BACKGROUND AND AIM:Colorectal cancer (CRC) screening is based on colonoscopy or fecal occult blood tests, but is imperfect and costly. The Asia Pacific Colorectal Screening Score (APCS) is derived from age, sex, family history of CRC, and smoking history and has been validated in Asian populations. Validation in a Western population is, however, yet to be tested. METHODS:In a teaching hospital, patients underwent colonoscopy for standard indications and screening over 18 months. Data was collected on age, sex, family history of CRC, smoking, weight, ethnicity, and symptoms. Evaluation of the APCS to predict colonoscopy findings (polyps, adenoma, high risk adenoma, and CRC) was performed. RESULTS:A total of 645 patients were prospectively recruited (46.7% male, median age 57 years); 17.8% were average risk (AR), 50.9% were moderate risk (MR), and 31.3% high risk (HR) on APCS. High risk adenomas (AA) were seen in 14.9% of the HR, 5.2% MR, and 0.9% LR patients, P < 0.0001. Comparing HR and MR to AR patients demonstrated significantly elevated relative risk (RR) for AA: 17.1 (95% confidence interval [CI] 2.4-123; P = 0.0001), and adenoma 6.0 (0.80-44.3; P = 0.044). Comparing HR to MR groups for AA, the RR was 2.87 (1.62-5.06; P = 0.0001). Symptoms did not predict findings (odds ratio [OR]: 1.06 [0.75-1.48]; P = 0.75). Body mass index (BMI) <20 kg/m(2) was protective against colonic polyps (OR: 0.28, 95%CI: 0.11-0.74; P = 0.010), adenoma (0.08, 0.01-0.62; P = 0.015), and AA (perfect prediction, OR 2.35 × 10(-8)). CONCLUSIONS:APCS predicts colonic findings in a Western population, to a greater extent than in Asians, independent to symptoms. Low body weight carries a strong protective effect against colonic neoplasia.

Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip-sample interactions in time (microseconds) and controlling force in the low pico-Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM-Live Cell probe, having a short cantilever with a 17-µm-long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico-Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions.

© 2014 Shaw et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Background: Deposition of amyloid-β (Aβ) in blood vessel walls as cerebral amyloid angiopathy (CAA) is observed in the majority of Alzheimer's disease (AD) brains. Inhibition of the 5-lipoxygenase (5-LOX) pathway has recently been suggested to play a role in reducing parenchymal Aβ deposition. However, products of the 5-LOX pathway also activate the peroxisome proliferator-activated receptor (PPAR) family, which promotes clearance of Aβ from the brain. Methods: In the present study, we investigated the effect of MK886, a 5-LOX-activating protein (FLAP) inhibitor and PPARα antagonist, on CAA severity in TgCRND8 mice overexpressing the human Swedish and Indiana amyloid precursor protein mutations. Results: We found that MK886 significantly reduced brain levels of nicastrin and PPARα, but did not affect levels of β-secretase, apolipoprotein E or low-density lipoprotein receptor-related protein-1. CAA severity and parenchymal plaque load was significantly decreased in both the cortex and hippocampus of mice treated with MK886 compared to control mice. Conclusion: These data suggest that 5-LOX and FLAP inhibitors may be useful in the treatment of CAA and AD.

To identify a lead skeleton structure for optimization of scyllo-inositol-based inhibitors of amyloid-beta peptide (Aβ) aggregation, we have synthesized aldoxime, hydroxamate, carbamate, and amide linked scyllo-inositol derivatives. These structures represent backbones that can be readily expanded into a wide array of derivatives. They also provide conservative modifications of the scyllo-inositol backbone, as they maintain the display of the equatorial polar atoms, preserving the stereochemical requirement necessary for maximum inhibition of Aβ(1-42) fiber formation. In addition, a reliable work plan for screening derivatives was developed in order to preferentially identify a backbone(s) structure that prevents fibrillogenesis and stabilizes nontoxic small molecular weight oligomers, as we have previously reported for scyllo-inositol. In the present studies, we have adapted a high throughput ELISA-based oligomerization assay followed by atomic force microscopy to validate the results screen compounds. The lead compounds were then tested for toxicity and ability to rescue Aβ(1-42) induced toxicity in vitro and the affinity of the compounds for Aβ(1-42) compared by mass spectrometry. The data to suggest that compounds must maintain a planar conformation to exhibit activity similar to scyllo-inositol and that the oxime derivative represents the lead backbone for future development.

