Atomic Force Microscopy Study of the Long-Term Effect of the Glycerol Flow, Stopped in a Coiled Heat Exchanger, on Horseradish Peroxidase.

Glycerol is employed as a functional component of heat-transfer fluids, which are of use in both bioreactors and various biosensor devices. At the same time, flowing glycerol was reported to cause considerable triboelectric effects. Herein, by using atomic force microscopy (AFM), we have revealed the long-term effect of glycerol flow, stopped in a ground-shielded coiled heat exchanger, on horseradish peroxidase (HRP) adsorption on mica. Namely, the solution of HRP was incubated in the vicinity of the side of the cylindrical coil with stopped glycerol flow, and then HRP was adsorbed from this solution onto a mica substrate. This incubation has been found to markedly increase the content of aggregated enzyme on mica-as compared with the control enzyme sample. We explain the phenomenon observed by the influence of triboelectrically induced electromagnetic fields of non-trivial topology. The results reported should be further considered in the development of flow-based heat exchangers of biosensors and bioreactors intended for operation with enzymes.

Sialic acids in infection and their potential use in detection and protection against pathogens.

In structural terms, the sialic acids are a large family of nine carbon sugars based around an alpha-keto acid core. They are widely spread in nature, where they are often found to be involved in molecular recognition processes, including in development, immunology, health and disease. The prominence of sialic acids in infection is a result of their exposure at the non-reducing terminus of glycans in diverse glycolipids and glycoproteins. Herein, we survey representative aspects of sialic acid structure, recognition and exploitation in relation to infectious diseases, their diagnosis and prevention or treatment. Examples covered span influenza virus and Covid-19, Leishmania and Trypanosoma, algal viruses, Campylobacter, Streptococci and Helicobacter, and commensal Ruminococci.

Selection of Aptamers for Use as Molecular Probes in AFM Detection of Proteins.

Currently, there is great interest in the development of highly sensitive bioanalytical systems for diagnosing diseases at an early stage, when pathological biomarkers are present in biological fluids at low concentrations and there are no clinical manifestations. A promising direction is the use of molecular detectors-highly sensitive devices that detect signals from single biomacromolecules. A typical detector in this class is the atomic force microscope (AFM). The high sensitivity of an AFM-based bioanalysis system is determined by the size of the sensing element of an atomic force microscope-the cantilever-the radius of the curvature of which is comparable to that of a biomolecule. Biospecific molecular probe-target interactions are used to ensure detection system specificity. Antibodies, aptamers, synthetic antibodies, and peptides can be used as molecular probes. This study has demonstrated the possibility of using aptamers as molecular probes for AFM-based detection of the ovarian cancer biomarker CA125. Antigen detection in a nanomolar solution was carried out using AFM chips with immobilized aptamers, commercially available or synthesized based on sequences from open sources. Both aptamer types can be used for antigen detection, but the availability of sequence information enables additional modeling of the aptamer structure with allowance for modifications necessary for immobilization of the aptamer on an AFM chip surface. Information on the structure and oligomeric composition of aptamers in the solution was acquired by combining small-angle X-ray scattering and molecular modeling. Modeling enabled pre-selection, before the experimental stage, of aptamers for use as surface-immobilized molecular probes.

Registration of Functioning of a Single Horseradish Peroxidase Macromolecule with a Solid-State Nanopore.

Currently, nanopore-based technology for the determination of the functional activity of single enzyme molecules continues its development. The use of natural nanopores for studying single enzyme molecules is known. At that, the approach utilizing artificial solid-state nanopores is also promising but still understudied. Herein, we demonstrate the use of a nanotechnology-based approach for the investigation of the enzymatic activity of a single molecule of horseradish peroxidase with a solid-state nanopore. The artificial 5 nm solid-state nanopore has been formed in a 40 nm thick silicon nitride structure. A single molecule of HRP has been entrapped into the nanopore. The activity of the horseradish peroxidase (HRP) enzyme molecule inserted in the nanopore has been monitored by recording the time dependence of the ion current through the nanopore in the course of the reaction of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) oxidation reaction. We have found that in the process of ABTS oxidation in the presence of 2.5 mM hydrogen peroxide, individual HRP enzyme molecules are able to retain activity for approximately 700 s before a decrease in the ion current through the nanopore, which can be explained by structural changes of the enzyme.

