Detection of Lumbar Spondylolisthesis from X-ray Images Using Deep Learning Network.

Spondylolisthesis refers to the displacement of a vertebral body relative to the vertrabra below it, which can cause radicular symptoms, back pain or leg pain. It usually occurs in the lower lumbar spine, especially in women over the age of 60. The prevalence of spondylolisthesis is expected to rise as the global population ages, requiring prudent action to promptly identify it in clinical settings. The goal of this study was to develop a computer-aided diagnostic (CADx) algorithm, LumbarNet, and to evaluate the efficiency of this model in automatically detecting spondylolisthesis from lumbar X-ray images. Built upon U-Net, feature fusion module (FFM) and collaborating with (i) a P-grade, (ii) a piecewise slope detection (PSD) scheme, and (iii) a dynamic shift (DS), LumbarNet was able to analyze complex structural patterns on lumbar X-ray images, including true lateral, flexion, and extension lateral views. Our results showed that the model achieved a mean intersection over union (mIOU) value of 0.88 in vertebral region segmentation and an accuracy of 88.83% in vertebral slip detection. We conclude that LumbarNet outperformed U-Net, a commonly used method in medical image segmentation, and could serve as a reliable method to identify spondylolisthesis.

Dynamic Interconversions of HCV Helicase Binding Modes on the Nucleic Acid Substrate.

The dynamics involved in the interaction between hepatitis C virus nonstructural protein 3 (NS3) C-terminal helicase and its nucleic acid substrate have been the subject of interest for some time given the key role of this enzyme in viral replication. Here, we employed fluorescence-based techniques and focused on events that precede the unwinding process. Both ensemble Förster resonance energy transfer (FRET) and ensemble protein induced fluorescence enhancement (PIFE) assays show binding on the 3' single-stranded overhang of model DNA substrates (>5 nucleotides) with no preference for the single-stranded/double-stranded (ss/ds) junction. Single-molecule PIFE experiments revealed three enhancement levels that correspond to three discrete binding sites at adjacent bases. The enzyme is able to transition between binding sites in both directions without dissociating from the nucleic acid. In contrast, the NS3 mutant W501A, which is unable to engage in stacking interactions with the DNA, is severely compromised in this switching activity. Altogether our data are consistent with a model for NS3 dynamics that favors ATP-independent random binding and sliding by one and two nucleotides along the overhang of the loading strand.

Interactions of the Disordered Domain II of Hepatitis C Virus NS5A with Cyclophilin A, NS5B, and Viral RNA Show Extensive Overlap.

Domain II of the nonstructural protein 5 (NS5A) of the hepatitis C virus (HCV) is involved in intermolecular interactions with the viral RNA genome, the RNA-dependent RNA polymerase NS5B, and the host factor cyclophilin A (CypA). However, domain II of NS5A (NS5ADII) is largely disordered, which makes it difficult to characterize the protein-protein or protein-nucleic acid interfaces. Here we utilized a mass spectrometry-based protein footprinting approach in attempts to characterize regions forming contacts between NS5ADII and its binding partners. In particular, we compared surface topologies of lysine and arginine residues in the context of free and bound NS5ADII. These experiments have led to the identification of an RNA binding motif (305RSRKFPR311) in an arginine-rich region of NS5ADII. Furthermore, we show that K308 is indispensable for both RNA and NS5B binding, whereas W316, further downstream, is essential for protein-protein interactions with CypA and NS5B. Most importantly, NS5ADII binding to NS5B involves a region associated with RNA binding within NS5B. This interaction down-regulated RNA synthesis by NS5B, suggesting that NS5ADII modulates the activity of NS5B and potentially regulates HCV replication.

Exploiting Conjugated Polyelectrolyte Photophysics toward Monitoring Real-Time Lipid Membrane-Surface Interaction Dynamics at the Single-Particle Level.

