Proximity Probes Research Articles

Two mosaic terminal inverted duplications arising post-zygotically: Evidence for possible formation of neo-telomeres

ObjectiveTo elucidate the structure of terminal inverted duplications and to investigate potential mechanisms of formation in two cases where there was mosaicism with cells of apparently normal karyotype.ResultsA karyotype [46,XY,inv dup(4)(p16.3p15.1)/46,XY] performed on blood lymphocytes from a patient referred for developmental delay (case 1) demonstrated a normal karyotype in 60% of cells with a terminal inverted duplication 4p in the remainder. In case 2, referred for multiple fetal anomalies on an ultrasound scan, 33% of amniocyte colonies were karyotypically normal, with a terminal inv dup 10p in the remainder [46,XX,inv dup(10)(p15.3p11)/46,XX]. Duplicated FISH signals for GATA3 and NEBL loci (in case 2), and for the Wolf-Hirschhorn locus (case 1) confirmed the inverted structure of both duplications. In the GTL banded normal cells from both cases, there was a cryptic deletion detected by FISH of one copy of the subtelomere 4p (case 1, probe GS-36P21), and subtelomere 10p (case 2, probe GS-306F7). At pter on both inv dup chromosomes there was no FISH signal present for the specific subtelomere probe. However, a positive pantelomeric probe signal was detected at 4 pter and 10 pter in both the cryptically-deleted chromosomes and the inv dup chromosomes in the respective cell lines of both cases.ConclusionAn inv dup structure was evident for both cases on GTL bands, and confirmed by the various FISH studies. The presence of telomere (TTAGGG repeat) sequences at pter on the inv dup chromosomes (where more proximal chromosome specific subtelomeric probes were negative) was indicated by the pantelomeric probe signals in both cases. We conclude the most likely mechanism of origin in both cases was by sub-telomeric breakage in the zygote at pter, and delayed repair/rearrangement until after one or more subsequent mitotic divisions. In these divisions, at least one breakage-fusion-bridge cycle occurred, to produce inverted duplications. It is proposed then that two differently "repaired" daughter cells proliferated in parallel. In one daughter cell line (with an overtly normal karyotype) there was deletion of the subtelomere and presumed repair through capping by a neo-telomere (i.e. "healing", as initially proposed by McClintock). This occurred in both cases presented. In the other daughter cell of each case, it is proposed that chromosome stabilization was achieved (after replication) by sister chromatid reunion to form a dicentric, which broke at a subsequent anaphase, to form an inverted duplication (with loss of the reciprocal product, and the other daughter cell line). One inv dup was repaired without an interstitial specific subtelomere (case 1) and one was repaired with a duplicated specific interstitial subtelomere (case 2). After repair TTAGGG repeats were detected by FISH at each respective new pter.

Femoral artery‐vein shunt steals collateral flow and promotes collateral arteriogenesis

To study high flow‐stimulated collateral artery enlargement, a side to side femoral artery‐vein anastomosis was established in Yucatan pigs (~40 kg, n=7, limited to cage activity). The femoral artery was occluded proximally to the a‐v shunt by an Ameroid occluder placed between the a‐v shunt and a proximal transonic flow probe. Femoral artery blood flows (BF) were monitored daily to evaluate the magnitude and time‐course of BF change. Before femoral artery occlusion, BF through the proximal flow probe increased from ~400 to 700 ml/min, while BF through the distal flow probe (distal to the shunt) were ~100 to 200 ml/min. Following femoral artery occlusion (~ 2 wks post surgery), proximal BF reduced to only ~50 ml/min, while distal BF (i.e., arising from collateral dependent BF delivered distal to the shunt) gradually increased from ~200 to 400 ml/min by 4 wks post‐surgery. Thus, a significant­ly greater collateral‐depen­dent BF was shunted back to the central circulation over time. This placed increasing flow demands on the collateral circuit. Angiograms showed extensive collateral vessel development in the upper thigh area in the leg with the a‐v shunt, primarily arising from the profunda artery. We attribute this collateral vessel enlarge­ment to the apparent high shear stress induced in these vessels that would occur to support flow through the low‐resistance shunt created by the by‐pass. Thus, we conclude that an exceptional enlargement of the collateral circuit can be induced over a rather brief period of time, if the stimulus for collateral development is sufficient.Supported by NIH grant P01HL 52490‐12

