Rapid Detection of Bacillus anthracis Bloodstream Infections by Use of a Novel Assay in the GeneXpert System.

Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non-B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log10 dynamic range. Assay specificity was 100% for tests of non-B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.

Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use.

Extensively drug-resistant (XDR) tuberculosis (TB) cannot be easily or quickly diagnosed. We developed a rapid, automated assay for the detection of XDR-TB plus resistance to the drug isoniazid (INH) for point-of-care use. Using a simple filter-based cartridge with an integrated sample processing function, the assay identified a wide selection of wild-type and mutant sequences associated with XDR-TB directly from sputum. Four new large-Stokes-shift fluorophores were developed. When these four Stokes-shift fluorophores were combined with six conventional fluorophores, 10-color probe detection in a single PCR tube was enabled. A new three-phase, double-nested PCR approach allowed robust melting temperature analysis with enhanced limits of detection (LODs). Finally, newly designed sloppy molecular beacons identified many different mutations using a small number of probes. The assay correctly distinguished wild-type sequences from 32 commonly occurring mutant sequences tested in gyrA, gyrB, katG, and rrs genes and the promoters of inhA and eis genes responsible for resistance to INH, the fluoroquinolone (FQ) drugs, amikacin (AMK), and kanamycin (KAN). The LOD was 300 CFU of Mycobacterium tuberculosis in 1 ml sputum. The rate of detection of heteroresistance by the assay was equivalent to that by Sanger sequencing. In a blind study of 24 clinical sputum samples, resistance mutations were detected in all targets with 100% sensitivity, with the specificity being 93.7 to 100%. Compared to the results of phenotypic susceptibility testing, the sensitivity of the assay was 75% for FQs and 100% each for INH, AMK, and KAN and the specificity was 100% for INH and FQ and 94% for AMK and KAN. Our approach could enable testing for XDR-TB in point-of-care settings, potentially identifying highly drug-resistant TB more quickly and simply than currently available methods.

Single-molecule probing of amyloid nano-ensembles using the polymer nanoarray approach.

Soluble amyloid-beta (Aß) oligomers are the prime causative agents of cognitive deficits during early stages of Alzheimer's disease (AD). The transient nature of the oligomers makes them difficult to characterize by traditional techniques, suggesting that advanced approaches are necessary. Previously developed fluorescence-based tethered approach for probing intermolecular interactions (TAPIN) and AFM-based single-molecule force spectroscopy are capable of probing dimers of Aß peptides. In this paper, a novel polymer nanoarray approach to probe trimers and tetramers formed by the Aß(14-23) segment of Aß protein at the single-molecule level is applied. By using this approach combined with TAPIN and AFM force spectroscopy, the impact of pH on the assembly of these oligomers was characterized. Experimental results reveal that pH affects the oligomer assembly process. At neutral pH, trimers and tetramers assemble into structures with a similar stability, while at acidic conditions (pH 3.7), the oligomers adopt a set of structures with different lifetimes and strengths. Models for the assembly of Aß(14-23) trimers and tetramers based on the results obtained is proposed.

A flexible nanoarray approach for the assembly and probing of molecular complexes.

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid ß peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid ß peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers' rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.

Left ventricular pseudoaneurysm in Lemierre's syndrome: a case report.

Background: Lemierre's syndrome is an infectious phenomenon characterized by oropharyngeal infection with bacteraemia, thrombophlebitis, and distant septic emboli. Septic emboli are a recognized cause of a Type 2 myocardial infarction, with a left ventricular pseudoaneurysm being a rare but important complication of this. Case summary: A 19-year-old male presented with acute confusion, fevers, and a cough. Blood cultures were positive for Fusobacterium necrophorum and initial imaging showed a cavitating pneumonia. Further evaluation revealed septic emboli in the distal digits and brain. The patient initially responded to antibiotic therapy but developed chest pain with increased troponin levels. An electrocardiogram showed inferolateral ST elevation. A transthoracic echocardiogram (TTE) showed hypokinaesia of the mid to apical lateral wall, and a computed tomography (CT) scan showed a pericardial effusion with a possible purulent effusion or abscess. The patient underwent surgical drainage of a sterile effusion. A post-operative TTE and CT demonstrated a left ventricular pseudoaneurysm that was surgically repaired. The venous thrombus was encountered intra-operatively confirming a diagnosis of Lemierre's syndrome. The patient completed the regimen of antibiotics and showed a good post-operative recovery. Discussion: This is the first case described of left ventricular pseudoaneurysm as a complication of Lemierre's syndrome. It highlights not only the importance of serial, multimodality imaging in both diagnostic workup and identification of complications, but also the importance of a multidisciplinary team in the management of patients with complex and rare presentations.