Herein we report the real-time observation of the interaction dynamics between cationic liposomes flowing in solution and a surface-immobilized charged scaffolding formed by the deposition of conjugated polyanion poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene (MPS-PPV) onto 100-nm-diameter SiO2 nanoparticles (NPs). Contact of the freely floating liposomes with the polymer-coated surfaces led to the formation of supported lipid bilayers (SLBs). The interaction of the incoming liposomes with MPS-PPV adsorbed on individual SiO2 nanoparticles promoted the deaggregation of the polymer conformation and led to large emission intensity enhancements. Single-particle total internal reflection fluorescence microscopy studies exploited this phenomenon as a way to monitor the deformation dynamics of liposomes on surface-immobilized NPs. The MPS-PPV emission enhancement (up to 25-fold) reflected on the extent of membrane contact with the surface of the NP and was correlated with the size of the incoming liposome. The time required for the MPS-PPV emission to reach a maximum (ranging from 400 to 1000 ms) revealed the dynamics of membrane deformation and was also correlated with the liposome size. Cryo-TEM experiments complemented these results by yielding a structural view of the process. Immediately following the mixing of liposomes and NPs the majority of NPs had one or more adsorbed liposomes, yet the presence of a fully formed SLB was rare. Prolonged incubation of liposomes and NPs showed completely formed SLBs on all of the NPs, confirming that the liposomes eventually ruptured to form SLBs. We foresee that the single-particle studies we report herein may be readily extended to study membrane dynamics of other lipids including cellular membranes in live cell studies and to monitor the formation of polymer-cushioned SLBs.

Dye lipophilicity and retention in lipid membranes: implications for single-molecule spectroscopy.

Fluorescence studies of individual lipid vesicles rely on the proper positioning of probes in the lipid milieu. This is true for both positional tags and chemoselective fluorogenic probes that undergo chemical modification following reaction with an analyte of interest within the lipid environment. The present report describes lipophilicity and localization estimations for a series of BODIPY dyes bearing substituents of varying hydrophobicity. We also studied fluorogenic trap-reporter probes that undergo fluorescence emission enhancement upon trapping of reactive oxygen species (ROS), including lipid peroxyl radicals. We show that caution has to be taken to extrapolate ensemble partition measurements of dyes to the single-molecule regime as a result of the dramatically different lipid concentration prevailing in ensemble versus single-molecule experiments. We show that the mole fraction of dyes that remains embedded in liposomes during a typical single-molecule experiment may be accurately determined from a ratiometric single-particle imaging analysis. We further demonstrate that fluorescence correlation spectroscopy (FCS) provides a very rapid and reliable estimate of the lipophilic nature of a given dye under highly dilute single-molecule-like conditions. Our combined single-particle spectroscopy and FCS experiments suggest that the minimal mole fraction of membrane-associated dyes (x(m)) as determined from FCS experiments is about 0.5 for adequate dye retention during single-molecule imaging in lipid membranes. Our work further highlights the dramatic effect that chemical modifications can have on chemoselective fluorogenic probe localization.

Rapid atmospheric pressure plasma jet processed reduced graphene oxide counter electrodes for dye-sensitized solar cells.

In this work, we present the use of reduced graphene oxide (rGO) as the counter electrode materials in dye-sensitized solar cells (DSSCs). rGO was first deposited on a fluorine-doped tin oxide glass substrate by screen-printing, followed by post-treatment to remove excessive organic additives. We investigated the effect of atmospheric pressure plasma jet (APPJ) treatment on the DSSC performance. A power conversion efficiency of 5.19% was reached when DSSCs with an rGO counter electrode were treated by APPJs in the ambient air for a few seconds. For comparison, it requires a conventional calcination process at 400 °C for 15 min to obtain comparable efficiency. Scanning electron micrographs show that the APPJ treatment modifies the rGO structure, which may reduce its conductivity in part but simultaneously greatly enhances its catalytic activity. Combined with the rapid removal of organic additives by the highly reactive APPJ, DSSCs with APPJ-treated rGO counter electrode show comparable efficiencies to furnace-calcined rGO counter electrodes with greatly reduced process time. This ultrashort process time renders an estimated energy consumption per unit area of 1.1 kJ/cm(2), which is only one-third of that consumed in a conventional furnace calcination process. This new methodology thus saves energy, cost, and time, which is greatly beneficial to future mass production.

Dynamics of hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B in complex with RNA.