Nanoform of Phospholipid Composition: Investigation of the Morphological Features by Atomic Force Microscopy.

Morphological features of the nanoform of a phospholipid composition (NFPh), which can be used as an individual pharmaceutic agent or as a platform for designing drug delivery systems, have been studied using atomic force microscopy (AFM). NFPh has been developed, and its characteristics have been investigated using conventional drug analysis methods, including the determination of the mean diameter of nanosized vesicles in the emulsion via dynamic light scattering (DLS). Using DLS, the mean diameter of the vesicles was found to be ~20 nm. AFM imaging of the surface has revealed four types of objects related to NFPh: (1) compact objects; (2) layer fragments; (3) lamellar structures; and (4) combined objects containing the compact and extended parts. For type (4) objects, it has been found that the geometric ratio of the volume of the convex part to the total area of the entire object is constant. It has been proposed that these objects formed owing to fusion of vesicles of the same size (with the same surface-to-volume ratio). It has been shown that this is possible for vesicles with diameters of 20 nm. This diameter is in good coincidence with the value obtained using DLS.

[Progeroid syndromes : Aging, skin aging, and mechanisms of progeroid syndromes]. / Progeroide Syndrome : Alterung, Hautalterung und Mechanismen progeroider Erkrankungen.

Progeroid syndromes (PSs) are characterized by the premature onset of age-related pathologies. PSs display a wide range of heterogeneous pathological symptoms that also manifest during natural aging, including vision and hearing loss, atrophy, hair loss, progressive neurodegeneration, and cardiovascular defects. Recent advances in molecular pathology have led to a better understanding of the underlying mechanisms of these diseases. The genetic mutations underlying PSs are functionally linked to genome maintenance and repair, supporting the causative role of DNA damage accumulation in aging. While some of those genes encode proteins with a direct involvement in a DNA repair machinery, such as nucleotide excision repair (NER), others destabilize the genome by compromising the stability of the nuclear envelope, when lamin A is dysfunctional in Hutchinson-Gilford progeria syndrome (HGPS) or regulate the DNA damage response (DDR) such as the ataxia telangiectasia-mutated (ATM) gene. Understanding the molecular pathology of progeroid diseases is crucial in developing potential treatments to manage and prevent the onset of symptoms. This knowledge provides insight into the underlying mechanisms of premature aging and could lead to improved quality of life for individuals affected by progeroid diseases.

Stopped Flow of Glycerol Induces the Enhancement of Adsorption and Aggregation of HRP on Mica.

Glycerol is a usable component of heat-transfer fluids, and is thus suitable for the use in microchannel-based heat exchangers in biosensors and microelectronic devices. The flow of a fluid can lead to the generation of electromagnetic fields, which can affect enzymes. Herein, by means of atomic force microscopy (AFM) and spectrophotometry, a long-term effect of stopped flow of glycerol through a coiled heat exchanger on horseradish peroxidase (HRP) has been revealed. Samples of buffered HRP solution were incubated near either the inlet or the outlet sections of the heat exchanger after stopping the flow. It has been found that both the enzyme aggregation state and the number of mica-adsorbed HRP particles increase after such an incubation for 40 min. Moreover, the enzymatic activity of the enzyme incubated near the inlet section has been found to increase in comparison with that of the control sample, while the activity of the enzyme incubated near the outlet section remained unaffected. Our results can find application in the development of biosensors and bioreactors, in which flow-based heat exchangers are employed.

Calcium-dependent modulation of BKCa channel activity induced by plasmonic gold nanoparticles in pulmonary artery smooth muscle cells and hippocampal neurons.