Chorioamnionitis disrupts erythropoietin and melatonin homeostasis through the placental-fetal-brain axis during critical developmental periods.

Introduction: Novel therapeutics are emerging to mitigate damage from perinatal brain injury (PBI). Few newborns with PBI suffer from a singular etiology. Most experience cumulative insults from prenatal inflammation, genetic and epigenetic vulnerability, toxins (opioids, other drug exposures, environmental exposure), hypoxia-ischemia, and postnatal stressors such as sepsis and seizures. Accordingly, tailoring of emerging therapeutic regimens with endogenous repair or neuro-immunomodulatory agents for individuals requires a more precise understanding of ligand, receptor-, and non-receptor-mediated regulation of essential developmental hormones. Given the recent clinical focus on neurorepair for PBI, we hypothesized that there would be injury-induced changes in erythropoietin (EPO), erythropoietin receptor (EPOR), melatonin receptor (MLTR), NAD-dependent deacetylase sirtuin-1 (SIRT1) signaling, and hypoxia inducible factors (HIF1α, HIF2α). Specifically, we predicted that EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α alterations after chorioamnionitis (CHORIO) would reflect relative changes observed in human preterm infants. Similarly, we expected unique developmental regulation after injury that would reveal potential clues to mechanisms and timing of inflammatory and oxidative injury after CHORIO that could inform future therapeutic development to treat PBI. Methods: To induce CHORIO, a laparotomy was performed on embryonic day 18 (E18) in rats with transient uterine artery occlusion plus intra-amniotic injection of lipopolysaccharide (LPS). Placentae and fetal brains were collected at 24 h. Brains were also collected on postnatal day 2 (P2), P7, and P21. EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α levels were quantified using a clinical electrochemiluminescent biomarker platform, qPCR, and/or RNAscope. MLT levels were quantified with liquid chromatography mass spectrometry. Results: Examination of EPO, EPOR, and MLTR1 at 24 h showed that while placental levels of EPO and MLTR1 mRNA were decreased acutely after CHORIO, cerebral levels of EPO, EPOR and MLTR1 mRNA were increased compared to control. Notably, CHORIO brains at P2 were SIRT1 mRNA deficient with increased HIF1α and HIF2α despite normalized levels of EPO, EPOR and MLTR1, and in the presence of elevated serum EPO levels. Uniquely, brain levels of EPO, EPOR and MLTR1 shifted at P7 and P21, with prominent CHORIO-induced changes in mRNA expression. Reductions at P21 were concomitant with increased serum EPO levels in CHORIO rats compared to controls and variable MLT levels. Discussion: These data reveal that commensurate with robust inflammation through the maternal placental-fetal axis, CHORIO impacts EPO, MLT, SIRT1, and HIF signal transduction defined by dynamic changes in EPO, EPOR, MLTR1, SIRT1, HIF1α and HIF2α mRNA, and EPO protein. Notably, ligand-receptor mismatch, tissue compartment differential regulation, and non-receptor-mediated signaling highlight the importance, complexity and nuance of neural and immune cell development and provide essential clues to mechanisms of injury in PBI. As the placenta, immune cells, and neural cells share many common, developmentally regulated signal transduction pathways, further studies are needed to clarify the perinatal dynamics of EPO and MLT signaling and to capitalize on therapies that target endogenous neurorepair mechanisms.

Placental mediated mechanisms of perinatal brain injury: Evolving inflammation and exosomes.

Pregnancy is an inflammatory process that is carefully regulated by the placenta via immunomodulation and cell-to-cell communication of maternal and fetal tissues. Exosomes, types of extracellular vesicles, facilitate the intercellular communication and traffic biologically modifying cargo within the maternal-placental-fetal axis in normal and pathologic pregnancies. Chorioamnionitis is characterized by inflammation of chorioamniotic membranes that produces systemic maternal and fetal inflammatory responses of cytokine dysregulation and has been associated with brain injury and neurodevelopmental disorders. This review focuses on how pathologic placental exosomes propagate acute and chronic inflammation leading to brain injury. The evidence reviewed here highlights the need to investigate exosomes from pathologic pregnancies and those with known brain injury to identify new diagnostics, biomarkers, and potential therapeutic targets.

