Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA’s receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus–cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA’s receptor binding preference does not necessarily reflect virus–cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.

Bone is a composite material consisting of organic and inorganic components that are organized into hierarchical structures to provide load-bearing functions. This paper presents the results of PeakForce Tapping atomic force microscopy (AFM) scans on cut and polished bovine cortical bone specimens that were submerged in water. The elastic modulus map and surface morphology were obtained for various bone hierarchical structures from submillimeter- to submicron-scales. The elastic modulus of osteons (20.51 +/- 6.85 GPa) was slightly lower than the interstitial bone (21.87 +/- 5.48 GPa); they were both much greater than that of the cement lines (7.49 +/- 4.23 GPa). The elastic modulus in the lamella structures varied periodically from higher values in thick sub-lamellae (21.49 +/- 6.58 GPa) to the lower values in thin sub-lamellae (9.67 +/- 2.69 GPa). The results also show relatively softer mineralized collagen fibril bundle arrays (12.94 +/- 2.71 GPa) embedded in harder matrix materials (28.39 +/- 5.75 GPa). The variations in the elastic modulus suggest different degrees of mineralization or different fibril orientations. The histograms of elastic modulus indicate the dominating compositions or dominating fibril orientations. (C) 2020 Elsevier Ltd. All rights reserved.

Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.

Human mtDNA contains three promoters, suggesting a need for differential expression of the mitochondrial genome. Studies of mitochondrial transcription have used a reductionist approach, perhaps masking differential regulation. Here we evaluate transcription from light-strand (LSP) and heavy-strand (HSP1) promoters using templates that mimic their natural context. These studies reveal sequences upstream, hypervariable in the human population (HVR3), and downstream of the HSP1 transcription start site required for maximal yield. The carboxy-terminal tail of TFAM is essential for activation of HSP1 but not LSP. Images of the template obtained by atomic force microscopy show that TFAM creates loops in a discrete region, the formation of which correlates with activation of HSP1; looping is lost in tail-deleted TFAM. Identification of HVR3 as a transcriptional regulatory element may contribute to between-individual variability in mitochondrial gene expression. The unique requirement of HSP1 for the TFAM tail may enable its regulation by post-translational modifications.

The 2009 influenza pandemic originated from a swine-origin H1N1 virus, which, although less pathogenic than anticipated, may acquire additional virulence-associated mutations in the future. To estimate the potential risk, we sequentially passaged the isolate A/Hamburg/04/2009 in A549 human lung epithelial cells. After passage 6, we observed a 100-fold increased replication rate. High-throughput sequencing of viral gene segments identified five dominant mutations, whose contribution to the enhanced growth was analyzed by reverse genetics. The increased replication rate was pinpointed to two mutations within the hemagglutinin (HA) gene segment (HA1 D130E, HA2 I91L), near the receptor binding site and the stem domain. The adapted virus also replicated more efficiently in mice in vivo. Enhanced replication rate correlated with increased fusion pH of the HA protein and a decrease in receptor affinity. Our data might be relevant for surveillance of pre-pandemic strains and development of high titer cell culture strains for vaccine production.

We describe microarrays of receptor molecules that capture viral particles with high specificity and at high density. Patches of viral receptors were generated by first modifying microscale gold squares on glass substrates with alkanethiol derivatives and then immobilizing the His -tagged very-low-density lipoprotein 6 (VLDL) receptor ligand binding domain via metal-chelate complexes to the gold surfaces. Free glass areas surrounding the gold squares were passivated with a dense film of poly(ethylene glycol) (PEG). As assessed by atomic force microscopy, human rhinovirus particles were captured onto the VLDL-receptor patches with a high surface coverage but were effectively repelled by the PEG layer, resulting in a 330 000-fold higher density of the particles on the gold as compared to the glass surfaces. The metal chelate-based coupling strategy was found to be superior to two alternative routes, which used the covalent coupling of viral particles or viral receptors to the substrate surface. The high density receptor arrays were employed for sensing and characterizing viral particles with so far unprecedented selectivity. Furthermore, an alternative route to create arrays in the nanometer range for the site specific binding of proteins, nanoparticles and pathogens is shown. These arrays are results of AFM based nanolithography performed on Mica substrates passivated by repellent protein- or PEG films. We could show that with the help of this method we are able to create nanometer-sized structures in the range of an AFM tip radius. The structures created in this manner were subsequently refilled with proteins which serve as a basis for the specific binding of other proteins, particles or pathogens of interest and could furthermore be analyzed by AFM under near physiological conditions.

Background H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. Methodology/Principal Findings We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH≤5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. Conclusion/Significance Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.

Single-molecule characterization is essential for ascertaining the structural and functional properties of bottom-up DNA nanostructures. Here we enlist three atomic force microscopy (AFM) techniques to examine tetrahedron-shaped DNA nanostructures that are functionally enhanced with small chemical tags. In line with their application for biomolecule immobilization in biosensing and biophysics, the tetrahedra feature three disulfide-modified vertices to achieve directed attachment to gold surfaces. The remaining corner carries a single bioligand that can capture and present individual cargo biomolecules at defined lateral nanoscale spacing. High-resolution AFM topographic imaging confirmed the directional surface attachment as well as the highly effective binding of individual receptor molecules to the exposed bioligands. Insight into the binding behavior at the single-molecule level was gained using molecular recognition force spectroscopy using an AFM cantilever tip with a tethered molecular receptor. Finally, simultaneous topographic and recognition imaging demonstrated the specific receptor-ligand interactions on Individual tetrahedra. In summary, AFM characterization verified that the rationally designed DNA nanostructures feature characteristics to serve as valuable immobilization agents in biosensing, biophysics, and cell biology.

