Effect of Oxidation on the Structure of Human Low- and High-Density Lipoproteins

This work presents a controlled study of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) structural changes due to in vitro oxidation with copper ions. The changes were studied by small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS) techniques in the case of LDL and by SAXS, DLS, and Z-scan (ZS) techniques in the case of HDL. SAXS data were analyzed with a to our knowledge new deconvolution method. This method provides the electron density profile of the samples directly from the intensity scattering of the monomers. Results show that LDL particles oxidized for 18 h show significant structural changes when compared to nonoxidized particles. Changes were observed in the electrical density profile, in size polydispersity, and in the degree of flexibility of the APO-B protein on the particle. HDL optical results obtained with the ZS technique showed a decrease of the amplitude of the nonlinear optical signal as a function of oxidation time. In contrast to LDL results reported in the literature, the HDL ZS signal does not lead to a complete loss of nonlinear optical signal after 18 h of copper oxidation. Also, the SAXS results did not indicate significant structural changes due to oxidation of HDL particles, and DLS results showed that a small number of oligomers formed in the sample oxidized for 18 h. All experimental results for the HDL samples indicate that this lipoprotein is more resistant to the oxidation process than are LDL particles.     

High Spatiotemporal-Resolution Magnetic Tweezers: Calibration and Applications for DNA Dynamics

The observation of biological processes at the molecular scale in real time requires high spatial and temporal resolution. Magnetic tweezers are straightforward to implement, free of radiation or photodamage, and provide ample multiplexing capability, but their spatiotemporal resolution has lagged behind that of other single-molecule manipulation techniques, notably optical tweezers and AFM. Here, we present, to our knowledge, a new high-resolution magnetic tweezers apparatus. We systematically characterize the achievable spatiotemporal resolution for both incoherent and coherent light sources, different types and sizes of beads, and different types and lengths of tethered molecules. Using a bright coherent laser source for illumination and tracking at 6 kHz, we resolve 3 Å steps with a 1 s period for surface-melted beads and 5 Å steps with a 0.5 s period for double-stranded-dsDNA-tethered beads, in good agreement with a model of stochastic bead motion in the magnetic tweezers. We demonstrate how this instrument can be used to monitor the opening and closing of a DNA hairpin on millisecond timescales in real time, together with attendant changes in the hairpin dynamics upon the addition of deoxythymidine triphosphate. Our approach opens up the possibility of observing biological events at submillisecond timescales with subnanometer resolution using camera-based detection.     

High-resolution,High-speed,Three-dimensional Video Imaging with Digital Fringe Projection Techniques

Digital fringe projection (DFP) techniques provide dense 3D measurements of dynamically changing surfaces. Like the human eyes and brain, DFP uses triangulation between matching points in two views of the same scene at different angles to compute depth. However, unlike a stereo-based method, DFP uses a digital video projector to replace one of the cameras¹. The projector rapidly projects a known sinusoidal pattern onto the subject, and the surface of the subject distorts these patterns in the camera’s field of view. Three distorted patterns (fringe images) from the camera can be used to compute the depth using triangulation.Unlike other 3D measurement methods, DFP techniques lead to systems that tend to be faster, lower in equipment cost, more flexible, and easier to develop. DFP systems can also achieve the same measurement resolution as the camera. For this reason, DFP and other digital structured light techniques have recently been the focus of intense research (as summarized in^1-5). Taking advantage of DFP, the graphics processing unit, and optimized algorithms, we have developed a system capable of 30 Hz 3D video data acquisition, reconstruction, and display for over 300,000 measurement points per frame^6,7. Binary defocusing DFP methods can achieve even greater speeds⁸.Diverse applications can benefit from DFP techniques. Our collaborators have used our systems for facial function analysis⁹, facial animation¹⁰, cardiac mechanics studies¹¹, and fluid surface measurements, but many other potential applications exist. This video will teach the fundamentals of DFP techniques and illustrate the design and operation of a binary defocusing DFP system.     