Self-Assembly of Proximity Probes for Flexible and Modular Proximity Ligation Assays

Proximity ligation assay (PLA) is a recently developed strategy for protein analysis in which antibody-based detection of a target protein via a DNA ligation reaction of oligonucleotides linked to the antibodies results in the formation of an amplifiable DNA strand suitable for analysis. Here we describe a faster and more cost-effective strategy to construct the antibody-based proximity ligation probes used in PLA that is based on the noncovalent interaction of biotinylated oligonucleotides with streptavidin followed by the interaction of this complex with biotinylated antibodies.

On the technical diagnostics of ball-bearing based on modelling of rotor-bearing systems by vibration method

In this paper, after analysing deformations of loading ball-bearing it is able to investigate vibrations of rotor-bearing system as the system with non-linear and periodically varying stiffness. The modelling of the systems is depent on properties of the shaft (rigid or flexible) and on measurement techniques for system vibrations (by eddy current proximity probe for rotating shaft or on non-rotating parts of the rotor-bearing systems) it can be described by a single degree-of-freedom or two-degree-of freedom nonlinear systems subjected to parametric and external excitations. Therefore it is possible to obtain different characteristics of system vibrations with respect to different defects of ball-bearing. These symptoms help us to identify and estimate the bearing quality by measurement and analysis of system vibrations.

In Situ Detection of Phosphorylated Platelet-derived Growth Factor Receptor β Using a Generalized Proximity Ligation Method

Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.

Manipulation of thrombin exosite I, by ligand-directed covalent modification.

For many enzymes, substrate specificity is directed by secondary binding sites (exosites) that are remote from the active site. Peptide inhibition studies of protein-protein interactions are useful to identify exosite functions. To develop an approach to manipulate these exosites using ligand-directed covalent modification of the enzyme. To demonstrate this strategy, we have engineered an exosite-deficient variant of human plasma-derived thrombin (FIIa) . Desulfato-hirugen (Hir(55-65)) analogs were synthesized with a fluorescent label, photocrosslinker, and an optional cleavable linker conjugated to the N-terminus of the peptide, specifically fluorescein-benzoyl-phenylalanyl-(Fl-bF-)glycyl-Hir(55-65), Fl-bF-mercaptopropionyl-Hir(55-65) and Fl-bF-lactyl-Hir(55-65) were synthesized. Each analog was bound and photocrosslinked to FIIa, and the resulting covalent complex was purified. This modified enzyme, FIIa-Hir(55-65), hydrolyzed small substrates as efficiently as native FIIa, but was significantly inhibited in fibrinogen clotting and in thrombomodulin-mediated PC activation, implying that the active site was unaffected by labeling but exosite I was blocked. In addition, this approach was used to transfer a fluorescein label from the exosite I binding peptide Hir(55-65) to a site proximal to but not obstructing exosite I. The activity of this fluorescently labeled FIIa (Fl-FIIa) could be inhibited by unlabeled Hir(55-65), suggesting that exosite I is unmodified. Importantly, this interaction could be followed spectroscopically by fluorescence, demonstrating that the exosite I proximal probe can be used to monitor specific ligand binding interactions. Our results show that exosites of clotting factors (e.g. thrombin) can be specifically inhibited and labeled with fluorescent reporters. This novel technology may have broad applicability for studies of protein-protein interactions that regulate coagulation.

