Sequence-Dependent Melting and Refolding Dynamics of RNA UNCG Tetraloops Using Temperature-Jump/Drop Infrared Spectroscopy.

Time-resolved temperature-jump/drop infrared (IR) spectroscopy has been used to measure the impact of stem base sequence on the melting and refolding dynamics of ribonucleic acid (RNA) tetraloops. A series of three 12-nucleotide RNA hairpin sequences were studied, each featuring a UACG tetraloop motif and a double-stranded stem containing four base pairs. In each case, the stem comprised three GC pairs plus a single AU base pair inserted at the closing point of the loop (RNAloop), in the middle of the stem (RNAmid), or at the stem terminus (RNAend). Results from analogous DNA tetraloop (TACG) sequences were also obtained. Inclusion of AU or AT base pairs in the stem leads to faster melting of the stem-loop structure compared to a stem sequence featuring four GC base pairs while refolding times were found to be slower, consistent with a general reduction in stem-loop stability caused by the AU/AT pair. Independent measurement of the dynamic timescales for melting and refolding of ring vibrational modes of guanine (GR) and adenine (AR) provided position-specific insight into hairpin dynamics. The GR-derived data showed that DNA sequences melted more quickly (0.5 ± 0.1 to 0.7 ± 0.1 µs at 70 °C) than analogous RNA sequences (4.3 ± 0.4 to 4.4 ± 0.3 µs at 70 °C). Position-sensitive data from the AR modes suggests that DNA hairpins begin melting from the terminal end of the stem toward the loop while RNA sequences begin melting from the loop. Refolding timescales for both RNA and DNA hairpins were found to be similar (250 ± 50 µs at 70 °C) except for RNAend and DNAloop which refolded much more slowly (746 ± 36 and 430 ± 31 µs, respectively), showing that the refolding pathway is significantly impaired by the placement of AU/AT pairs at different points in the stem. We conclude that conformational changes of analogous pairs of RNA and DNA tetraloops proceed by different mechanisms.

Measuring RNA UNCG Tetraloop Refolding Dynamics Using Temperature-Jump/Drop Infrared Spectroscopy.

Determining the structural dynamics of RNA and DNA is essential to understanding their cellular function, but direct measurement of strand association or folding remains experimentally challenging. Here we illustrate a temperature-jump/drop method able to reveal refolding dynamics. Time-resolved temperature-jump/drop infrared spectroscopy is used to measure the melting and refolding dynamics of a 12-nucleotide RNA sequence comprising a UACG tetraloop and a four-base-pair double-stranded GC stem, comparing them to an equivalent DNA (TACG) sequence. Stem-loop melting occurred an order of magnitude more slowly in RNA than DNA (6.0 ± 0.1 µs versus 0.8 ± 0.1 µs at 70 °C). In contrast, the refolding dynamics of both sequences occurred on similar time scales (200 µs). While the melting and refolding dynamics of RNA and DNA hairpins both followed Arrhenius temperature dependences, refolding was characterized by an apparent negative activation energy, consistent with a mechanism involving multiple misfolded intermediates prior to zipping of the stem base pairs.

A 100 kHz Pulse Shaping 2D-IR Spectrometer Based on Dual Yb:KGW Amplifiers.

A high-speed, high-sensitivity and compact two-dimensional infrared (2D-IR) spectrometer based on 100 kHz Yb:KGW regenerative amplifier technology is described and demonstrated. The setup is three color, using an independent pump OPA and two separately tunable probe OPAs. The spectrometer uses 100 kHz acousto-optic pulse shaping on the pump beam for rapid 2D-IR acquisitions. The shot-to-shot stability of the laser system yields excellent signal-to-noise figures (∼10 µOD noise on 5000 laser shots). We show that the reduced bandwidth of the Yb:KGW amplifiers in comparison with conventional Ti:sapphire systems does not compromise the ability of the setup to generate high-quality 2D-IR data. Instrument responses of <300 fs are demonstrated and 2D-IR data presented for several systems of interest to physical chemists, showing spectral diffusion in NaSCN, amide I and II bands of a ß sheet protein and DNA base-pair-backbone couplings. Overall, the increased data acquisition speed, intrinsic stability, and robustness of the Yb:KGW lasers are a significant step forward for 2D-IR spectroscopy.