Optimization of a Widefield Structured Illumination Microscope for Non-Destructive Assessment and Quantification of Nuclear Features in Tumor Margins of a Primary Mouse Model of Sarcoma

Cancer is associated with specific cellular morphological changes, such as increased nuclear size and crowding from rapidly proliferating cells. In situ tissue imaging using fluorescent stains may be useful for intraoperative detection of residual cancer in surgical tumor margins. We developed a widefield fluorescence structured illumination microscope (SIM) system with a single-shot FOV of 2.1×1.6 mm (3.4 mm²) and sub-cellular resolution (4.4 µm). The objectives of this work were to measure the relationship between illumination pattern frequency and optical sectioning strength and signal-to-noise ratio in turbid (i.e. thick) samples for selection of the optimum frequency, and to determine feasibility for detecting residual cancer on tumor resection margins, using a genetically engineered primary mouse model of sarcoma. The SIM system was tested in tissue mimicking solid phantoms with various scattering levels to determine impact of both turbidity and illumination frequency on two SIM metrics, optical section thickness and modulation depth. To demonstrate preclinical feasibility, ex vivo 50 µm frozen sections and fresh intact thick tissue samples excised from a primary mouse model of sarcoma were stained with acridine orange, which stains cell nuclei, skeletal muscle, and collagenous stroma. The cell nuclei were segmented using a high-pass filter algorithm, which allowed quantification of nuclear density. The results showed that the optimal illumination frequency was 31.7 µm⁻¹ used in conjunction with a 4×0.1 NA objective ( = 0.165). This yielded an optical section thickness of 128 µm and an 8.9×contrast enhancement over uniform illumination. We successfully demonstrated the ability to resolve cell nuclei in situ achieved via SIM, which allowed segmentation of nuclei from heterogeneous tissues in the presence of considerable background fluorescence. Specifically, we demonstrate that optical sectioning of fresh intact thick tissues performed equivalently in regards to nuclear density quantification, to physical frozen sectioning and standard microscopy.     

Single-Molecule Vibrational Spectroscopy Adds Structural Resolution to the Optical Trap

The ability to apply forces on single molecules with an optical trap is combined with the endogenous structural resolution of Raman spectroscopy in an article in this issue, and applied to measure the Raman spectrum of ds-DNA during force-extension.The resounding success of single-molecule biophysical techniques has encouraged the development of additional tools for more detailed exploration. The unique ability of single-molecule methods to apply force and torque, to disentangle heterogeneity, and to watch equilibrium kinetics would pair beautifully with the ultrafast time resolution and atomistic structural sensitivity of vibrational spectroscopy. However, the weak signal levels endemic to vibrations have left them mostly in the domain of bulk spectroscopy; cross-sections for Raman scattering are typically 10¹⁴ times smaller than for fluorescence emission. In this issue, Rao et al. (1) overcome this gap using surface-enhanced Raman spectroscopy (SERS) (2,3) to add vibrational spectroscopic resolution to their optical trap. In this experiment, a single DNA strand is brought into the near-field vicinity of a silver nanoparticle-coated silica bead that enhances its Raman scattering, and the spectrum is recorded as the DNA is extended in the optical trap. The authors find that applying force shifts the phosphate-stretching vibrational frequency. Molecular dynamics and density functional theory calculations were used to explain these results by showing that external load applied to the DNA backbone induces Ångstrom-level displacements in the P-O bonds.This work is immediately relevant to the communities interested in DNA mechanics and single-molecule Raman spectroscopy. While the authors’ results may refine our structural models for DNA in the low-force regime (1–9 pN), the ongoing debate about the molecular nature of the transition into overstretched DNA (≥65 pN) (4) would be well served by additional structural resolution. For the SERS community, the optical trap provides a fantastic control as it allows one to unambiguously verify that a single-molecule is probed and systematically control its distance and orientation to the metal surface, which may finally resolve long-standing mysteries about the mechanism of SERS. Ideally, both methods will be advanced in concert at the expense of coercing as much information as possible out of a single molecule.While this work is groundbreaking, the real excitement is in its potential. One limitation in most implementations of both single-molecule force and fluorescence spectroscopy is acute sensitivity to distance changes >5 nm, which diminishes upon approaching the subnanometer scale. Raman scattering and infrared absorption vibrational spectroscopies offer a complementary distance sensitivity as molecular oscillators sense their local environment and couple to one another on scales of ∼0.1 nm; see Fig. 1 for a comparison. The optical trap can now be used to initiate specific structural changes to be probed by SERS. In such mechanistic studies, one benefits from the fact that the vibrational spectrum is an endogenous probe, arising from oscillations in all the different bonds present (enzyme as well as substrate), that directly encodes the kinetics and dynamics of structural changes. Such a detailed view of hydrogen-bond rearrangements, covalent-bond formation/breaking, and symmetry changes can offer subtle details that are impossible to tag with fluorophores or directly monitor via a force measurement. As vibrational spectroscopy is rapidly approaching the molecular fingerprinting level with DNA base resolution (5) and protein identification (6), there is an optimistic future for this apparently new multiplexed technique across the various divisions of biophysics.Open in a separate windowFigure 1(A) Examples of mesoscopic structural changes typically underlying single-molecule experiments, such as unfolding of DNA and proteins; translocation of enzymes on a scaffold such as the motor proteins, dynein and kinesin, and replication proteins; and binding of substrates such as ATP and FAD (7,8). (B) Examples of microscopic structural changes probed by bulk vibrational spectroscopy, which may complement single-molecule studies such as hydrogen bonding, isomerization, subtle secondary structural changes such as α-helix rotation and β-sheet reordering, and ligand-binding geometry and kinetics (9–12).     