Capturing and detecting viruses require specialized platforms and analysis techniques. The atomic force microscope (AFM) offers not only tremendously high spatial and topographic resolution, but also allows localizing single molecules and determining their interaction on a molecular level. AFM makes possible (a) generating nanopatterned surfaces, (b) detecting protein and viral pathogen captured in these specific regions over time, and (c) gathering information about binding parameters. A section of this chapter describes, for instance, the measurement of kinetic parameters governing the first step of viral infection the attachment of a human rhinovirus to a cell at the single-molecule level. We review the principles of AFM and its applications in the analysis of nanopatterned surfaces and protein arrays, in the detection of proteins and viruses, and in the characterization of the interactions of these viruses with their cognate receptors under physiological conditions.

Microwave measurements combined with scanning probe microscopy is a novel tool to explore high-localized mechanical and electrical properties of biological species. Complex permittivities and permeabilities are detected through slight variations of an incident microwave signal. Here we report the high-frequency dependence of the electromagnetic dynamic characteristics in human monocytic leukemia cells (THP1) through local measurements by scanning microwave microscopy (SMM). The amplitude and phase images were shown to depend on the applied resonance frequency. While the amplitude yields information about the resistivity determined by the water and the ionic strength, the phase information reflects the dielectric losses arising from the fluid density.

Tabelle 3 auf Seite S8 der Hintergrundinformationen für diese Zuschrift enthielt eine fehlerhafte Bezeichnung, die in der nun verfügbaren Version der Hintergrundinformationen korrigiert wurde. Die Autoren entschuldigen sich für dieses Versehen und versichern, dass es keine Auswirkungen auf die Interpretation der Resultate hatte.

We describe microarrays of receptors on gold/glass substrates for the selective capturing of viral particles at high density. Microscale gold squares were surface-modified with alkanethiol derivatives which enabled the immobilization of the HiS(6)-tagged virus-binding domain from the very-low density lipoprotein (VLDL) receptor. The free glass areas surrounding the gold squares were passivated with a dense film of poly(ethylene glycol) (PEG). As assessed by atomic force microscopy, human rhinovirus particles were captured onto the VLDL-receptor patches with a high surface coverage but were effectively repelled by the PEG layer, resulting in a 330 000-fold higher density of the particles on the gold as compared to the glass surfaces. The metal chelate-based coupling strategy was found to be superior to two alternative routes, which used the covalent coupling of viral particles or viral receptors to the substrate surface. The high-density receptor arrays were employed for sensing and characterizing viral particles with so far unprecedented selectivity.

Native-protein nanolithography is combined with topography and recognition imaging to synergistically use AFM tips to write and image nanoscale protein patterns on a surface (see picture). The approach is validated with different feedback modes, using surface-bound biotinylated bovine serum albumin (BSA) protein and AFM tips carrying streptavidin.

Native-protein nanolithography (NPNL) was used to fabricate stable bioactive arrays of viral receptor spots. The arrays were specific for the cognate virus and devoid of nonspecific protein and virus adsorption under physiologic conditions. The spot size ranged from 200 nm x 200 nm to 2 microm x 2 microm and up to 3 x 3 spots were arranged per array. With proper force adjustment in the patterning experiments, His(6)-tagged bovine serum albumin (BSA) molecules were selectively removed from the underlying self-assembled monolayer (SAM) while leaving the latter intact. Injection of His(6)-tagged very low density lipoprotein receptor (VLDLR-His(6)) constructs resulted in specific, oriented binding to the Ni(2+)-loaded bis-(nitrolotriacetic acid) (bis-NTA) groups to the re-exposed SAM areas. The arrays of viral receptors were used for the detection of human rhinovirus particles (serotype 2; HRV2) under native conditions by topographical imaging at high signal-to-noise ratio. The kinetic on-rate of the HRV2-VLDLR interaction was derived from the time-dependent binding of the virions to the VLDL receptor spots. No significant binding was observed for the major group virus HRV14 that uses the unrelated receptor ICAM-1.

Enamel bond strength is an important factor in restorative dentistry and crucially depends on the enamel roughness. To increase roughness, different etching procedures are employed and profilometric estimations, with probe profilometers, including atomic force microscopy (AFM), have been made. However, no correlation between roughness and bond strength has been found. To search for a possible error source leading to the underestimation of enamel roughness when utilizing probe profilometers, the authors compared scanning electron microscopy and AFM images of acid-etched tooth enamel. The results showed that AFM imaging cannot correctly depict the acid-etched enamel surface, because of the high steepness of the enamel crystallites and the generation of convolute images. This leads to a large underestimation of the profilometric parameters measured with AFM, or other profilometers, on acid-etched tooth enamel surfaces.