Repeated mechanical damage enhanced Aquilaria sinensis resistance to Heortia vitessoides through jasmonic acid.

Introduction: The leaf-chewing pest Heortia vitessoides severely threatens the growth and development of Aquilaria sinensis. In our previous study, we found that mechanical damage (MD) to stem enhanced A. sinensis sapling resistance to H. vitessoides larvae. Methods: To reveal the defense mechanisms underlying this observation, we analyzed the types and contents of volatile organic compounds (VOCs), phytohormone contents, and expression of phytohormone-related genes in response to MD and herbivory wounding(HW). Results: Here, we identified several VOCs, such as the pesticides fenobucarb and 2,4-di-tert-butylphenol, in mature leaf (ML) of MD-treated plants. Compared with salicylic acid (SA) or the ethylene (ET) pathway, jasmonic acid (JA) content and JA-related genes were more strongly upregulated. Interestingly, we found a dramatic difference between JA-related upstream and downstream genes expression in YL and ML, which confirmed that JA-Ile accumulation in MD-ML and HW-ML could be derived from local damaged site. Discussion: Taken together, we provide evidence that the JA pathway plays a dominant role in the A. sinensis response to MD and HW.

Melting Away Pain: Decay of Thermal Nociceptor Transduction during Heat-Induced Irreversible Desensitization of Ion Channels.

Thermal transient receptor potential channels play a key role in thermal sensation. Although predictive models exist for temperature-dependent transduction through these channels and for the associated sensations of pain, the ability to predict irreversible desensitization has been lacking. We explored the role of irreversible ion channel desensitization in pain sensation and hypothesized that desensitization of ion channels would follow the kinetics similar to the denaturation of catalytic enzymes. We therefore proposed a three-state model to simulate the kinetic of temperature-sensitive ion channels from the closed state through opening and irreversible thermal desensitization. We tested the model against data obtained in vivo from a feline model. The theoretical model predicts all experimental data with reasonable accuracy, and represents an important step toward the ability for understanding of the molecular basis of nociceptor signaling providing the possibility to design local anesthesia regimens and to the prediction of postoperative pain.

Comparison of unbiased estimation of neuronal number in the rat hippocampus with different staining methods.

BACKGROUND: NeuN and Nissl staining (toluidine blue, cresyl violet staining) are routinely used methods in unbiased stereological estimation of the total number of hippocampal neurons. NEW METHOD: In the present study, we stained serial frozen coronal sections from 5 normal adult male Sprague-Dawley rat brains with different methods, measured the deformation of hippocampal area in brain sections and estimated the total number of hippocampal neurons using the optical fractionator. RESULTS: The deformation in x, y-axis was not obviously different with different staining protocols, but shrinkage in z-axis was significant after staining (p < 0.001). NeuN staining produced significant higher estimate number than cresyl violet staining by 24% (p = 0.002), however, NeuN and Cresyl Violet staining showed a high degree of correlation in quantification of total neuronal numbers and both methods are suitable for unbiased stereological estimation. COMPARISON WITH EXISTING METHOD (S): NeuN is more reliable but if time is limited or the number of animals used in experiments is high, cresyl violet staining may be a feasible method. CONCLUSIONS: Compared with previous estimates of the neurons number in rat hippocampus, our present data is reliable and the stereological analysis based on our system is a cost-effective unbiased method for estimation of neuron number.

N-acetyl-L-tryptophan delays disease onset and extends survival in an amyotrophic lateral sclerosis transgenic mouse model.