New RAD51 Inhibitors to Target Homologous Recombination in Human Cells.

Targeting DNA repair proteins with small-molecule inhibitors became a proven anti-cancer strategy. Previously, we identified an inhibitor of a major protein of homologous recombination (HR) RAD51, named B02. B02 inhibited HR in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. Here, using a medicinal chemistry approach, we aimed to improve the potency of B02. We identified the B02 analog, B02-isomer, which inhibits HR in human cells with significantly higher efficiency. We also show that B02-iso sensitizes triple-negative breast cancer MDA-MB-231 cells to the PARP inhibitor (PARPi) olaparib.

Quantitative evaluation and comparison of coronary artery characteristics by 3D coronary volume reconstruction.

Non-atherosclerotic abnormalities of vessel calibre, aneurysm and ectasia, are challenging to quantify and are often overlooked in qualitative reporting. Utilising a novel 3-dimensional (3D) quantitative coronary angiography (QCA) application, we have evaluated the characteristics of normal, diabetic and aneurysmal or ectatic coronary arteries. We selected 131 individuals under 50 years-of-age, who had undergone coronary angiography for suspected myocardial ischaemia between 1st January 2011 and 31st December 2015, at the Bristol Heart Institute, Bristol, UK. This included 42 patients with angiographically normal coronary arteries, 36 diabetic patients with unobstructed coronaries, and 53 patients with abnormal coronary dilatation (aneurysm and ectasia). A total of 1105 coronary segments were analysed using QAngio XA 3D (Research Edition, Medis medical imaging systems, Leiden, The Netherlands). The combined volume of the major coronary arteries was significantly different between each group (1240 ± 476 mm3 diabetic group, 1646 ± 391 mm3 normal group, and 2072 ± 687 mm3 abnormal group). Moreover, the combined coronary artery volumes correlated with patient body surface area (r = 0.483, p < 0.01). Inter-observer variability was assessed and intraclass correlation coefficient of the total coronary artery volume demonstrated a low variability of 3D QCA (r = 0.996, p < 0.001). Dedicated 3D QCA facilitates reproducible coronary artery volume estimation and allows discrimination of normal and diseased vessels.

Starvation ketoacidosis on the acute medical take.

Starvation ketoacidosis (SKA) represents one of three metabolic acidoses caused by the accumulation of ketone bodies within the bloodstream. While easily treated, it is a diagnosis that can be easily missed in patients with an unexplained metabolic acidosis.In this case report, we discuss two patients presenting with a starvation ketoacidosis and psychiatric illness. This link between SKA and psychiatric disease is especially pertinent as this is a cohort of patients who may not be able to give an accurate history, which can potentially lead to a delay in diagnosis and treatment.Furthermore, the patient cohort described here have higher rates of alcohol dependence and are therefore at risk of alcoholic ketoacidosis. It is important to recognise that these conditions may coexist and should be managed as such, with thiamine prior to carbohydrate replacement in all at-risk patients.

Atomic force microscopy imaging and probing of DNA, proteins, and protein DNA complexes: silatrane surface chemistry.

Despite their rather recent invention, atomic force microscopes are widely available commercially. AFM and its special modifications (tapping mode and noncontact operation in solution) have been successfully used for topographic studies of a large number of biological objects including DNA, RNA, proteins, cell membranes, and even whole cells. AFM was also successfully applied to studies of nucleic acids and various protein DNA complexes. Part of this success is due to the development of reliable sample preparation procedures. This chapter describes one of the approaches based on chemical functionalization of mica surface with 1-(3-aminopropyl) silatrane (APS). One of the most important properties of APS-mica approach is that the sample can be deposited on the surface in a wide range of ionic strengths, in the absence of divalent cations and a broad range of pH. In addition to imaging of dried sample, APS-mica allows reliable and reproducible time lapse imaging in aqueous solutions. Finally, APS mica is terminated with reactive amino groups that can be used for covalent and ionic attachment of molecules for AFM force spectroscopy studies. The protocols for the preparation of APS, functionalization with APS mica and AFM probes, preparation of samples for imaging in air and in aqueous solutions, and force spectroscopy studies are outlined. All these applications are illustrated with a few examples.