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay.

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu(2+)) to cuprous ions (Cu(1+)), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.

Symptoms of heat illness in surface mine workers.

OBJECTIVE: To assess the symptoms of heat illness experienced by surface mine workers. METHODS: Ninety-one surface mine workers across three mine sites in northern Australia completed a heat stress questionnaire evaluating their symptoms for heat illness. A cohort of 56 underground mine workers also participated for comparative purposes. Participants were allocated into asymptomatic, minor or moderate heat illness categories depending on the number of symptoms they reported. Participants also reported the frequency of symptom experience, as well as their hydration status (average urine colour). RESULTS: Heat illness symptoms were experienced by 87 and 79 % of surface and underground mine workers, respectively (p = 0.189), with 81-82 % of the symptoms reported being experienced by miners on more than one occasion. The majority (56 %) of surface workers were classified as experiencing minor heat illness symptoms, with a further 31 % classed as moderate; 13 % were asymptomatic. A similar distribution of heat illness classification was observed among underground miners (p = 0.420). Only 29 % of surface miners were considered well hydrated, with 61 % minimally dehydrated and 10 % significantly dehydrated, proportions that were similar among underground miners (p = 0.186). Heat illness category was significantly related to hydration status (p = 0.039) among surface mine workers, but only a trend was observed when data from surface and underground miners was pooled (p = 0.073). Compared to asymptomatic surface mine workers, the relative risk of experiencing minor and moderate symptoms of heat illness was 1.5 and 1.6, respectively, when minimally dehydrated. CONCLUSIONS: These findings show that surface mine workers routinely experience symptoms of heat illness and highlight that control measures are required to prevent symptoms progressing to medical cases of heat exhaustion or heat stroke.

Time-resolved multiple probe spectroscopy.

Time-resolved multiple probe spectroscopy combines optical, electronic, and data acquisition capabilities to enable measurement of picosecond to millisecond time-resolved spectra within a single experiment, using a single activation pulse. This technology enables a wide range of dynamic processes to be studied on a single laser and sample system. The technique includes a 1 kHz pump, 10 kHz probe flash photolysis-like mode of acquisition (pump-probe-probe-probe, etc.), increasing the amount of information from each experiment. We demonstrate the capability of the instrument by measuring the photolysis of tungsten hexacarbonyl (W(CO)(6)) monitored by IR absorption spectroscopy, following picosecond vibrational cooling of product formation through to slower bimolecular diffusion reactions on the microsecond time scale.

Use of near infrared femtosecond lasers as sub-micron radiation microbeam for cell DNA damage and repair studies.

Laser induced radiation microbeam technology for radiobiology research is undergoing rapid growth because of the increased availability and ease of use of femtosecond laser sources. The main processes involved are multiphoton absorption and/or plasma formation. The high peak powers these lasers generate make them ideal tools for depositing sub-micrometer size radiant energy within a region of a living cell nucleus to activate ionising and/or photochemically driven processes. The technique allows questions relating to the effects of low doses of radiation, the propagation and treatment of deoxyribonucleic acid (DNA) damage and repair in individual live cells as well as non-targeted cell to cell effects to be addressed. This mini-review focuses on the use of near infrared (NIR) ca. 800nm radiation to induce damage that is radically different from the early and subsequent ultraviolet microbeam techniques. Ultrafast pulsed NIR instrumentation has many benefits including the ability to eliminate issues of unspecific UV absorption by the many materials prevalent within cells. The multiphoton interaction volume also permits energy deposition beyond the diffraction limit. Work has established that the fundamental process of the damage induced by the ultrashort laser pulses is different to those induced from continuous wave light sources. Pioneering work has demonstrated that NIR laser microbeam radiation can mimic ionising radiation via multiphoton absorption within the 3D femtolitre volume of the highly focused Gaussian beam. This light-matter interaction phenomenon provides a novel optical microbeam probe for mimicking both complex ionising and UV radiation-type cell damage including double strand breaks (DSBs) and base damage. A further advantage of the pulsed laser technique is that it provides further scope for time-resolved experiments. Recently the NIR laser microbeam technique has been used to investigate the recruitment of repair proteins to the sub-micrometre size area of damage in viable cells using both immuno-fluorescent staining of gamma-H2AX (a marker for DSBs) and real-time imaging of GFP-labelled repair proteins including ATM, p53 binding protein 1 (53BP1), RAD51 and Ku 70/80 to elucidate the interaction of the two DNA DSB repair pathways, homologous recombination and the non-homologous end joining pathway.