BACKGROUND: Whether L-NAT, a cytochrome c release inhibitor and an antagonist of NK-1R, provides protection in ALS is not known. RESULTS: L-NAT delays disease onset and mortality in mSOD1(G93A) ALS mice by inhibiting mitochondrial cell death pathways, inflammation, and NK-1R downregulation. CONCLUSION: L-NAT offers protection in a mouse model of ALS. SIGNIFICANCE: Data suggest the potential of L-NAT as a novel therapeutic strategy for ALS and provide insight into its action mechanisms. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, while inflammation has been implicated in its pathogenesis. Both inhibitors of cytochrome c release and antagonists of the neurokinin 1 receptor (NK-1R) have been reported to provide neuroprotection in ALS and/or other neurodegenerative diseases by us and other researchers. However, whether N-acetyl-L-tryptophan (L-NAT), an inhibitor of cytochrome c release and an antagonist of NK-1R, provides neuroprotection in ALS remains unknown. Here we demonstrate that the administration of L-NAT delayed disease onset, extended survival, and ameliorated deteriorations in motor performance in mSOD1(G93A) ALS transgenic mice. Our data showed that L-NAT reached the spinal cord, skeletal muscle, and brain. In addition, we demonstrate that L-NAT reduced the release of cytochrome c/smac/AIF, increased Bcl-xL levels, and inhibited the activation of caspase-3. L-NAT also ameliorated motor neuron loss and gross atrophy, and suppressed inflammation, as shown by decreased GFAP and Iba1 levels. Furthermore, we found gradually reduced NK-1R levels in the spinal cords of mSOD1(G93A) mice, while L-NAT treatment restored NK-1R levels. We propose the use of L-NAT as a potential therapeutic invention against ALS.

Ox-LDL induces dysfunction of endothelial progenitor cells via activation of NF-κB.

Dyslipidemia increases the risks for atherosclerosis in part by impairing endothelial integrity. Endothelial progenitor cells (EPCs) are thought to contribute to endothelial recovery after arterial injury. Oxidized low-density lipoprotein (ox-LDL) can induce EPC dysfunction, but the underlying mechanism is not well understood. Human EPCs were cultured in endothelial growth medium supplemented with VEGF (10 ng/mL) and bFGF (10 ng/mL). The cells were treated with ox-LDL (50 µg/mL). EPC proliferation was assayed by using CCK8 kits. Expression and translocation of nuclear factor-kabba B (NF-κB) were evaluated. The level of reactive oxygen species (ROS) in cells was measured using H2DCF-DA as a fluorescence probe. The activity of NADPH oxidase activity was determined by colorimetric assay. Ox-LDL significantly decreased the proliferation, migration, and adhesion capacity of EPCs, while significantly increased ROS production and NADPH oxidase expression. Ox-LDL induced NF-κB P65 mRNA expression and translocation in EPCs. Thus ox-LDL can induce EPC dysfunction at least by increasing expression and translocation of NF-κB P65 and NADPH oxidase activity, which represents a new mechanism of lipidemia-induced vascular injury.

Macrophage migration inhibitory factor polymorphism is associated with susceptibility to inflammatory coronary heart disease.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine. This study explored the association of 173G/C polymorphism of the MIF gene with coronary heart disease (CHD). METHODS: Sequencing was carried out after polymerase chain reaction with DNA specimens from 186 volunteers without CHD and 70 patients with CHD. Plasma MIF levels on admission were measured by ELISA. Patients were classified into either stable angina pectoris (SAP) or unstable angina pectoris (UAP). Genotype distribution between cases and controls and the association of patients' genotypes with MIF level and plaque stability were statistically evaluated (ethical approval number: 2012-01). RESULTS: The frequency of the C genotype was higher in CHD patients than in the control (P = 0.014). The frequency of the 173*CC genotype was higher in CHD patients than in the control (P = 0.005). The plasma MIF level was higher in MIF173*C carriers than in MIF173*G carriers (P = 0.033). CHD patients had higher plasma MIF levels than the control (P = 0.000). Patients with UAP had higher plasma MIF levels than patients with SAP (P = 0.014). CONCLUSIONS: These data suggest that MIF -173G/C polymorphism may be related to the development of CHD in a Chinese population. Plasma MIF level is a predictor of plaque stability. This trial is registered with NCT01750502.

Neuroprotective agents target molecular mechanisms of disease in ALS.