Light Scattering at Various Angles: Theoretical Predictions of the Effects of Particle Volume Changes

The Mie theory of scattering is used to provide new information on how changes in particle volume, with no change in dry weight, should influence light scattering for various scattering angles and particle sizes. Many biological cells (e.g., algal cells, erythrocytes) and large subcellular structures (e.g., chloroplasts, mitochondria) in suspension undergo this type of reversible volume change, a change which is related to changes in the rates of cellular processes. A previous study examined the effects of such volume changes on total scattering. In this paper scattering at 10° is found to follow total scattering closely, but scattering at 45°, 90°, 135°, and 170° behaves differently. Small volume changes can cause very large observable changes in large angle scattering if the sample particles are uniform in size; however, the natural particle size heterogeneity of most samples would mask this effect. For heterogeneous samples of most particle size ranges, particle shrink-age is found to increase large angle scattering.     

Multimodal Optical Microscopy Methods Reveal Polyp Tissue Morphology and Structure in Caribbean Reef Building Corals

An integrated suite of imaging techniques has been applied to determine the three-dimensional (3D) morphology and cellular structure of polyp tissues comprising the Caribbean reef building corals Montastraeaannularis and M. faveolata. These approaches include fluorescence microscopy (FM), serial block face imaging (SBFI), and two-photon confocal laser scanning microscopy (TPLSM). SBFI provides deep tissue imaging after physical sectioning; it details the tissue surface texture and 3D visualization to tissue depths of more than 2 mm. Complementary FM and TPLSM yield ultra-high resolution images of tissue cellular structure. Results have: (1) identified previously unreported lobate tissue morphologies on the outer wall of individual coral polyps and (2) created the first surface maps of the 3D distribution and tissue density of chromatophores and algae-like dinoflagellate zooxanthellae endosymbionts. Spectral absorption peaks of 500 nm and 675 nm, respectively, suggest that M. annularis and M. faveolata contain similar types of chlorophyll and chromatophores. However, M. annularis and M. faveolata exhibit significant differences in the tissue density and 3D distribution of these key cellular components. This study focusing on imaging methods indicates that SBFI is extremely useful for analysis of large mm-scale samples of decalcified coral tissues. Complimentary FM and TPLSM reveal subtle submillimeter scale changes in cellular distribution and density in nondecalcified coral tissue samples. The TPLSM technique affords: (1) minimally invasive sample preparation, (2) superior optical sectioning ability, and (3) minimal light absorption and scattering, while still permitting deep tissue imaging.     

