β‑Carba-Fapy-dG Lesion
The addition of hydroxyl radicals to the C8 position
of guanine
can lead to the formation of a 2,6-diamino-4-hydroxy-5-formamido-2′-deoxypyrimidine
(Fapy-dG) lesion, whose endogenous levels in cellular DNA rival those
of 8-oxo-7,8-dihydroxy-2′-deoxyguanosine. Despite its prevalence,
the structure of duplex DNA containing Fapy-dG is unknown. We have
prepared an undecameric duplex containing a centrally located β-cFapy-dG
residue paired to dC and determined its solution structure by high-resolution
NMR spectroscopy and restrained molecular dynamic simulations. The
damaged duplex adopts a right-handed helical structure with all residues
in an <i>anti</i> conformation, forming Watson–Crick
base pair alignments, and 2-deoxyribose conformations in the C2′-endo/C1′-exo
range. The formamido group of Fapy rotates out of the pyrimidine plane
and is present in the <i>Z</i> and <i>E</i> configurations
that equilibrate with an approximate 2:1 population ratio. The two
isomeric duplexes show similar lesion-induced deviations from a canonical
B-from DNA conformation that are minor and limited to the central
three-base-pair segment of the duplex, affecting the stacking interactions
with the 5-lesion-neighboring residue. We discuss the implications
of our observations for translesion synthesis during DNA replication
and the recognition of Fapy-dG by DNA glycosylases
Chemical Strategies for Enhancing Activity and Charge Transfer in Ultrathin Pt Nanowires Immobilized onto Nanotube Supports for the Oxygen Reduction Reaction
Multiwalled carbon nanotubes (MWNTs)
represent a promising support medium for electrocatalysts, especially
Pt nanoparticles (NPs). The advantages of using MWNTs include their
large surface area, high conductivity, as well as long-term stability.
Surface functionalization of MWNTs with various terminal groups, such
as −COOH, −SH, and −NH<sub>2</sub>, allows for
rational electronic tuning of catalyst–support interactions.
However, several issues still need to be addressed for such systems.
First, over the course of an electrochemical run, catalyst durability
can decrease, due in part to metal NP dissolution, a process facilitated
by the inherently high surface defect concentration within the support.
Second, the covalent functionalization treatment of MWNTs adopted
by most groups tends to lead to a loss of structural integrity of
the nanotubes (NTs). To mitigate for all of these issues, we have
utilized two different attachment approaches (i.e., covalent versus
noncovalent) to functionalize the outer walls of pristine MWNTs and
compared the catalytic performance of as-deposited ultrathin (<2
nm) 1D Pt nanowires with that of conventional Pt NPs toward the oxygen
reduction reaction (ORR). Our results demonstrated that the electrochemical
activity of Pt nanostructures immobilized onto functionalized carbon
nanotube (CNT) supports could be dramatically improved by using ultrathin
Pt nanowires (instead of NPs) with noncovalently (as opposed to covalently)
functionalized CNT supports. Spectroscopic evidence corroborated the
definitive presence of charge transfer between the metal catalysts
and the underlying NT support, whose direction and magnitude are a
direct function of (i) the terminal chemistry as well as (ii) the
attachment methodology, both of which simultaneously impact upon the
observed electrocatalytic performance. Specifically, the use of a
noncovalent π–π stacking method coupled with a
−COOH terminal moiety yielded the highest performance results,
reported to date, for any similar system consisting of Pt (commercial
NPs or otherwise) deposited onto carbon-based supports, a finding
of broader interest toward the fabrication of high-performing electrocatalysts
in general