The hepatitis C virus (HCV) non-structural protein 5B (NS5B) is an RNA-dependent RNA polymerase that is essentially required for viral replication. Although previous studies revealed important properties of static NS5B-RNA complexes, the nature and relevance of dynamic interactions have yet to be elucidated. Here, we devised a single molecule Förster Resonance Energy Transfer (SM-FRET) assay to monitor temporal changes upon binding of NS5B to surface immobilized RNA templates. The data show enzyme association-dissociation events that occur within the time resolution of our setup as well as FRET-fluctuations in association with stable binary complexes that extend over prolonged periods of time. Fluctuations are shown to be dependent on the length of the RNA substrate, and enzyme concentration. Mutations in close proximity to the template entrance (K98E, K100E), and in the center of the RNA binding channel (R394E), reduce both the population of RNA-bound enzyme and the fluctuations associated to the binary complex. Similar observations are reported with an allosteric nonnucleoside NS5B inhibitor. Our assay enables for the first time the visualization of association-dissociation events of HCV-NS5B with RNA, and also the direct monitoring of the interaction between HCV NS5B, its RNA template, and finger loop inhibitors. We observe both a remarkably low dissociation rate for wild type HCV NS5B, and a highly dynamic enzyme-RNA binary complex. These results provide a plausible mechanism for formation of a productive binary NS5B-RNA complex, here NS5B slides along the RNA template facilitating positioning of its 3' terminus at the enzyme active site.

Binding kinetics and affinities of heterodimeric versus homodimeric HIV-1 reverse transcriptase on DNA-DNA substrates at the single-molecule level.

During viral replication, HIV-1 reverse transcriptase (RT) plays a pivotal role in converting genomic RNA into proviral DNA. While the biologically relevant form of RT is the p66-p51 heterodimer, two recombinant homodimer forms of RT, p66-p66 and p51-p51, are also catalytically active. Here we investigate the binding of the three RT isoforms to a fluorescently labeled 19/50-nucleotide primer/template DNA duplex by exploiting single-molecule protein-induced fluorescence enhancement (SM-PIFE). PIFE, which does not require labeling of the protein, allows us to directly visualize the binding/unbinding of RT to a double-stranded DNA substrate. We provide values for the association and dissociation rate constants of the RT homodimers p66-p66 and p51-p51 with a double-stranded DNA substrate and compare those to the values recorded for the RT heterodimer p66-p51. We also report values for the equilibrium dissociation constant for the three isoforms. Our data reveal great similarities in the intrinsic binding affinities of p66-p51 and p66-p66, with characteristic Kd values in the nanomolar range, much smaller (50-100-fold) than that of p51-p51. Our data also show discrepancies in the association/dissociation dynamics among the three dimeric RT isoforms. Our results further show that the apparent binding affinity of p51-p51 for its DNA substrate is to a great extent time-dependent when compared to that of p66-p66 and p66-p51, and is more likely determined by the dimer dissociation into its constituent monomers rather than the intrinsic binding affinity of dimeric RT.

Flexible silver nanowire meshes for high-efficiency microtextured organic-silicon hybrid photovoltaics.

Hybrid organic-silicon heterojunction solar cells promise a significant reduction on fabrication costs by avoiding energy-intensive processes. However, their scalability remains challenging without a low-cost transparent electrode. In this work, we present solution-processed silver-nanowire meshes that uniformly cover the microtextured surface of hybrid heterojunction solar cells to enable efficient carrier collection for large device area. We systematically compare the characteristics and device performance with long and short nanowires with an average length/diameter of 30 µm/115 nm and 15 µm/45 nm, respectively, to those with silver metal grids. A remarkable power conversion efficiency of 10.1% is achieved with a device area of 1 × 1 cm(2) under 100 mW/cm(2) of AM1.5G illumination for the hybrid solar cells employing long wires, which represents an enhancement factor of up to 36.5% compared to the metal grid counterpart. The high-quality nanowire network displays an excellent spatial uniformity of photocurrent generation via distributed nanowire meshes and low dependence on efficient charge transport under a high light-injection condition with increased device area. The capability of silver nanowires as flexible transparent electrodes presents a great opportunity to accelerate the mass deployment of high-efficiency hybrid silicon photovoltaics via simple and rapid soluble processes.

Enhancing the emissive properties of poly(p-phenylenevinylene)-conjugated polyelectrolyte-coated SiO2 nanoparticles.