Comparison between CUUG and UUCG tetraloops: thermodynamic stability and structural features analyzed by UV absorption and vibrational spectroscopy

CUUG loop is one of the most frequently occurring tetraloops in bacterial 16S rRNA. This tetraloop has a high thermodynamic stability as proved by previous UV absorption and NMR experiments. Here, we present our results concerning the thermodynamic and structural features of the 10mer 5′-r(GCG-CUUG-CGC)-3′, forming a highly stable CUUG tetraloop hairpin in aqueous solution, by means of several optical techniques (UV and FT-IR absorption, Raman scattering). UV melting profile of this decamer provides a high melting temperature (60.7°C). A set of Raman spectra recorded at different temperatures allowed us to analyze the order-to-disorder (hairpin-to-random coil) transition. Assignment of vibrational markers led us to confirm the particular nucleoside conformation, and to get information on the base stacking and base pairing in the hairpin structure. Moreover, comparison of the data obtained from two highly stable CUUG and UUCG tetraloops containing the same nucleotides but in a different order permitted an overall discussion of their structural features on the basis of Raman marker evidences.     

Simultaneous Carbon and Nitrogen Removal from Domestic Wastewater using High Rate Vermifilter

Being a cost-effective and environmentally benign technology, vermifiltration has significantly replaced the available conventional wastewater remediation methods in many cases over the last few decades. The present work emphasizes on the investigation of the nitrogen transformation dynamics, in addition to the organic carbon abatement in the designed high rate hybrid vermifilter. Moreover, the economical sustainability of the vermifiltration technology has also been enlightened by creating a bridge with the concept of circular bio-economy. The designed high rate macrophyte-assisted vermifilter (MAVF) ascertained significant high nitrogen and organic carbon removal efficiencies from the real domestic sewage, considering the chemical oxygen demand (COD) of the influent and hydraulic loading rate (HLR) as the input variables. The designed MAVF facilitated the maximum ammonium nitrogen (NH₄⁺-N), organic nitrogen, and total kjeldahl nitrogen removal efficiencies up to 98.2 ± 0.70%, 100%, and 99 ± 0.47%, respectively when COD of the influent and HLR were 200 ± 25 mg/L and 3 ± 0.1 m³/m²-d, respectively. On the other hand, substantial enhancement in the nitrate nitrogen (NO₃⁻-N) in the effluent (73 ± 10.55 times its influent concentration) was observed with influent COD of 200 ± 25 mg/L and HLR of 7 ± 0.2 m³/m²-d. When the influent COD and HLR were maintained at 700 ± 45 mg/L and 3 ± 0.1 m³/m²-d, respectively, the highest total nitrogen removal of 87 ± 2.25% was obtained. Alternatively, the influent COD of 200 ± 25 mg/L and HLR of 3 ± 0.1 m³/m²-d yielded the highest COD removal efficiency of 77 ± 1.59%. Hence, the outcome of the present research work strengthens the suitability of the vermifiltration technology as an economically and ecologically sound natural wastewater bio-remediation technology for the treatment of domestic wastewater.     

Alteration of Water Structure by Peptide Clusters Revealed by Neutron Scattering in the Small-Angle Region (below 1???1)

Solution scattering of neutrons and x-rays can provide direct information on local interactions of importance for biomolecular folding and structure. Here, neutron scattering experiments are combined with molecular-dynamics simulation to interpret the scattering signal of a series of dipeptides with varying degrees of hydrophobicity (GlyAla, GlyPro, and AlaPro) in concentrated aqueous solution (1:20 solute/water ratio) in which the peptides form large segregates (up to 50–60 amino acids). Two main results are found: 1), the shift to lower Q of the so-called water-ring peak (Q ≈ 2 Å⁻¹) arises mainly from an overlap of water-peptide and peptide-peptide correlations in the region of 1.3 < Q < 2 Å⁻¹, rather than from a shift of the water signal induced by the presence of the clusters; and 2), in the low-Q region (Q ≈ 0.6 Å⁻¹) a positive peak is observed originating from both the solute-solute correlations and changes in the water structure induced by the formation of the clusters. In particular, the water molecules are found to be more connected than in the bulk with hydrogen-bonding directions tangential to the exposed hydrophobic surfaces, and this effect increases with increasing peptide hydrophobicity. This work demonstrates that important information on the (hydrophobic) hydration of biomolecules can be obtained in the very-small-angle region.     