Gastroesophageal Flap Valve Is Associated with Gastroesophageal and Gastropharyngeal Reflux

Background: The endoscopic grading of the gastroesophageal flap valve (GEFV) is suggested to be a good predictor of the reflux status. The aim of this study was to investigate the association of the GEFV and gastroesophageal and gastropharyngeal reflux. Methods: A total of 364 patients (151 men and 233 women; mean age 52.2 years) who underwent endoscopy, esophageal manometry, and ambulatory 24-hour dual probe pH monitoring were included. GEFV was graded I through IV using Hill's classification: the GEFV was largely classified into 2 groups: the normal GEFV group (grade I and II) and the abnormal GEFV group (grade III and IV). Findings of endoscopy, esophageal manometry, and ambulatory pH monitoring were compared among the groups. Results: Increased GEFV grade was significantly associated with an increased prevalence of both reflux esophagitis and Barrett's epithelium (p < 0.001). LES pressure was significantly lower in the abnormal GEFV group compared to the normal GEFV group (p < 0.001). All variables showing gastroesophageal reflux in the distal probe were significantly higher in the abnormal GEFV group compared to the normal GEFV group (p < 0.001). In addition, all variables, except the supine time of pH below 4, showing the gastropharyngeal reflux in the proximal probe were significantly higher in the abnormal GEFV group compared to the normal GEFV group (p < 0.001). The frequency of gastroesophageal reflux disease and gastropharyngeal reflux disease was significantly higher in the abnormal GEFV group compared to the normal GEFV group (p < 0.001). Conclusions: Endoscopic grading of the GEFV is easy and provides useful information about the status of gastroesophageal and gastrophryngeal reflux.

Piezoresistive and self-actuated 128-cantilever arrays for nanotechnology applications

A major limitation for future nanotechnology, particularly for bottom-up manufacturing is the non-availability of 2-dimensional massively parallel probe arrays. Scanning proximity probes are uniquely powerful tools for analysis, manipulation and bottom-up synthesis: they are capable of addressing and engineering surfaces at the atomic level and are the key to unlocking the full potential of Nanotechnology. Generic massively parallel intelligent cantilever-probe platforms is demonstrated through a number of existing and ground-breaking techniques. A packaged VLSI NEMS-chip (Very Large Scale Integrated Nano Electro Mechanical System) incorporating 128 proximal probes, fully addressable with control and readout interconnects and advanced software will be presented.

Esophageal Mucosal Sensitivity: Possible Links with Clinical Presentations in Patients with Erosive Esophagitis and Laryngopharyngeal Reflux

The goal of this study was to assess whether mucosal sensitivity is related to different combinations of symptoms and lesions in laryngopharyngeal reflux (LPR) and erosive esophagitis and to determine whether reflux pattern is important in the development of the symptoms and lesions of gastroesophageal reflux disease (GERD) or LPR. We conducted a prospective study of 55 unselected new patients: 27 erosive esophagitis patients and 28 LPR patients. The erosive esophagitis group had a significantly higher rate of Bernstein test positivity than the LPR group. The proportions of patients with pathologic acid reflux at the proximal and distal probe were similar in the erosive esophagitis group and in the LPR group. These results were inconsistent with the endoscopic findings. Both retrosternal heartburn and regurgitation symptoms were higher in erosive esophagitis patients. The paucity of classical GERD symptoms and lesions in LPR patients could be attributable to mucosal insensitivity but not to reflux pattern.