Here we describe single-particle imaging studies conducted on the conjugated polyelecrolyte poly[5-methoxy-2-(3-sulfopropoxy)-1,4-phenylenevinylene] (MPS-PPV) supported on SiO(2) nanoparticles. The particles are subjected to a time-programmed sequence involving addition and removal of different additives, including excited-triplet-state quenchers and scavengers of singlet oxygen as well as ground-state oxygen. Our studies show that these additives enhance the emission intensity and photostability of the nanoparticles and may further repair photodamaged conjugated polymer. The ability to monitor the emission from individual particles along multiple cycles under a range of conditions provides a mechanistic insight into the action of these additives.

Microfluidic device for single-molecule experiments with enhanced photostability.

A microfluidic device made of polydimethylsiloxane (PDMS) addresses key limitations in single-molecule fluorescence experiments by providing high dye photostability and low sample sticking. Photobleaching is dramatically reduced by deoxygenation via gas diffusion through porous channel walls. Rapid buffer exchange in a laminar sheath flow followed by optical interrogation minimizes surface-sample contacts and allows the in situ addition and combination of other reagents.

Single-molecule study of the inhibition of HIV-1 transactivation response region DNA/DNA annealing by argininamide.

Single-molecule spectroscopy was used to examine how a model inhibitor of HIV-1, argininamide, modulates the nucleic acid chaperone activity of the nucleocapsid protein (NC) in the minus-strand transfer step of HIV-1 reverse transcription, in vitro. In minus-strand transfer, the transactivation response region (TAR) RNA of the genome is annealed to the complementary "TAR DNA" generated during minus-strand strong-stop DNA synthesis. Argininamide and its analogs are known to bind to the hairpin bulge region of TAR RNA as well as to various DNA loop structures, but its ability to inhibit the strand transfer process has only been implied. Here, we explore how argininamide modulates the annealing kinetics and secondary structure of TAR DNA. The studies reveal that the argininamide inhibitory mechanism involves a shift of the secondary structure of TAR, away from the NC-induced "Y" form, an intermediate in reverse transcription, and toward the free closed or "C" form. In addition, more potent inhibition of the loop-mediated annealing pathway than stem-mediated annealing is observed. Taken together, these data suggest a molecular mechanism wherein argininamide inhibits NC-facilitated TAR RNA/DNA annealing in vitro by interfering with the formation of key annealing intermediates.

Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer.

Reverse transcription of the HIV-1 genome involves several nucleic acid rearrangement steps that are catalyzed (chaperoned) by the nucleocapsid protein (NC), including the annealing of the transactivation response region (TAR) RNA of the genome to the complementary sequence (TAR DNA) in minus-strand strong-stop DNA. It has been extremely challenging to obtain unambiguous mechanistic details on the annealing process at the molecular level because of the kinetic involvement of a complex and heterogeneous set of nucleic acid/protein complexes of variable structure and variable composition. Here, we investigate the in vitro annealing mechanism using a multistep single-molecule spectroscopy kinetic method. In this approach, an immobilized hairpin is exposed to a multistep programmed concentration sequence of NC, model complementary targeted-oligonucleotides, and buffer-only solutions. The sequence controllably "drags" single immobilized TAR hairpins among the kinetic stable states of the reaction mechanism; i.e., reactants, intermediates, and products. This single-molecule spectroscopy method directly probes kinetic reversibility and the chaperone (catalytic) role of NC at various stages along the reaction sequence, giving access to previously inaccessible kinetic processes and rate constants. By employing target oligonucleotides for specific TAR regions, we kinetically trap and investigate structural models for putative nucleation complexes for the annealing process. The new results lead to a more complete and detailed understanding of the ability of NC to promote nucleic acid/nucleic acid rearrangement processes. This includes information on the ability of NC to chaperone "reverse annealing" in single-strand transfer and the first observation of partially annealed, conformational substates in the annealing mechanism.

Insights on the role of nucleic acid/protein interactions in chaperoned nucleic acid rearrangements of HIV-1 reverse transcription.