Dynamic position and force measurement for multiple optically trapped particles using a high-speed active pixel sensor.

A high frame rate active pixel sensor designed to track the position of up to six optically trapped objects simultaneously within the field of view of a microscope is described. The sensor comprises 520 x 520 pixels from which a flexible arrangement of six independent regions of interest is accessed at a rate of up to 20 kHz, providing the capability to measure motion in multiple micron scale objects to nanometer accuracy. The combined control of both the sensor and optical traps is performed using unique, dedicated electronics (a field programmable gate array). The ability of the sensor to measure the dynamic position and the forces between six optically trapped spheres, down to femtonewton level, is demonstrated paving the way for application in the physical and life sciences.

Prediction of sublayer depth in turbid media using spatially offset Raman spectroscopy.

We demonstrate experimentally the feasibility of monitoring the depth of optically thick layers within turbid media using spatially offset Raman spectroscopy (SORS) in combination with multivariate analysis. The method uses the deep penetration capability of SORS to characterize significantly thicker (by at least a factor of 2) layers than possible with conventional Raman spectroscopy. Typical relative accuracies were between 5 and 10%. The incorporation of depth information into a SORS experiment as an additional dimension allows pure spectra of each individual layer to be resolved using three-dimensional multivariate techniques (parallel factor analysis, PARAFAC) to accuracies comparable with the results of a two-dimensional analysis.

Structure and vibrational dynamics of model compounds of the [FeFe]-hydrogenase enzyme system via ultrafast two-dimensional infrared spectroscopy.

Ultrafast two-dimensional infrared (2D) spectroscopy has been applied to study the structure and vibrational dynamics of (mu-S(CH2)3S)Fe2(CO)6, a model compound of the active site of the [FeFe]-hydrogenase enzyme system. Comparison of 2D-IR spectra of (mu-S(CH2)3S)Fe2(CO)6 with density functional theory calculations has determined that the solution-phase structure of this molecule is similar to that observed in the crystalline phase and in good agreement with gas-phase simulations. In addition, vibrational coupling and rapid (<5 ps) solvent-mediated equilibration of energy between vibrationally excited states of the carbonyl ligands of the di-iron-based active site model are observed prior to slower (approximately 100 ps) relaxation to the ground state. These dynamics are shown to be solvent-dependent and form a basis for the future determination of the vibrational interactions between active site and protein.

Solvent effects on the charge transfer excited states of 4-dimethylaminobenzonitrile (DMABN) and 4-dimethylamino-3,5-dimethylbenzonitrile (TMABN) studied by time-resolved infrared spectroscopy: a direct observation of hydrogen bonding interactions.

Time-resolved infrared absorption spectra of the C[triple bond]N bands of photoexcited TMABN and DMABN have been measured in non-polar hexane, polar aprotic THF and polar protic butanol with high temporal and spectral resolution (<0.5 ps and 5 cm(-1), respectively). In butanol, the intramolecular charge transfer (ICT) state C[triple bond]N infrared absorption bands of DMABN and TMABN both develop from an initial singlet into a doublet, demonstrating the co-existence of two charge transfer excited states, one of which is hydrogen-bonded and the other similar to the state formed in aprotic solvents. The ICT C[triple bond]N absorption band of TMABN is already strong at the earliest measurement time of 2 ps in THF, hexane, and butanol, indicating prompt population of ICT by a barrierless process, as expected from the pre-twisted structure of this molecule. There are little or no subsequent fast kinetics in hexane and THF but the signal observed in butanol continues to grow substantially at later times, prior to decay, indicating population transfer from a second state excited at 267 nm. No CN absorption band attributable to this state is observed, consistent with it being similar to the LE state of DMABN. The kinetics of the later stages of the hydrogen-bonding of both DMABN and TMABN in butanol takes place on timescales consistent with known values for dipolar solvation relaxation and result in a ratio of the hydrogen-bonded to non-bonded species of approximately 3 : 1 at equilibrium for both molecules. The contrast between the prompt population of the charge transfer state of TMABN in all three solvents and charge transfer rates in DMABN limited to 13 ps(-1) in THF and 9 ps(-1) in butanol is fully consistent with the TICT description for the ICT state structure.