beta-Amyloid (A beta) peptides are thought to play a major role in the pathogenesis of Alzheimer's disease. Compounds that disrupt the kinetic pathways of A beta aggregation may be useful in elucidating the role of oligomeric, protofibrillar and fibrillar A beta in the etiology of the disease. We have previously reported that scyllo-inositol inhibits A beta(42) fibril formation but the mechanism(s) by which this occurs has not been investigated in detail. Using a series of scyllo-inositol derivatives in which one or two hydroxyl groups were replaced with hydrogen, chlorine or methoxy substituents, we examined the role of hydrogen bonding and hydrophobicity in the structure-function relationship of scyllo-inositol-A beta binding. We report here that all scyllo-inositol derivatives demonstrated reduced effectiveness in preventing A beta(42) fibrillization compared with scyllo-inositol, suggesting that scyllo-inositol interacts with A beta(42) via key hydrogen bonds that are formed by all hydroxyl groups. Increasing the hydrophobicity of scyllo-inositol by the addition of two methoxy groups (1,4-di-O-methyl-scyllo-inositol) produced a derivative that stabilized A beta(42) protofibrils in vitro. Prophylactic administration of 1,4-di-O-methyl-scyllo-inositol to TgCRND8 mice attenuated spatial memory impairments and significantly decreased cerebral amyloid pathology. These results suggest that A beta aggregation can be targeted at multiple points along the kinetic pathway for the improvement of Alzheimer's disease-like pathology.

Structural insight into the conformational changes associated with aggregation and assembly of fibrils has provided a number of targets for therapeutic intervention. Solid-state NMR, hydrogen/deuterium exchange and mutagenesis strategies have been used to probe the secondary and tertiary structure of amyloid fibrils and key intermediates. Rational design of peptide inhibitors directed against key residues important for aggregation and stabilization of fibrils has demonstrated effectiveness at inhibiting fibrillogenesis. Studies on the interaction between Aβ and cell membranes led to the discovery that inositol, the head group of phosphatidylinositol, inhibits fibrillogenesis. As a result, scyllo-inositol is currently in clinical trials for the treatment of AD. Additional small-molecule inhibitors, including polyphenolic compounds such as curcumin, (−)-epigallocatechin gallate (EGCG), and grape seed extract have been shown to attenuate Aβ aggregation through distinct mechanisms, and have shown effectiveness at reducing amyloid levels when administered to transgenic mouse models of AD. Although the results of ongoing clinical trials remain to be seen, these compounds represent the first generation of amyloid-based therapeutics, with the potential to alter the progression of AD and, when used prophylactically, alleviate the deposition of Aβ.