Self Crowding of Globular Proteins Studied by Small-Angle X-Ray Scattering

Small-angle x-ray scattering (SAXS) was used to study the behavior of equine metmyoglobin (Mb) and bovine pancreatic trypsin inhibitor (BPTI) at concentrations up to 0.4 and 0.15 g/mL, respectively, in solutions also containing 50% D₂O and 1 M urea. For both proteins, significant effects because of interference between x-rays scattered by different molecules (interparticle interference) were observed, indicating nonideal behavior at high concentrations. The experimental data were analyzed by comparison of the observed scattering profiles with those predicted by crystal structures of the proteins and a hard-sphere fluid model used to represent steric exclusion effects. The Mb scattering data were well fit by the hard-sphere model using a sphere radius of 18 Å, only slightly smaller than that estimated from the three-dimensional structure (20 Å). In contrast, the scattering profiles for BPTI in phosphate buffer displayed substantially less pronounced interparticle interference than predicted by the hard-sphere model and the radius estimated from the known structure of the protein (15 Å). Replacing the phosphate buffer with 3-(N-morpolino)propane sulfonic acid (MOPS) led to increased interparticle interference, consistent with a larger effective radius and suggesting that phosphate ions may mediate attractive intermolecular interactions, as observed in some BPTI crystal structures, without the formation of stable oligomers. The scattering data were also used to estimate second virial coefficients for the two proteins: 2.0 ×10^-4 cm³mol/g² for Mb in phosphate buffer, 1.6 ×10^-4 cm³mol/g² for BPTI in phosphate buffer and 9.2 ×10^-4 cm³mol/g² for BPTI in MOPS. The results indicate that the behavior of Mb, which is nearly isoelectric under the conditions used, is well described by the hard-sphere model, but that of BPTI is considerably more complex and is likely influenced by both repulsive and attractive electrostatic interactions. The hard-sphere model may be a generally useful tool for the analysis of small-angle scattering data from concentrated macromolecular solutions.     

Multifocal Fluorescence Microscope for Fast Optical Recordings of Neuronal Action Potentials

In recent years, optical sensors for tracking neural activity have been developed and offer great utility. However, developing microscopy techniques that have several kHz bandwidth necessary to reliably capture optically reported action potentials (APs) at multiple locations in parallel remains a significant challenge. To our knowledge, we describe a novel microscope optimized to measure spatially distributed optical signals with submillisecond and near diffraction-limit resolution. Our design uses a spatial light modulator to generate patterned illumination to simultaneously excite multiple user-defined targets. A galvanometer driven mirror in the emission path streaks the fluorescence emanating from each excitation point during the camera exposure, using unused camera pixels to capture time varying fluorescence at rates that are ∼1000 times faster than the camera’s native frame rate. We demonstrate that this approach is capable of recording Ca²⁺ transients resulting from APs in neurons labeled with the Ca²⁺ sensor Oregon Green Bapta-1 (OGB-1), and can localize the timing of these events with millisecond resolution. Furthermore, optically reported APs can be detected with the voltage sensitive dye DiO-DPA in multiple locations within a neuron with a signal/noise ratio up to ∼40, resolving delays in arrival time along dendrites. Thus, the microscope provides a powerful tool for photometric measurements of dynamics requiring submillisecond sampling at multiple locations.     