Amyotrophic lateral sclerosis (ALS) is a debilitating disease characterized by progressive loss of voluntary motor neurons leading to muscle atrophy, weight loss and respiratory failure. Evidence suggests that inflammation, oxidative stress, mitochondrial dysfunction, apoptosis, glutamate excitotoxicity and proteasomal dysfunction are all responsible for ALS pathogenesis. We review neuroprotective agents with the ability to reduce ALS-related bodyweight loss, summarize the various therapies tested on animal models targeting the proposed molecular mechanisms, compare their effects on bodyweight loss, muscle damage, disease onset, duration and survival, and analyze their structure-activity relationships, with the overall goal of creating a screening strategy for further clinical application.

Necroptosis mediates TNF-induced toxicity of hippocampal neurons.

Tumor necrosis factor-α (TNF-α) is a critical proinflammatory cytokine regulating neuroinflammation. Elevated levels of TNF-α have been associated with various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. However, the signaling events that lead to TNF-α-initiated neurotoxicity are still unclear. Here, we report that RIP3-mediated necroptosis, a form of regulated necrosis, is activated in the mouse hippocampus after intracerebroventricular injection of TNF-α. RIP3 deficiency attenuates TNF-α-initiated loss of hippocampal neurons. Furthermore, we characterized the molecular mechanism of TNF-α-induced neurotoxicity in HT-22 hippocampal neuronal cells. HT-22 cells are sensitive to TNF-α only upon caspase blockage and subsequently undergo necrosis. The cell death is suppressed by knockdown of CYLD or RIP1 or RIP3 or MLKL, suggesting that this necrosis is necroptosis and mediated by CYLD-RIP1-RIP3-MLKL signaling pathway. TNF-α-induced necroptosis of HT-22 cells is largely independent of both ROS accumulation and calcium influx although these events have been shown to be critical for necroptosis in certain cell lines. Taken together, these data not only provide the first in vivo evidence for a role of RIP3 in TNF-α-induced toxicity of hippocampal neurons, but also demonstrate that TNF-α promotes CYLD-RIP1-RIP3-MLKL-mediated necroptosis of hippocampal neurons largely bypassing ROS accumulation and calcium influx.

Factors associated with decision time for patients with ST-segment elevation acute myocardial infarction.

Increased delay in visiting a hospital for patients with ST-segment elevation myocardial infarction (STEMI) is often associated with poor outcomes. The factors associated with the decision time were analyzed by comparing the characteristics of patients with delays longer or shorter than the median of 60 min. Pre-hospital delay tended to be longer for patients living in suburban areas compared to those in urban areas (P=0.015). Shorter decision time was more likely among older patients. Being married, medical insurance coverage, and the level of educational qualification did not affect decision time. More efforts should be paid to educate the patients with high risk in suburban areas in order to effectively reduce pre-hospital delays.

[The change of transient receptor potential vanilloid-1 and interleukin-1α in periodontal tissue during experimental tooth movement].

PURPOSE: To investigate the expression of TRPV1 and IL-1α in Sprague-Dawley rats' periodontal ligament (PLD) during experimental tooth movement of different orthodontic force and duration, and explore the role of TRPV1 and IL-1α in orthodontic pain. METHODS: 66 Sprague-Dawley rats were divided into control group (n=6), sham operation group (n=6) and experimental group (n=54) randomly. Orthodontic force was applied on right first maxillary molar in rats and the changes of TRPV1 and IL-1α expression were detected by real-time PCR at 4 h, 8 h, 1 d (three subgroups were added according to different forces: 1 d-30 g, 1 d-50 g, 1 d-80 g ), 3 d, 5 d, 7 d, 14 d after tooth movement. The data was analyzed using SPSS 13.0 software pakage. RESULTS: Following the experimental tooth movement, the expression of TRPV1 and IL-1α in periodontal tissues were significantly up-regulated from 4 h to 7 d, with a peak at 1 d and returned to normal level at 1 week. The greater force applied during experimental tooth movement at 1 d, the higher expression of TRPV1 and IL-1α were detected in periodontal tissues. CONCLUSIONS: Experimental tooth movement leads to regular change of TRPV1 and IL-1α expression in periodontal tissues, which indicates that TRPV1 and IL-1α may play an important role in orthodontic pain.