Endoscopic Grading of Atrophic Gastritis is Inversely Associated with Gastroesophageal Reflux and Gastropharyngeal Reflux

BackgroundReflux esophagitis is inversely associated with the presence of atrophic gastritis, and endoscopic grading of atrophic gastritis correlates with histological evaluation. The aim of this study was to investigate the association of the endoscopic grade of atrophic gastritis with gastroesophageal and gastropharyngeal reflux.MethodsA total of 627 patients, who underwent endoscopy and ambulatory 24-hour dual-probe pH monitoring, were included in this study. The grade of atrophic gastritis was endoscopically classified into 2 types with the atrophic pattern system: the closed-type (C-type) and the open-type (O-type). We compared the findings from endoscopy and ambulatory pH monitoring for these 2 types.ResultsThe O-type was significantly associated with a lower prevalence of reflux esophagitis (p=0.001). All variables showing gastroesophageal reflux in the distal probe were significantly lower in the O-type than in the C-type (p<0.05). Similarly for the proximal probe, all variables, except the supine time of pH<4, were significantly lower in the O-type than in the C-type (p<0.05). The frequency of gastroesophageal reflux disease and gastropharyngeal reflux disease was in significantly lower in the O-type than in the C-type (p<0.001, p=0.012, respectively).ConclusionsEndoscopic grading of atrophic gastritis is easy and is inversely associated with gastroesophageal and gastropharyngeal reflux.

Organization of the interferon-inducible 2',5'-oligoadenylate-dependent ribonuclease L (RNase L) gene of mouse.

Interferons (IFNs) induce a 2',5'-oligoadenylate (2-5A)-dependent ribonuclease L (RNase L) following virus-infection of mammalian cells. RNase L degrades both viral and cellular RNAs and restricts virus-proliferation. We have studied organization of RNase L gene in genomic DNA from the mouse liver by Southern blot analysis. Several BamHI, BglII, EcoRI, HincII, HindIII, NcoI, PstI, SacI, and XbaI restriction fragments hybridized to (32)P-labeled mouse RNase L cDNA and the 5'-proximal exon probes. Mouse RNase L gene exists as a single copy (>16 kb DNA) gene. A 5 kb HindIII and a 2.5 kb EcoRI DNA were detected as 5'-upstream DNA of the gene which may contain mouse RNase L promoter. Our results will help studying mouse RNase L gene promoter in further details.

Micromachined piezoresistive proximal probe with integrated bimorph actuator for aligned single ion implantation

The authors report a microfabrication procedure of self-actuated piezoresistive scanning probes (SAPSPs). They are designed for a SAPSP instrument that is integrated with an ion beam for aligned single ion implantation in ultrahigh vacuum. The novelty of the design is an integrated hollow pyramid, instead of a previously mechanically hand mounted pyramid [J. Vac. Sci. Technol. B 23, 2798 (2005)]. The pyramid has dual purpose. First it collimates the ion beam and suppresses secondary particles from the back side of the cantilever, so that secondary particles from the target material can be used for single ion detection. Second the pyramid also provides an atomic force microscope tip for the scanning probe. A crucial step in the fabrication is the back side opening via etching for the hollow pyramid. The fabrication procedure will be discussed in detail.

Proximal Probes Based Nanorobotic Drawing of Polymer Micro/Nanofibers

This paper proposes a nanorobotic fiber fabrication method which uses proximal probes to draw polymer fibers down to few hundred nanometers in diameter and several hundred micrometers in length. Using proximal probes such as Atomic Force Microscope (AFM) and Scanning Tunneling Microscope (STM) or glass micropipettes, liquid polymers dissolved in a solvent are drawn. During drawing, the solvent evaporates in real-time which solidifies the fiber. Controlling the drawn fibers trajectory and solidification in three-dimensions (3-D), suspended fibers, fiber cantilevers, custom 3-D fibers, and fiber networks, are proposed to be fabricated. Poly(methyl methacrylate) (PMMA) polymer dissolved in chlorobenzene is used to form a variety of suspended polymer fibers with diameters from few microns to 200nm. Fabrication of crossed and linear networks of fibers is also demonstrated. Viscoelastic modeling of polymer fiber drawing is realized using a finite element method to test the significance of the drawing speed and velocity profile on the extensional behavior of the drawn fiber. Since the mechanical properties of the drawn micro/nanofibers could vary from the bulk polymer material significantly, mechanical characterization of suspended fibers using an AFM and a Nanoindenter setup is proposed. Extending this technique to a variety of nonconductive and electroactive polymer fibers, many novel applications in micro/nanoscale sensors, actuators, fibrillar structures, and optical and electronic devices would become possible