AIM: Gold nanoparticles are widely used for biomedical applications, but the precise molecular mechanism of their interaction with cellular structures is still unclear. Assuming that intracellular calcium fluctuations associated with surface plasmon-induced calcium entry could modulate the activity of potassium channels, we studied the effect of 5 nm gold nanoparticles on calcium-dependent potassium channels and associated calcium signaling in freshly isolated rat pulmonary artery smooth muscle cells and cultured hippocampal neurons. METHODS: Outward potassium currents were recorded using patch-clamp techniques. Changes in intracellular calcium concentration were measured using the high affinity Ca2+ fluorescent indicator fluo-3 and laser confocal microscope. RESULTS: In pulmonary artery smooth muscle cells, plasmonic gold nanoparticles increased the amplitude of currents via large-conductance Ca2+ -activated potassium channels, which was potentiated by green laser irradiation near plasmon resonance wavelength (532 nm). Buffering of intracellular free calcium with ethylene glycol-bis-N,N,N',N'-tetraacetic acid (EGTA) abolished these effects. Furthermore, using confocal laser microscopy it was found that application of gold nanoparticles caused oscillations of intracellular calcium concentration that were decreasing in amplitude with time. In cultured hippocampal neurons gold nanoparticles inhibited the effect of EGTA slowing down the decline of the BKCa current while partially restoring the amplitude of the slow after hyperpolarizing currents. CONCLUSION: We conclude that fluctuations in intracellular calcium can modulate plasmonic gold nanoparticles-induced gating of BKCa channels in smooth muscle cells and neurons through an indirect mechanism, probably involving the interaction of plasmon resonance with calcium-permeable ion channels, which leads to a change in intracellular calcium level.

AFM Investigation of the Influence of Steam Flow through a Conical Coil Heat Exchanger on Enzyme Properties.

The present study is aimed at the revelation of subtle effects of steam flow through a conical coil heat exchanger on an enzyme, incubated near the heat exchanger, at the nanoscale. For this purpose, atomic force microscopy (AFM) has been employed. In our experiments, horseradish peroxidase (HRP) was used as a model enzyme. HRP is extensively employed as a model in food science in order to determine the influence of electromagnetic fields on enzymes. Adsorption properties of HRP on mica have been studied by AFM at the level of individual enzyme macromolecules, while the enzymatic activity of HRP has been studied by spectrophotometry. The solution of HRP was incubated either near the top or at the side of the conically wound aluminium pipe, through which steam flow passed. Our AFM data indicated an increase in the enzyme aggregation on mica after its incubation at either of the two points near the heat exchanger. At the same time, in the spectrophotometry experiments, a slight change in the shape of the curves, reflecting the HRP-catalyzed kinetics of ABTS oxidation by hydrogen peroxide, has also been observed after the incubation of the enzyme solution near the heat exchanger. These effects on the enzyme adsorption and kinetics can be explained by alterations in the enzyme hydration caused by the influence of the electromagnetic field, induced triboelectrically by the flow of steam through the heat exchanger. Our findings should thus be considered in the development of equipment involving conical heat exchangers, intended for either research or industrial use (including miniaturized bioreactors and biosensors). The increased aggregation of the HRP enzyme, observed after its incubation near the heat exchanger, should also be taken into account in analysis of possible adverse effects from steam-heated industrial equipment on the human body.

Direct Identification of Zeolite Beta Polymorphs by Solid-State 29Si Nuclear Magnetic Resonance Spectroscopy.

Stacking disorder and polymorphism in zeolite and zeolite-like materials hinder their structural characterization. In this work, we propose an advanced approach based on applying "pure shift" solid-state 29Si nuclear magnetic resonance (NMR) spectroscopy for the structural investigation of zeolitic materials containing intergrown polymorphs. The approach developed in the case study of zeolite beta allows for the resolution of 21 29Si signals, attributing them to non-equivalent T sites in polymorphs A, B, and C, reconstruction of individual 29Si magic angle spinning NMR spectra for each polymorph, and determination of the polymorph composition with higher accuracy than X-ray diffraction. The results reveal that two widely used synthetic routes for zeolite beta, alkaline and fluoride synthesis, lead to different polymorph compositions. These findings indicate that "pure shift" solid-state 29Si NMR can serve as a superior tool for the elucidation of polymorphism in zeolites.