scyllo-Inositol, ELND005, is a small molecule that is presently in phase II clinical trials for AD. Although our preclinical data demonstrated the efficacy in mouse models of AD, the direct binding of scyllo-inositol with Aβ is not well understood. Our previous in vitro data suggest that scyllo-inositol inhibits Aβ42 fibrillogenesis and stabilizes a small Aβ conformer but not Aβ40. To further understand this interaction, we examined the physical characteristics of this conformer using several biophysical approaches. Atomic force microscopy, neutral loss screening method using a triple quadrupole mass spectrometer and X-ray diffraction were utilized. By in situ atomic force microscopy we confirmed earlier electron microscopy data that Aβ42 fibres are not formed in the presence of scyllo-inositol, but rather form a mixture of conformer sizes. By collision-induced dissociation mass spectrometry, which incorporates a capillary ultra high pressure HPLC system coupled to a triple quadrupole mass spectrometer, we were able to measure the binding stoichiometry of the Aβ42-scyllo-inositol complex. We also demonstrated that scyllo-inositol preferentially binds to Aβ42 rather than Aβ40 under physiological conditions. Comparison of x-ray diffraction patterns from Aβ in the presence and absence of scyllo-inositol demonstrated an absence of the intersheet reflection for Aβ42 in the presence of scyllo-inositol whilst no changes could be detected for Aβ40. Altogether, our results suggest that scyllo-inositol binds to Aβ42 in a 2:1 ratio thereby inhibiting aggregation through interaction between the two C-terminal residues and the neighbouring ones that stabilizes the β-sheet formation of Aβ42.

Mass transfer between flowing blood and arterial mural cells (including vascular endothelial cells) may play an important role in atherogenesis. Endothelial cells are known to have an apical surface topography that is not flat, and hence mass transfer patterns to individual endothelial cells are likely affected by the local cellular topography. The purpose of this paper is to investigate the relationship between vascular endothelial cell surface topography and cellular level mass transfer. Confluent porcine endothelial monolayers were cultured under both shear and static conditions and atomic force microscopy was used to measure endothelial cell topography. Using finite element methods and the measured cell topography, flow and concentration fields were calculated for a typical, small, blood-borne solute. A relative Sherwood number was defined as the difference between the computed Sherwood number and that predicted by the Leveque solution for mass transfer over a flat surface: this eliminates the effects of axial location on mass transfer efficiency. The average intracellular relative Sherwood number range was found to be dependent on cell height and not dependent on cell elongation due to shear stress in culture. The mass flux to individual cells reached a maximum at the highest point on the endothelial cell surface, typically corresponding to the nucleus of the cell. Therefore, for small receptor-mediated solutes, increased solute uptake efficiency can be achieved by concentrating receptors near the nucleus. The main conclusion of the work is that although the rate of mass transfer varies greatly over an individual cell, the average mass transfer rate to a cell is close to that predicted for a flat cell. In comparison to other hemodynamic factors, the topography of endothelial cells therefore seems to have little effect on mass transfer rates and is likely physiologically insignificant.

Lsr2 is a small, basic protein present in Mycobacterium and related actinomycetes. Recent studies suggest that Lsr2 is a regulatory protein involved in multiple cellular processes including cell wall biosynthesis and antibiotic resistance. However, the underlying molecular mechanisms remain unknown. In this article, we performed biochemical studies of Lsr2-DNA interactions and structure-function analysis of Lsr2. Analysis by atomic force microscopy revealed that Lsr2 has the ability to bridge distant DNA segments, suggesting that Lsr2 plays a role in the overall organization and compactness of the nucleoid. Mutational analysis identified critical residues and selection of dominant negative mutants demonstrated that both DNA binding and protein oligomerization are essential for the normal functions of Lsr2 in vivo. These results provide strong evidence that Lsr2 is a DNA bridging protein, which represents the first identification of such proteins in bacteria phylogenetically distant from the Enterobacteriaceae. DNA bridging by Lsr2 also provides a mechanism of transcriptional regulation by Lsr2.

scyllo-Inositol has shown promise as a potential therapeutic for Alzheimer's disease, by directly interacting with the amyloid β (Aβ) peptide to inhibit Aβ42 fiber formation. To explore the molecular details of the inositol-Aβ42 interaction, a series of scyllo-inositol derivatives have been synthesized which contain deoxy, fluoro, chloro, and methoxy substitutions. The effects of these compounds on the aggregation cascade of Aβ42 have been investigated using electron microscopy (EM). EM analyses revealed that the 1-deoxy-1-fluoro- and 1,4-dimethyl-scyllo-inositols significantly inhibit the formation of Aβ42 fibers. The other derivatives showed some alterations in the morphology of the Aβ42 fibers produced. These findings indicate the importance of all of the hydroxyl groups of scyllo-inositol for complete inhibition of Aβ aggregation.