The role of gastric Helicobacter pylori infection in laryngopharyngeal reflux disease

To investigate the role of Helicobacter pylori (H pylori) infection in laryngopharyngeal reflux (LPR). A prospective multidisciplinary clinical study. Forty-four adult patients who applied to our ENT clinic with LPR symptoms were evaluated. Then these patients underwent upper gastrointestinal system endoscopy and double probe pH monitoring. In addition, during the endoscopy multiple biopsies from the stomach were obtained to detect H pylori. Results from 32 LPR positive patients were assessed (10 male and 22 female). There were no statistically significant differences between the presence of H pylori and sex, age, degree of gastritis and esophagitis, and also the number of reflux, fractional acid exposure time regarding proximal probe readings. Similarly for both proximal and distal probe readings, the average score of esophageal acid clearance was not statistically significant (P > 0.05). In addition, no statistically significant difference was found between the presence of H pylori and GERD (P > 0.05). The results of this study demonstrated that there is no relationship between gastric H pylori infection and LPR. B-3b.

Gastroesophageal flap valve is associated with gastroesophageal and gastropharyngeal reflux

The endoscopic grade of the gastroesophageal flap valve (GEFV) is suggested to be a good predictor of reflux status. The aim of this study was to investigate the association of the GEFV and gastroesophageal and gastropharyngeal reflux. A total of 364 patients (151 men and 233 women; mean age, 52.2 years) who underwent endoscopy, esophageal manometry, and ambulatory 24-h dual-probe pH monitoring were included. GEFV was graded I through IV using Hill's classification; then, GEFV was classified into two groups: a normal GEFV group (grades I and II) and an abnormal GEFV group (grades III and IV). Findings of endoscopy, esophageal manometry, and ambulatory pH monitoring were compared between the groups. Increased GEFV grade was significantly associated with an increased prevalence of both reflux esophagitis and Barrett's epithelium (P < 0.001). Lower esophageal sphincter pressure was significantly lower in the abnormal GEFV group than in the normal GEFV group (P < 0.001). All variables showing gastroesophageal reflux in the distal probe were significantly higher in the abnormal GEFV group than in the normal GEFV group (P < 0.001). In addition, all variables, except the supine time of pH < 4, showing gastropharyngeal reflux in the proximal probe were significantly higher in the abnormal GEFV group than in the normal GEFV group (P < 0.001). The frequency of gastroesophageal reflux disease and of gastropharyngeal reflux disease was significantly higher in the abnormal GEFV group than in the normal GEFV group (P < 0.001). Endoscopic grading of the GEFV is easy and provides useful information about the status of gastroesophageal and gastropharyngeal reflux.

Measurement of the load behaviour in a dry pilot mill using an inductive proximity probe

Load behaviour in a dry pilot mill has been successfully measured as a function of mill speed and load filling using an inductive proximity probe. The inductive proximity probe detects the presence of metallic objects in the proximity of its sensing face. Static and dynamic test demonstrate that it is suitable in measuring load behaviour. The shoulder position is measured more reliably by the inductive proximity probe due to this region being well behaved and less variable as compared to the toe region. The shoulder and toe angular positions of the inductive proximity probe signal vary with mill speed and load filling. Cataracting of balls onto the mill shell at high mill speeds is detected. The probe is sensitive to changes in mill operating conditions and load packing. A comparison between the force probe and the inductive proximity probe reveals that the inductive proximity probe is more superior in measuring load behaviour.