Cellular Uptake of Gold Nanoparticles and Their Behavior as Labels for Localization Microscopy

Gold nanoparticles (GNPs) enhance the damaging absorbance effects of high-energy photons in radiation therapy by increasing the emission of Auger-photoelectrons in the nm-μm range. It has been shown that the incorporation of GNPs has a significant effect on radiosensitivity of cells and their dose-dependent clonogenic survival. One major characteristic of GNPs is also their diameter-dependent cellular uptake and retention. In this article, we show by means of an established embodiment of localization microscopy, spectral position determination microscopy (SPDM), that imaging with nanometer resolution and systematic counting of GNPs becomes feasible, because optical absorption and plasmon resonance effects result in optical blinking of GNPs at a size-dependent wavelength. To quantify cellular uptake and retention or release, SPDM with GNPs that have diameters of 10 and 25 nm was performed after 2 h and after 18 h. The uptake of the GNPs in HeLa cells was either achieved via incubation or transfection via DNA labeling. On average, the uptake by incubation after 2 h was approximately double for 10 nm GNPs as compared to 25 nm GNPs. In contrast, the uptake of 25 nm GNPs by transfection was approximately four times higher after 2 h. The spectral characteristics of the fluorescence of the GNPs seem to be environment-dependent. In contrast to fluorescent dyes that show blinking characteristics due to reversible photobleaching, the blinking of GNPs seems to be stable for long periods of time, and this facilitates their use as an appropriate dye analog for SPDM imaging.     

Direct Visualization of the Murine Dorsal Cochlear Nucleus for Optogenetic Stimulation of the Auditory Pathway

Investigation into the use of virus-mediated gene transfer to arrest or reverse hearing loss has largely been relegated to the peripheral auditory system. Few studies have examined gene transfer to the central auditory system. The dorsal cochlear nucleus (DCN) of the brainstem, which contains second order neurons of the auditory pathway, is a potential site for gene transfer. In this protocol, a technique for direct and maximal exposure of the murine DCN via a posterior fossa approach is demonstrated. This approach allows for either acute or survival surgery. Following direct visualization of the DCN, a host of experiments are possible, including injection of opsins into the cochlear nucleus and subsequent stimulation by an optical fiber coupled to a blue light laser. Other neurophysiology experiments, such as electrical stimulation and neural injector tracings are also feasible. The level of visualization and the duration of stimulation achievable make this approach applicable to a wide range of experiments.     

Fingerprinting of prokaryotic 16S rRNA genes using oligodeoxyribonucleotide microarrays and virtual hybridization

An oligonucleotide microarray hybridization system to differentiate microbial species was designed and tested. Seven microbial species were studied, including one Bacillus and six Pseudomonas strains. DNA sequences near the 5′ end of 16S rRNA genes were aligned and two contiguous regions of high variability, flanked by highly conserved sequences, were found. The conserved sequences were used to design PCR primers which efficiently amplified these polymorphic regions from all seven species. The amplicon sequences were used to design 88 9mer hybridization probes which were arrayed onto glass slides. Single-stranded, fluorescence-tagged PCR products were hybridized to the microarrays at 15°C. The experimental results were compared with the ΔG° values for all matched and mismatched duplexes possible between the synthetic probes and the 16S target sequences of the seven test species, calculated using a ‘virtual hybridization’ software program. Although the observed hybridization patterns differed significantly from patterns predicted solely on the basis of perfect sequence matches, a unique hybridization fingerprint was obtained for each of the species, including closely related Pseudomonas species, and there was a reasonable correlation between the intensity of observed hybridization signals and the calculated ΔG° values. The results suggest that both perfect and mismatched pairings can contribute to microbial identification by hybridization fingerprinting.     

Resolution and Probabilistic Models of Components in CryoEM Maps of Mature P22 Bacteriophage