Atomic Force Microscopy Study of the Effect of an Electric Field, Applied to a Pyramidal Structure, on Enzyme Biomolecules.

The influence of an external constant strong electric field, formed using a pyramidal structure under a high electric potential, on an enzyme located near its apex, is studied. Horseradish peroxidase (HRP) is used as a model. In our experiments, a 27 kV direct current (DC) voltage was applied to two electrodes with a conducting pyramidal structure attached to one of them. The enzyme particles were visualized by atomic force microscopy (AFM) after the adsorption of the enzyme from its 0.1 µM solution onto mica AFM substrates. It is demonstrated that after the 40 min exposure to the electric field, the enzyme forms extended structures on mica, while in control experiments compact HRP particles are observed. After the exposure to the electric field, the majority of mica-adsorbed HRP particles had a height of 1.2 nm (as opposed to 1.0 nm in the case of control experiments), and the contribution of higher (>2.0 nm) particles was also considerable. This indicates the formation of high-order HRP aggregates under the influence of an applied electric field. At that, the enzymatic activity of HRP against its substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) remains unaffected. These results are important for studying macroscopic effects of strong electromagnetic fields on enzymes, as well as for the development of cellular structure models.

The Effect of a Rotating Cone on Horseradish Peroxidase Aggregation on Mica Revealed by Atomic Force Microscopy.

Our study reported herein aims to determine whether an electromagnetic field, induced triboelectrically by a metallic cone, rotating at a frequency of 167 Hz, has an effect on the properties of the horseradish peroxidase (HRP) enzyme. Atomic force microscopy (AFM) was employed to detect even the most subtle effects on single enzyme molecules. In parallel, a macroscopic method (spectrophotometry) was used to reveal whether the enzymatic activity of HRP in solution was affected. An aqueous solution of the enzyme was incubated at a distance of 2 cm from the rotating cone. The experiments were performed at various incubation times. The control experiments were performed with a non-rotating cone. The incubation of the HRP solution was found to cause the disaggregation of the enzyme. At longer incubation times, this disaggregation was found to be accompanied by the formation of higher-order aggregates; however, no change in the HRP enzymatic activity was observed. The results of our experiments could be of interest in the development of enzyme-based biosensors with rotating elements such as stirrers. Additionally, the results obtained herein are important for the correct interpretation of data obtained with such biosensors.

The Effect of a Dodecahedron-Shaped Structure on the Properties of an Enzyme.

In this research, the influence of a dodecahedron-shaped structure on the adsorption behavior of a horseradish peroxidase (HRP) enzyme glycoprotein onto mica substrates was studied. In the experiments, samples of an aqueous HRP solution were incubated at various distances (0.03 m, 2 m, 5 m, and control at 20 m) from the dodecahedron surface. After the incubation, the direct adsorption of HRP onto mica substrates immersed in the solutions was performed, and the mica-adsorbed HRP particles were visualized via atomic force microscopy (AFM). The effect of the increased HRP aggregation was only observed after the incubation of the enzyme solution at the 2 m distance from the dodecahedron. In addition, with respect to the control sample, spectrophotometric measurements revealed no change in the HRP enzymatic activity after the incubation at any of the distances studied. The results reported herein can be of use in the modeling of the possible influences of various spatial structures on biological objects in the development of biosensors and other electronic equipment.

Atomic Force Microscopy Study of the Temperature and Storage Duration Dependencies of Horseradish Peroxidase Oligomeric State.

This paper presents an investigation of the temperature dependence of the oligomeric state of the horseradish peroxidase (HRP) enzyme on the temperature of its solution, and on the solution storage time, at the single-molecule level. Atomic force microscopy has been employed to determine how the temperature and the storage time of the HRP solution influence its aggregation upon direct adsorption of the enzyme from the solution onto bare mica substrates. In parallel, spectrophotometric measurements have been performed in order to estimate whether the HRP enzymatic activity changes over time upon the storage of the enzyme solution. The temperature dependence of the HRP oligomeric state has been studied within a broad (15-40 °C) temperature range. It has been demonstrated that the storage of the HRP solution for 14 days does not have any considerable effect on the oligomeric state of the enzyme, neither does it affect its activity. At longer storage times, AFM has allowed us to reveal a tendency of HRP to oligomerization during the storage of its buffered solution, while the enzymatic activity remains virtually unchanged even after a 1-month-long storage. By AFM, it has been revealed that after the incubation of a mica substrate in the HRP solution at various temperatures, the content of the mica-adsorbed oligomers increases insignificantly owing to a high-temperature stability of the enzyme.