HIV-1 reverse transcription requires several nucleic acid rearrangement steps that are "chaperoned" by the nucleocapsid protein (NC), including minus-strand transfer, in which the DNA transactivation response element (TAR) is annealed to the complementary TAR RNA region of the viral genome. These various rearrangement processes occur in NC bound complexes of specific RNA and DNA structures. A major barrier to the investigation of these processes in vitro has been the diversity and heterogeneity of the observed nucleic acid/protein assemblies, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules. Herein, we use a flow chamber approach involving rapid NC/nucleic acid mixing to substantially control aggregation for the NC chaperoned irreversible annealing kinetics of a model TAR DNA hairpin sequence to the complementary TAR RNA hairpin, i.e., to form an extended duplex. By combining the flow chamber approach with a broad array of fluorescence single-molecule spectroscopy (SMS) tools (FRET, molecule counting, and correlation spectroscopy), we have unraveled the complex, heterogeneous kinetics that occur during the course of annealing. The SMS results demonstrate that the TAR hairpin reactant is predominantly a single hairpin coated by multiple NCs with a dynamic secondary structure, involving equilibrium between a "Y" shaped conformation and a closed one. The data further indicate that the nucleation of annealing occurs in an encounter complex that is formed by two hairpins with one or both of the hairpins in the "Y" conformation.

Evidence for non-two-state kinetics in the nucleocapsid protein chaperoned opening of DNA hairpins.

In HIV-1 reverse transcription, the nucleocapsid protein, NC, induces secondary structure fluctuations in specific DNA and RNA hairpins. Time-resolved single-molecule fluorescence resonance energy transfer was used to study NC chaperoned opening of DNA hairpins over a broader range of conditions and in more depth than in previous studies. The experiments reveal a complex mechanism for secondary structure fluctuations with dynamic processes occurring over a wide time range, i.e., approximately 5 to >250 ms and with the involvement of long-lived intermediates. The dynamic role of DNA loop regions and NC binding/dissociation events are discussed.

Single-molecule FRET studies of important intermediates in the nucleocapsid-protein-chaperoned minus-strand transfer step in HIV-1 reverse transcription.

The minus-strand transfer step of HIV-1 reverse transcription is chaperoned by the nucleocapsid protein (NC), which has been shown to facilitate the annealing between the transactivation response element (TAR) RNA and complementary TAR DNA stem-loop structures. In this work, potential intermediates in the mechanism of NC-chaperoned TAR DNA/TAR RNA annealing have been examined using single-molecule fluorescence resonance energy transfer. The interaction between TAR DNA and various DNA oligonucleotides designed to mimic the initial annealing step was monitored to capture potential intermediates along the reaction pathway. Two possible mechanisms of annealing were examined, namely nucleation through the 3'/5' termini, termed the "zipper" complex, or nucleation through the hairpin loops in a "kissing" complex. Intermediates associated with both mechanisms were observed in the presence of NC, and the kinetics of formation of these intermediates were also measured. Thus, the single-molecule experiments support the notion that NC-assisted annealing of TAR DNA:TAR RNA may occur through multiple pathways.

Secondary structure and secondary structure dynamics of DNA hairpins complexed with HIV-1 NC protein.

Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. We report herein single-molecule fluorescence resonance energy transfer measurements on single immobilized TAR DNA hairpins and hairpin mutants complexed with NC (i.e., TAR DNA/NC). Using this approach we have explored the conformational distribution and dynamics of the hairpins in the presence and absence of NC protein. The data demonstrate that NC shifts the equilibrium secondary structure of TAR DNA hairpins from a fully "closed" conformation to essentially one specific "partially open" conformation. In this specific conformation, the two terminal stems are "open" or unwound and the other stems are closed. This partially open conformation is arguably a key TAR DNA intermediate in the NC-induced annealing mechanism of TAR DNA.

Multiple hydrogen bonds tuning guest/host excited-state proton transfer reaction: its application in molecular recognition.

A molecular recognition concept exploiting multiple-hydrogen-bond fine-tuned excited-state proton-transfer (ESPT) was conveyed using 3,4,5,6-tetrahydrobis(pyrido[3,2-g]indolo)[2,3-a:3',2'-j]acridine (1a). The catalytic type 1a/carboxylic acids hydrogen-bonding (HB) complexes undergo ultrafast ESPT, resulting in an anomalously large Stokes shifted tautomer emission (lambdamax approximately 600 nm). Albeit forming a quadruple HB complex, ESPT is prohibited in the noncatalytic-type 1a/urea complexes (lambdamax approximately 430 nm). The HB configuration tuning ESPT properties lead to a feasible design for sensing multiple-HB-site analytes of biological interest.