CryoEM continues to produce density maps of larger and more complex assemblies with multiple protein components of mixed symmetries. Resolution is not always uniform throughout a cryoEM map, and it can be useful to estimate the resolution in specific molecular components of a large assembly. In this study, we present procedures to 1) estimate the resolution in subcomponents by gold-standard Fourier shell correlation (FSC); 2) validate modeling procedures, particularly at medium resolutions, which can include loop modeling and flexible fitting; and 3) build probabilistic models that combine high-accuracy priors (such as crystallographic structures) with medium-resolution cryoEM densities. As an example, we apply these methods to new cryoEM maps of the mature bacteriophage P22, reconstructed without imposing icosahedral symmetry. Resolution estimates based on gold-standard FSC show the highest resolution in the coat region (7.6 Å), whereas other components are at slightly lower resolutions: portal (9.2 Å), hub (8.5 Å), tailspike (10.9 Å), and needle (10.5 Å). These differences are indicative of inherent structural heterogeneity and/or reconstruction accuracy in different subcomponents of the map. Probabilistic models for these subcomponents provide new insights, to our knowledge, and structural information when taking into account uncertainty given the limitations of the observed density.     

Comparison of Optical Quality and Intraocular Scattering after Posterior Chamber Phakic Intraocular Lens with and without a Central Hole (Hole ICL and Conventional ICL) Implantation Using the Double-Pass Instrument

Purpose

To objectively compare the optical quality and the intraocular scattering after implantation of the posterior chamber phakic implantable collamer lens (Visian ICL™, STAAR Surgical) with and without a central artificial hole for moderate to high ametropia.

Methods

This retrospective study comprised 28 eyes of 28 consecutive patients undergoing Hole ICL implantation (mean age ± standard deviation, 30.3±5.8 years), and 24 age-matched eyes of 24 patients undergoing conventional ICL implantation (age, 30.4±6.1 years). We quantitatively assessed the postoperative values of MTF cutoff frequency, Strehl ratio, objective scattering index (OSI), and OQAS values (OVs), using an Optical Quality Analysis System™. We compared these postoperative variables between the two groups.

Results

The mean MTF cutoff frequency, Strehl ratio, OSI, OV100%, OV 20%, and OV9%, were 26.21±8.32 cycles/degree, 0.16±0.04, 1.16±0.57, 0.87±0.28, 0.80±0.35, and 0.80±0.33, respectively, 3 months after Hole ICL implantation. We found no significant differences in the MTF cutoff frequency (Mann Whitney U test, p = 0.59), the Strehl ratio (p = 0.82), the OSI (p = 0.63), or the OVs at contrasts of 100% (p = 0.58), 20% (p = 0.40), and 9% (p = 0.87), between the two groups.

Conclusions

Both Hole ICL and conventional ICL implantation provides an excellent optical performance including intraocular scattering. Newly developed Hole ICL implantation appears to be essentially equivalent in the optical quality variables to conventional ICL implantation, suggesting that the presence of the central artificial hole does not significantly affect the optical quality and the intraocular scattering after surgery.     

Oxidation of single-stranded oligonucleotides by carbonate radical anions: generating intrastrand cross-links between guanine and thymine bases separated by cytosines

The carbonate radical anion is a biologically important one-electron oxidant that can directly abstract an electron from guanine, the most easily oxidizable DNA base. Oxidation of the 5′-d(CCTACGCTACC) sequence by photochemically generated CO₃^·− radicals in low steady-state concentrations relevant to biological processes results in the formation of spiroiminodihydantoin diastereomers and a previously unknown lesion. The latter was excised from the oxidized oligonucleotides by enzymatic digestion with nuclease P1 and alkaline phosphatase and identified by LC-MS/MS as an unusual intrastrand cross-link between guanine and thymine. In order to further characterize the structure of this lesion, 5′-d(GpCpT) was exposed to CO₃^·− radicals, and the cyclic nature of the 5′-d(G*pCpT*) cross-link in which the guanine C8-atom is bound to the thymine N3-atom was confirmed by LC-MS/MS, 1D and 2D NMR studies. The effect of bridging C bases on the cross-link formation was studied in the series of 5′-d(GpC_npT) and 5′-d(TpC_npG) sequences with n = 0, 1, 2 and 3. Formation of the G*-T* cross-links is most efficient in the case of 5′-d(GpCpT). Cross-link formation (n = 0) was also observed in double-stranded DNA molecules derived from the self-complementary 5′-d(TTACGTACGTAA) sequence following exposure to CO₃^·− radicals and enzymatic excision of the 5′-d(G^*pT^*) product.