Bulk Raman analysis of pharmaceutical tablets.

We compare and contrast two Raman collection geometries, backscattering and transmission, to identify their potential for monitoring the bulk chemical composition of turbid media. The experiments performed on pharmaceutical tablets confirm the expected strong bias of the backscattering Raman collection towards surface layers of the probed sample. However, this bias is largely absent with the transmission geometry, exhibiting gross insensitivity to the depth of impurities within the sample. The results are supported by Monte-Carlo simulations. The applicability of transmission geometry to tablets without any thinning is possible because of long migration times of Raman photons in non-absorbing powder media. The absolute measured intensity of the Raman signal was only 12 times lower in transmission geometry compared with backscattering geometry for a standard paracetamol tablet with a thickness of 3.9 mm. This makes detection relatively straightforward, and detectable Raman signals were observed even after propagation through three paracetamol tablets. Given its properties and instrumental simplicity, the transmission method is particularly well suited to the on-line analysis of bulk content of tablets in pharmaceutical applications.

Subsurface probing in diffusely scattering media using spatially offset Raman spectroscopy.

We describe a simple methodology for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The technique is based on the collection of Raman scattered light from surface regions that are laterally offset away from the excitation laser spot on the sample. The Raman spectra obtained in this way exhibit a variation in relative spectral intensities of the surface and subsurface layers of the sample being investigated. The data set is processed using a multivariate data analysis to yield pure Raman spectra of the individual sample layers, providing a method for the effective elimination of surface Raman scatter. The methodology is applicable to the retrieval of pure Raman spectra from depths well in excess of those accessible with conventional confocal microscopy. In this first feasibility study we have differentiated between surface and subsurface Raman signals within a diffusely scattering sample composed of two layers: trans-stilbene powder beneath a 1 mm thick over-layer of PMMA (poly(methyl methacrylate)) powder. The improvement in contrast of the subsurface trans-stilbene layer without numerical processing was 19 times. The potential applications include biomedical subsurface probing of specific tissues through different overlying tissues such as assessment of bone quality through skin, providing an effective noninvasive means of screening for bone degeneration, other skeletal disease diagnosis, and dermatology studies, as well as materials and catalyst research.

Identification and reactivity of the triplet excited state of 5-hydroxytryptophan.

Both the neurotransmitter serotonin and the unnatural amino acid 5-hydroxytryptophan (5HT), contain the 5-hydroxyindole chromophore. The photochemistry of 5HT is being investigated in relation to the multiphoton excitation of this chromophore to produce a characteristic photoproduct with green fluorescence ('hyperluminescence'). Laser flash photolysis (308 nm) of 5HT in aqueous solution at neutral pH produces both the neutral 5-indoloxyl radical (lambda(max) 400-420 nm) and another transient absorption with lambda(max) 480 nm and lifetime of 2 micros in deaerated solutions. Based on quenching by oxygen and beta-carotene, the species at 480 nm is identified as the triplet excited state of 5HT. In acidic solution a new oxygen-insensitive intermediate with lambda(max) 460 is assigned to the radical cation of 5HT. Time-resolved measurements of luminescence at 1270 nm have shown that the triplet state of 5HT is able to react with oxygen to form singlet excited oxygen (1O2*) with a quantum yield of approximately 0.1. However, 5HT has also been found to be an effective quencher of singlet oxygen with a second order rate constant of 1.3 x 10(8) dm3 mol(-1) s(-1). The results are discussed in the light of recent observations on the multiphoton-excited photochemistry of serotonin.

Depth profiling in diffusely scattering media using Raman spectroscopy and picosecond Kerr gating.