Scanning probe acceleration microscopy (SPAM) in fluids: Mapping mechanical properties of surfaces at the nanoscale

One of the major thrusts in proximal probe techniques is combination of imaging capabilities with simultaneous measurements of physical properties. In tapping mode atomic force microscopy (TMAFM), the most straightforward way to accomplish this goal is to reconstruct the time-resolved force interaction between the tip and surface. These tip-sample forces can be used to detect interactions (e.g., binding sites) and map material properties with nanoscale spatial resolution. Here, we describe a previously unreported approach, which we refer to as scanning probe acceleration microscopy (SPAM), in which the TMAFM cantilever acts as an accelerometer to extract tip-sample forces during imaging. This method utilizes the second derivative of the deflection signal to recover the tip acceleration trajectory. The challenge in such an approach is that with real, noisy data, the second derivative of the signal is strongly dominated by the noise. This problem is solved by taking advantage of the fact that most of the information about the deflection trajectory is contained in the higher harmonics, making it possible to filter the signal by "comb" filtering, i.e., by taking its Fourier transform and inverting it while selectively retaining only the intensities at integer harmonic frequencies. Such a comb filtering method works particularly well in fluid TMAFM because of the highly distorted character of the deflection signal. Numerical simulations and in situ TMAFM experiments on supported lipid bilayer patches on mica are reported to demonstrate the validity of this approach.

Effects of grinding media shapes on load behaviour and mill power in a dry ball mill

Little attention has been given to the study of grinding media shapes and how they affect load behaviour and power draw in grinding mills. To a large extent the focus has been on how grinding media shapes affect milling kinetics in terms of specific rate of breakage and breakage distribution parameterisation. This study investigated the effects of three media shapes (cylpebs, spherical and worn balls) on load behaviour and mill power draw at various mill speeds and load filling. An inductive proximity probe was used to determine the load orientation of the grinding media charge while a load beam enabled measurement of power draw. The variations in toe and shoulder positions among media shapes were observed. This was also reflected in the power draw. The power increases to a maximum with increasing mill speed for all media shapes. The maximum power draw was reached at different mill speeds for the three studied media shapes.

Scanning proximity probes for nanoscience and nanofabrication

The AFM-technology has undergone tremendous development during the past decade. This review is devoted to the realization of piezoresistive sensors used in scanning probe microscopy at University of Kassel. It is expected that in the near future major technological breakthroughs in scanning proximal probe nanotools will allow for key scientific impact on analysis and synthesis of nanostructures. All the piezoresistive cantilever sensors described here are based on advanced silicon micromachining and standard CMOS processing. Moreover, using a newly optimized piezoresistive detection scheme process comprising a Wheatstone bridge, we have designed and fabricated piezoresistive cantilevers for atomic force microscopy, which improve surface topography resolution by an order of magnitude to 0.1 nm. The elegance of this concept is that by using an almost identical detection principle and differently functionalized tips or cantilever surfaces, we can detect subtle sample interactions (mechanical, electrical, thermal, and chemical) with a significantly more compact system than with optical beam deflection techniques. For non-contact scanning force microscopy, we integrate a thermally driven bimorph actuator with the piezoresistive cantilever and make use of direct-oscillation in a higher flexural mode. The cantilever then operates in the phase shift atomic force microscopy (AFM) detection technique.

Plasmon interactions between gold nanoparticles in aqueous solution with controlled spatial separation

The effects of interparticle distance on the UV-visible absorption spectrum of gold nanocrystals aggregates in aqueous solution have been investigated. The aggregates were produced by ion-templated chelation of omega-mercaptocarboxylic acid ligands covalently attached to the nanoparticles surface. Variation of the ligand chain length provides control over the interparticle separation in the aggregates. The UV-visible spectra consist typically of a single particle band and a secondary band at higher wavelengths associated with the formation of aggregates in solution. The position of the latter depends on interparticle separation up to distances of approximately 8 nm, in accordance with existing models. Potential applications therefore include distance sensitive labels or proximity probes. Conversely, variation of the ligand length allows the preparation of nanostuctured materials with tuned optical properties.