Neuroprotection for amyotrophic lateral sclerosis: role of stem cells, growth factors, and gene therapy.

Various molecular mechanisms including apoptosis, inflammation, oxidative stress, mitochondrial dysfunction and excitotoxicity have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), though the exact mechanisms have yet to be specified. Furthermore, the underlying restorative molecular mechanisms resulting in neuronal and/or non-neuronal regeneration have to be yet elucidated. Therapeutic agents targeting one or more of these mechanisms to combat either initiation or progression of the disease are under research. Novel treatments including stem cell therapy, growth factors, and gene therapy might prolong survival and delay progression of symptoms. Harnessing the regenerative potential of the central nervous system would be a novel approach for the treatment of motor neuron death resulting from ALS. Endogenous neural replacement, if augmented with administration of exogenous growth factors or with pharmaceuticals that increase the rate of neural progenitor formation, neural migration, and neural maturation could slow the rate of cell loss enough to result in clinical improvement. In this review, we discuss the impact of therapeutic treatment involving stem cell therapy, growth factors, gene therapy, and combination therapy on disease onset and progression of ALS. In addition, we summarize human clinical trials of stem cell therapy, growth factor therapy, and gene therapy in individuals with ALS.

[The change of calcitonin gene- related peptide in periodontal tissue during orthodontic tooth movement].

PURPOSE: To establish a Sprague-Dawley rat model of tooth movement, detect the expression of calcitonin gene-related peptide (calcitonin gene related peptide, CGRP) in Sprague-Dawley rats' periodontal tissues during tooth movement and explore the role of the peripheral mechanisms of pain in orthodontics. METHODS: 60 Sprague-Dawley rats were randomly divided into experimental group (n=54) and control group (n=6). Force was applied on right maxillary first molar in rats and the changes of CGRP expression were detected by real-time PCR 4 h, 8 h, 1 d, 3 d, 5 d, 7 d, 14 d after tooth movement. SPSS 13.0 software package was selected and one-way ANOVA (least significant difference, LSD) was used for statistics analysis. RESULTS: Experimental tooth movement led to an increase in the expression of CGRP. In addition, following experimental tooth movement, the expression of CGRP in periodontal tissues was significantly up-regulated from 4 h, with a peak on 1d and returned to the control level at 2-week. 1 d after experimental tooth movement, the application of different forces to rats induced a significant increase in CGRP expression. CONCLUSION: The regular change of CGRP expression in periodontal tissues indicates that CGRP may play an important role in peripheral mechanism of orthodontic pain.

[Construction and identification of BIRC5 shRNA lentiviral expression vector].

AIM: Design and synthesis complementary DNA sequences of 3 pairs of short hairpin structure and a pair of negative control sequence by RNA interference technique and construct and identify a lentiviral interference vector with human BIRC5 gene as target gene. METHODS: The designed and synthesised Single-Stranded primer were annealed to Double-Stranded oligo sequences and subcloned into linear pMAGic lentiviral plasmid vector digested by enzyme Age I and EcoR I. Screening positive clone after transformed into DH5α competent cells and identified by PCR amplification and DNA sequencing. RESULTS: 335 bp straps of positive clone and 298 bp straps of negative clone form PCR amplification production have been obtained after gel electrophoresis, the designed and synthesised sequences have been contained in these clone straps confirmed by the result of DNA sequencing. CONCLUSION: Four pairs of BIRC5 shRNA recombinant lentiviral expression vector were constructed successfully, which laid the foundation for researching the inhibition of BIRC5 siRNA target against tumor cells proliferation, induction apoptosis and gene therapy.