We demonstrate how pulsed laser Raman excitation (approximately 1 ps) followed by fast optical Kerr gating (approximately 4 ps) can be used to effectively separate Raman signals originating from different depths in heterogeneous diffusely scattering media. The diffuse scattering slows down photon propagation through turbid samples enabling higher depth resolution than would be obtained for a given instrumental time resolution in an optically transparent medium. Two types of experiments on two-layer systems demonstrate the ability to differentiate between surface and sub-surface Raman signals. A Raman spectrum was obtained of stilbene powder buried beneath a 1 mm over-layer of PMMA (poly(methyl methacrylate)) powder. The signal contrasts of the lower stilbene layer and upper PMMA layer were improved by factors >or=5 and >or=180, respectively, by rejecting the Raman component of the counterpart layer. The ability to select the Raman signal of a thin top surface layer in preference to those from an underlying diffusely scattering substrate was demonstrated using a 100 mum thick optically transparent film of PET (poly(ethylene terephthalate)) on top of stilbene powder. The gating resulted in the suppression of the underlying stilbene Raman signal by a factor of 1200. The experiments were performed in back-scattering geometry using 400 nm excitation wavelength. The experimental technique should be well suited to biomedical applications such as disease diagnosis.

Numerical simulations of subsurface probing in diffusely scattering media using spatially offset Raman spectroscopy.

We present the first elementary model predicting how Raman intensities vary for a range of experimental variables for spatially offset Raman spectroscopy (SORS), a recently proposed technique for the effective retrieval of Raman spectra of subsurface layers in diffusely scattering media. The model was able to reproduce the key observations made from the first SORS experiments, namely the dependence of Raman signal intensities on the spatial offset between the illumination and collection points and the relative contributions to the overall spectrum from the top layer and sub-layer. The application of the SORS concept to a three-layer system is also discussed. The model also clearly indicates that an annular geometry, rather than a point-collection geometry, which was used in the earlier experiments, would yield much improved data.

Spectroscopy of photoinduced charge-transfer reactions between tetrasulfonated aluminium phthalocyanine and methyl viologen.

Interactions between tetrasulfonated aluminium phthalocyanine (AlPcTS4-) and methyl viologen (Mv2+) have been studied in water and ethanol solutions using several experimental techniques. UV-visible absorption and fluorescence spectroscopies show that ion-pair complexes occur in ethanol, disappearing in more polar environments such as water. Time-resolved fluorescence spectroscopy (picosecond timescale) reveals the existence of several emissive species in ethanol solutions, of which one of the components is attributed to the charge-transfer complex (AlPcTS4-)(Mv2+)2, another to higher-order aggregates and yet another to the isolated AlPcTS4- molecule. The AlPcTS4- emission is quenched by Mv2+, leading to transient diffusion in the fluorescence decay kinetics. On the other hand, the emissive complex has an exponential decay with a relatively long lifetime (above 1 ns). Time-resolved absorption measurements did not reveal the existence of radicals in aqueous solution, even on the picosecond timescale. The spectra reveal the presence of excited singlet state AlPcTS4-, which decays via the triplet excited state back to the ground state.

Measurement of long-range repulsive forces between charged particles at an oil-water interface.

Using a laser tweezers method, we have determined the long-range repulsive force as a function of separation between two charged, spherical polystyrene particles (2.7 microm diameter) present at a nonpolar oil-water interface. At large separations (6 to 12 microm between particle centers) the force is found to decay with distance to the power -4 and is insensitive to the ionic strength of the aqueous phase. The results are consistent with a model in which the repulsion arises primarily from the presence of a very small residual electric charge at the particle-oil interface. This charge corresponds to a fractional dissociation of the total ionizable (sulfate) groups present at the particle-oil surface of approximately 3 x 10(-4).

Structure of the radical from one-electron oxidation of 4-hydroxycinnamate.

Radicals from one-electron oxidation of 4-hydroxycinnamate, ferulate and 3,4-dihydroxycinnamate have been formed by reaction with the oxidising triplet state of duroquinone. All three compounds react with triplet duroquinone with second order rate constants close to the diffusion-controlled limit. The identity of the resulting radicals is confirmed by observation of their characteristic visible absorption spectra. Time-resolved resonance Raman (TR3) spectra of the radical from 4-hydroxycinnamate were measured using a probe laser wavelength of 600 nm, to be in resonance with the long wavelength absorption band of the radical. The TR3 spectra contain prominent bands ascribed to the C-O and ring C-C stretching vibrations. The spectra are interpreted as indicating strong delocalisation of the radical site to the double bond in conjugation with the aromatic ring in 4-hydroxycinnamate. This contributes to the low reduction potential of the radical and the antioxidant properties of hydroxycinnamates.