Polymer Nanoarray Approach for the Characterization of Biomolecular Interactions.

Pair-wise interactions at the single-molecule level can be done with nanoprobing techniques, such as AFM force spectroscopy, optical tweezers, and magnetic tweezers. These techniques can be used to probe interactions between well-characterized assemblies of biomolecules, such as monomer-dimer, dimer-dimer, and trimer-monomer. An important step of these techniques is the proper assembly of dimers, trimers, and higher oligomers to enable the interactions to be probed. We have developed a novel approach in which a defined number of peptides are assembled along a flexible polymeric molecule that serves as a linear matrix, termed as flexible nanoarray (FNA). The construct is synthesized with the use of phosphoramidite chemistry (PA), in which non-nucleoside PA spacers and standard oligonucleotide synthesis are used to grow the polymeric chain with the desired length. The reactive sites are incorporated during FNA synthesis. As a result, the FNA polymer contains a set of predesigned reactive sites to which the peptides are covalently conjugated. We describe the protocol for the synthesis of FNA and the application of this methodology to measure the molecular interactions between amyloid peptides of monomer-monomer, monomer-dimer, and dimer-dimer.

[Live Animals and Staged Nature : Drawing and Photography in German Popular Zoology between 1860 and 1910]. / Lebende Tiere und inszenierte Natur : Zeichnung und Fotografie in der populären Zoologie zwischen 1860 und 1910.

It is the central thesis of this paper that the "biological perspective" (Lynn Nyhart) typical for Germany, with its interest in living animals, not only influenced natural history practices in many ways during the second half of the 19th century, rather also shaped the illustrations of popular zoology publications, as for example those in Brehms Thierleben. The illustrators of this period preferred to use live animals as models, which they studied in zoos. These animals were often depicted in their "natural" habitats. Since the illustrators knew only very little about these habitats, they had to be imagined. Another fashionable genre within popular zoology was the portrayal of animals fighting, which attracted attention because of their drama. The first wildlife photographers oriented themselves on the zoological illustrations and, with the aid of manipulation, staging and retouching, gave their photographs the impression of natural surroundings and drama. Yet both the illustrators and the photographers emphasized their truth to nature and - based on this - the scientific value of their pictures. In so doing, they developed a "biological" kind of wildlife photography, which, after the turn of the 19th century, allowed dedicated amateurs to create a popular zoological oeuvre that was well received by broad audiences.

The New Xpert MTB/RIF Ultra: Improving Detection of Mycobacterium tuberculosis and Resistance to Rifampin in an Assay Suitable for Point-of-Care Testing.

The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection.IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R.

Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases.

Guanine (G)-rich oligodeoxyribonucleotides (ODNs) can form undesired complexes by self association through non-Watson-Crick interactions. These aggregates can compromise performance of DNA probes and make genetic analysis unpredictable. We found that the 8-aza-7-deazaguanine (PPG), a pyrazolo[3,4-d]pyrimidine analog, reduces guanine self association of G-rich ODNs. In the PPG heterocycle, the N-7 and C-8 atoms of G are interposed. This leaves the ring system with an electron density similar to G, but prevents Hoogsteen-bonding associated with N-7. ODNs containing multiple PPG bases were easily prepared using a dimethylformamidine-protected phosphoramidite reagent. Substitution of PPG for G in ODNs allowed formation of more stable DNA duplexes. When one or more PPGs were substituted for G in ODNs containing four or more consecutive Gs, G aggregation was eliminated. Substitution of PPG for G also improved discrimination of G/A, G/G and G/T mismatches in Watson-Crick hybrids. Use of PPG in fluorogenic minor groove binder probes was also explored. PPG prevented aggregation in MGB probes (MGB(TM) is a trademark of Epoch Biosciences) and allowed use of G-rich sequences. An increased signal was observed in 5'-PPG probes due to reduced quenching of fluorescein by PPG. In summary, substitution of PPG for G enhances affinity, specificity, sensitivity and predictability of G-rich DNA probes.

A Metal-free Click Chemistry Approach for the Assembly and Probing of Biomolecules.