Comparative analysis of RNA/protein dynamics for the arginine-rich-binding motif and zinc-finger-binding motif proteins encoded by HIV-1.

We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.

RNAi knockdown of C-erbB2 expression inhibits salivary gland adenoid cystic carcinoma SACC-83 cell growth in vitro.

OBJECTIVE: To knockdown the C-erbB2 gene in salivary gland adenoid cystic carcinoma SACC-83 cells using RNA interference, and determine the effect of silencing C-erbB2 on cell proliferation. METHODS: C-erbB2-siRNA was transfected into SACC-83 cells. RT-PCR and immunohistochemistry were used to detect C-erbB2 expression in SACC-83 cells. Cell proliferation was measured by the MTT assay and gene knockdown was achieved by RNA interference. Apoptosis was analyzed by flow cytometry. RESULTS: Compared with the control, C-erbB2 mRNA expression was decreased in the C-erbB2-siRNA transfection group, and immunohistochemical analysis indicated that C-erbB2 protein expression was decreased. After C-erbB2-siRNA was transfected for 48 h, absorbance at 570 nm (MTT) was 0.185±0.021 compared with 0.354±0.034, 0.299±0.053, and 0.314±0.049 in the blank control, liposome control and negative control siRNA groups, respectively. The differences were statistically significant (P < 0.05) between the C-erbB2-siRNA group and the control groups. Following the C-erbB2 knockdown, the percentage of apoptotic cells was 5.63% compared with 2.04%, 2.85%, and 2.98% in the three control groups, respectively. Proliferation of SACC-83 cells was inhibited, and early apoptotic cells were increased. CONCLUSION: RNA interference can effectively silence C-erbB2 gene expression and inhibit growth of SACC-83 cells, which indicates the potential of targeting this gene as a novel gene therapy approach for the treatment of salivary gland adenoid cystic carcinoma.

A one-dimensional free energy surface does not account for two-probe folding kinetics of protein alpha(3)D.

We present fluorescence-detected measurements of the temperature-jump relaxation kinetics of the designed three-helix bundle protein alpha(3)D taken under solvent conditions identical to previous infrared-detected kinetics. The fluorescence-detected rate is similar to the IR-detected rate only at the lowest temperature where we could measure it (326 K). The fluorescence-detected rate decreases by a factor of 3 over the 326-344 K temperature range, whereas the IR-detected rate remains nearly constant over the same range. To investigate this probe dependence, we tested an extensive set of physically reasonable one-dimensional (1D) free energy surfaces by Langevin dynamics simulation. The simulations included coordinate- and temperature-dependent roughness, diffusion coefficients, and IR/fluorescence spectroscopic signatures. None of these can reproduce the IR and fluorescence data simultaneously, forcing us to the conclusion that a 1D free energy surface cannot accurately describe the folding of alpha(3)D. This supports the hypothesis that alpha(3)D has a multidimensional free energy surface conducive to downhill folding at 326 K, and that it is already an incipient downhill folder with probe-dependent kinetics near its melting point.

Using an amino acid fluorescence resonance energy transfer pair to probe protein unfolding: application to the villin headpiece subdomain and the LysM domain.

Previously, we have shown that p-cyanophenylalanine (Phe CN) and tryptophan (Trp) constitute an efficient fluorescence resonance energy transfer (FRET) pair that has several advantages over commonly used dye pairs. Here, we aim to examine the general applicability of this FRET pair in protein folding-unfolding studies by applying it to the urea-induced unfolding transitions of two small proteins, the villin headpiece subdomain (HP35) and the lysin motif (LysM) domain. Depending on whether Phe CN is exposed to solvent, we are able to extract either qualitative information about the folding pathway, as demonstrated by HP35, which has been suggested to unfold in a stepwise manner, or quantitative thermodynamic and structural information, as demonstrated by LysM, which has been shown to be an ideal two-state folder. Our results show that the unfolding transition of HP35 reported by FRET occurs at a denaturant concentration lower than that measured by circular dichroism (CD) and that the loop linking helix 2 and helix 3 remains compact in the denatured state, which are consistent with the notion that HP35 unfolds in discrete steps and that its unfolded state contains residual structures. On the other hand, our FRET results on the LysM domain allow us to develop a model for extracting structural and thermodynamic parameters about its unfolding, and we find that our results are in agreement with those obtained by other methods. Given the fact that Phe CN is a non-natural amino acid and, thus, amenable to incorporation into peptides and proteins via existing peptide synthesis and protein expression methods, we believe that the FRET method demonstrated here is widely applicable to protein conformational studies, especially to the study of relatively small proteins.