Purpose: Polymeric membranes containing pH-sensitive nano-hydrogels and glucose oxidase were found to exhibit glucose-responsive insulin release. To verify that this glucose-responsiveness stemmed from the decrease in the internal pH of the membranes, we determined the spatial and temporal pH profiles inside the composite membranes in situ for the first time.Materials and Methods: A pH-sensitive fluorescent dye and an inert internal reference was incorporated in the membranes consisting of poly(N-isopropylacrylamide-co-methacrylic acid) nanoparticles in a polymer matrix, with or without glucose oxidase and catalase. The fluorescence intensity versus time was measured by laser scanning confocal microscopy. The intensity ratios of the two fluorescent dyes were used to determine the internal pH profiles of the membranes in buffer solutions of various pH or glucose levels.Results: The internal pH was found to be lower than the external pH of buffer solutions. The pH decreased with an increase in glucose concentration, incubation time and the distance towards the center of the membranes due to the relative rates of glucose oxidation and solute diffusion.Conclusions: The results provided direct experimental evidence of acidic internal pH that inversely related to external glucose concentration in an external medium of constant neutral pH.

Our understanding of how antimicrobial and cell-penetrating peptides exert their action at cell membranes would benefit greatly from direct visualization of their modes of action and possible targets within the cell membrane. We previously described how the cationic antimicrobial peptide, indolicidin, interacted with mixed zwitterionic planar lipid bilayers as a function of both peptide concentration and lipid composition [Shaw, J.E. et al., 2006. J. Struct. Biol. 154 (1), 42–58]. In the present report, in situ atomic force microscopy was used to characterize the interactions between three families of cationic peptides: (1) tryptophan-rich antimicrobial peptides—indolicidin and two of its analogues, (2) an amphiphilic α-helical membranolytic peptide—melittin, and (3) an arginine-rich cell-penetrating peptide—Tat with phase-separated planar bilayers containing 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC)/1,2-distearoyl-sn-glycerol-3-phosphocholine (DSPC) or DOPC/N-stearoyl-d-erythro-sphingosylphosphorylcholine (SM)/cholesterol. We found that these cationic peptides all induced remodelling of the model membranes in a concentration, and family-dependent manner. At low peptide concentration, these cationic peptides, despite their different biological roles, all appeared to reduce the interfacial line tension at the domain boundary between the liquid-ordered and liquid-disordered domains. Only at high peptide concentration was the membrane remodelling induced by these peptides morphologically distinct among the three families. While the transformation caused by indolicidin and its analogues were structurally similar, the concentration required to initiate the transformation was strongly dependent on the hydrophobicity of the peptide. Our use of lipid compositions with no net charge minimized the electrostatic interactions between the cationic peptides and the model supported bilayers. These results suggest that peptides within the same functional family have a common mechanism of action, and that membrane insertion of short cationic peptides at low peptide concentration may also alter membrane structure through a common mechanism regardless of the peptide’s origin.

Acid beta-glucosidase (GCase) is a soluble lysosomal enzyme responsible for the hydrolysis of glucose from glucosylceramide and requires activation by the small nonenzymatic protein saposin C (sapC) to gain access to the membrane-embedded glycosphingolipid substrate. We have used in situ atomic force microscopy(AFM) with simultaneous confocal and epifluorescence microscopies to investigate the interactions of GCase and sapC with lipid bilayers. GCase binds to sites on membranes transformed by sapC, and enzyme activity occurs at loci containing both GCase and sapC. Using FRET, we establish the presence of GCase/sapC and GCase/product contacts in the bilayer. These data support a mechanism in which sapC locally alters regions of bilayer for subsequent attack by the enzyme in stably bound protein complexes.