Probing of biomolecular complexes by single-molecule force spectroscopy (SMFS) methods including AFM requires proper and suitable coupling methods for immobilization of biomolecules onto the AFM tip and the surface. The use of flexible tethers for the coupling process has dual advantages. First, they allow the specific immobilization of interacting molecules, and second, their flexibility facilitates the proper orientation of the interacting partners. Recently, we developed an approach termed Flexible Nano Array (FNA) in which interacting partners are located on the same polymeric FNA molecule separated by a flexible segment with a defined length. In this paper, we modified the FNA tether approach by incorporating click chemistry with non-metal modification. FNA was synthesized using DNA synthesis chemistry, in which phosphoramidite (PA) spacers containing six ethylene glycol units were used instead of nucleoside triphosphates. During the synthesis, two T modifiers conjugated to two dibenzocyclooctyl (DBCO) residues were incorporated at selected positions within the FNA. The DBCO functionality allows for coupling azide labeled biomolecules via click chemistry. Amyloid peptide Aß(14-23) terminated with azide was incorporated into the FNA and the reaction was controlled with mass-spectrometry. Assembly of tethered Aß(14-23) peptides into dimers was characterized by AFM force spectroscopy experiments in which the AFM tip functionalized with FNA terminated with biotin probed a streptavidin-coated mica surface. The formation of the peptide dimer was verified with force spectroscopy that showed the appearance of a specific fingerprint for dimer dissociation followed by a rupture event for the biotin-streptavidin link. The developed approach is capable of multiple probing events to allow the collection of a large set of data for a quantitative analysis of the force spectroscopy events.

KP-1212/1461, a nucleoside designed for the treatment of HIV by viral mutagenesis.

We report the activities of a novel nucleoside analog against HIV. This nucleoside (KP-1212) is not a chain terminator but exerts its antiviral effects via mutagenesis of the viral genome. Serial passaging of HIV in the presence of KP-1212 causes an increase in the mutation rate of the virus leading to viral ablation. HIV strains resistant to KP-1212 have not yet been isolated. Quite to the contrary, virus treated with KP-1212 exhibited an increased sensitivity not only to KP-1212 but also to another nucleoside reverse transcriptase inhibitor (NRTI), zidovudine. HIV strains resistant to other NRTIs (e.g. zidovudine, lamivudine, stavudine, abacavir, etc.) exhibited no cross-resistance towards KP-1212. Multiple assays confirmed that KP-1212 has a favorable (low) genotoxicity profile when compared to some approved antiviral nucleosides. In addition, KP-1212 is not toxic to mitochondria nor does it exhibit any inhibitory effects on mitochondrial DNA synthesis.

Silatrane-based surface chemistry for immobilization of DNA, protein-DNA complexes and other biological materials.

The procedure of surface functionalization based on the use of 1-(3-Aminopropyl)silatrane (APS) instead of our early procedure utilizing aminopropyl triethoxy silane (APTES) is described. Unlike APTES, APS is less reactive and extremely resistant to hydrolysis and polymerization at neutral pH. The kinetics of DNA adsorption to APS-mica was studied. The results are consistent with a diffusion controlled mechanism suggesting that DNA molecules bind irreversibly with the surface upon immobilization. This conclusion is supported by the data on imaging of supercoiled DNA, the labile conformations of which are very sensitive to the conditions at the surface-liquid interface. In addition, we demonstrated directly that the segments of DNA molecules could move along the surface if the sample is imaged in aqueous solution without drying of the sample. Using the time-lapse mode of AFM imaging we visualized the transition of purine-pyrimidine sequence in supercoiled DNA from intramolecular triple-helical conformation (H-form) into the B-helix upon the change of pH from acidic (pH 5) to neutral. The mechanisms of the H-to-B transitions and the correlation of the local structural transitions with a global DNA conformation are discussed.

Visualization of DNA and protein-DNA complexes with atomic force microscopy.

This article describes sample preparation techniques for AFM imaging of DNA and protein-DNA complexes. The approach is based on chemical functionalization of the mica surface with aminopropyl silatrane (APS) to yield an APS-mica surface. This surface binds nucleic acids and nucleoprotein complexes in a wide range of ionic strengths, in the absence of divalent cations, and in a broad range of pH. The chapter describes the methodologies for the preparation of APS-mica surfaces and the preparation of samples for AFM imaging. The protocol for synthesis and purification of APS is also provided. The AFM applications are illustrated with examples of images of DNA and protein-DNA complexes.