Effect of hindlimb unloading on resting intracellular calcium in intrafusal fibers and ramp-and-hold stretches evoked responsiveness of soleus muscle spindles in conscious rats.

The present study was to investigate and evaluate the dynamic changes of calcium homeostasis of soleus muscle spindle for the exploration of the potential mechanisms of muscle spindle degeneration induced by hindlimb unloading. We systematically observed the changes in immunoreactivity of calbindin D28K (CaBP-D28K), intracellular resting calcium in intrafusal fibers of soleus muscle spindle, and the responsiveness of muscle spindles to ramp-and-hold stretches after short- and long-term (3, 7, 14 d) hindlimb unloading. The immunoreactivity of CaBP-D28K started to decrease after 7-d hindlimb unloading, while its decrease was obviously different compared with the control group 14 d following the hindlimb unloading. The resting calcium concentration was increased significantly at 3 d, and reached the peak level 14 d after the hindlimb unloading. The responsiveness of muscle spindles, assessed by investigating Index3-L(2), Index3-L(3), and Index5, to ramp-and-hold stretches started to decrease during the period of 7-d hindlimb unloading. All Indexs, in particular Index3-L(3) and Index5, were significantly decreased at 14 d after the hindlimb unloading. The data suggest the disturbance of calcium homeostasis in intrafusal fibers during the exposure to hindlimb unloading might gradually influence the structure and function of muscle spindles.

[Clinical and experimental retrospective analysis on acute leukemia with trisomy 4 cell].

OBJECTIVE: To explore the clinical and experimental features of acute leukemia (AL) with trisomy 4. METHODS: A retrospective analysis on the clinical and laboratory data of 21 cases of AL with trisomy 4 was performed. Chromosomes were prepared using direct method and/or short-term (24 h) cultures of bone marrow cells. Karyotypic analysis was carried out by using R-banding technique. Thirteen cases were studied by interphase fluorescence in situ hybridization (FISH) by using a chromosome 4-specific alpha -satellite DNA probe labeled by spectrum Green to ascertain the presence of a clone with trisomy 4. Five cases with t (8; 21) revealed by karyotypic analysis were detected by dual-color FISH using t (8; 21) translocation probe to confirm the AML1/ETO rearrangement. RESULTS: All the patients with AL and trisomy 4 were with de novo AL except two cases with secondary AL. M2 was the most frequent Franch-American-British(FAB) subtype in this series (9/21 cases). The initial leukocyte count more than 10x 10(9)/L was seen in 16 cases. An enlargement of liver, spleen and/or lymph nodes in varying degrees was found in 15 cases. Among 15 cases received immunophenotypic analysis, 11 cases showed CD34 positivity and 6 cases co-expressed myeloid and lymphocyte antigens. Karyotypic analysis disclosed clonal trisomy 4 in 18 cases and one cell with +4 in 3 cases. Isolated trisomy 4 was found in 7 cases, while 14 cases had other abnormalities besides trisomy 4 among which t (8; 21) was found in 8 cases. Dual-color FISH confirmed that all 13 cases including 3 cases having one cell with +4 on karyotypic analysis had clonal trisomy 4. Dual-color FISH confirmed that all 5 cases with t (8; 21) had AML1/ETO rearrangement. CONCLUSION: AL patients with trisomy 4 have unique clinical and experimental features and a poor prognosis.

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.