Evaluation of Zeolite Acidity by 31P MAS NMR Spectroscopy of Adsorbed Phosphine Oxides: Quantitative or Not?

31P magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy of adsorbed alkyl-substituted phosphine oxides has witnessed tremendous progress during the last years and has become one of the most informative and sensitive methods of zeolite acidity investigation. However, quantitative evaluation of the number of sites is still a challenge. This study clarifies the main origin of errors occurring during NMR experiments, introduces the appropriate standards (both internal and external), and determines the relaxation parameters and the conditions for the acquisition and integration of spectra. As a result, a methodology for the quantitative measurement of the content of Brønsted and Lewis sites and the amount of internal and external silanol groups is established. The application of probe molecules of different sizes (namely, trimethylphosphine oxide (TMPO), tri-n-butylphosphine oxide (TBPO), and tri-n-octylphosphine oxide (TOPO)) is shown to be a good tool for distinguishing between the active sites inside the zeolite pores, mesopores, and on the outer crystal surface. The methodology proposed is verified on BEA zeolites different in composition, texture, and morphology.

Application-Driven Material Design of Printable Strain Hardening Cementitious Composites (SHCC).

The creation of concrete shells from customized prefabricated modules is a novel approach that facilitates the construction of free-form surfaces considerably. In the framework of the Adaptive Concrete Diamond Construction (ACDC) project at TU Dresden, a material for 3D printing of the outer contours of such modules has been developed based on the principles of Strain Hardening Cementitious Composite (SHCC). In addition to its high ductility, the required material must also be suitable for 3D printing while enabling the achievement of high geometric accuracy in the manufacture of the modules. To gain the required performance, cellulose ether and starch ether were used specifically to extend the open time, for a longer period of maintaining initial workability, as well as for enhancing shape stability and surface quality. An extensive experimental program was carried out to evaluate the outcomes of the material modifications, including flow table tests, water retention tests, and several specific tests to determine the adhesiveness of the fresh SHCC. For hardened SHCC, surface roughness was assessed using a laser 3D scanner in addition to testing its mechanical properties.

Improved Spectral Purity of 222-nm Irradiation Eliminates Detectable Cyclobutylpyrimidine Dimers Formation in Skin Reconstructs even at High and Repetitive Disinfecting Doses.

UVC222 nm has germicidal effects with potential clinical applications. However, UVC irradiation is capable of inducing DNA damage like cyclobutylpyrimidine dimers (CPD). Although new devices have emission peaks in the short-wavelength region of UVC (~222 nm), the remaining "collateral" radiation at longer wavelengths could be harmful to human health. We investigated the DNA damage caused by far-UVC 222 nm KrCl exciplex radiation on human skin reconstructs after additional filtering using silica filters. The skin reconstructs were irradiated with 100 mJ cm-2 , 500 mJ cm-2 , and 3 × 500 mJ cm-2 unfiltered and filtered (230-270 nm suppressed) far-UVC or UVB (308 nm) radiation. UVB and non-filtered UVC irradiation induced a significant amount of CPDs, compared with the background. Filtered far-UVC lowered the CPD amount compared with unfiltered UVC and UVB treatments. Repetitive UVC irradiation did not result in the accumulation of CPDs compared with UVB treatment. Reduction in excess of 99.9% of E. coli, S. aureus and C. albicans was detected after applying far-UVC radiation. This identifies a therapeutic window in which microorganisms are killed but tissue is still alive and not damaged, which could give rise to new clinical applications.

COVID-19 Vaccinations: Summary Guidance for Cancer Patients in 28 Languages: Breaking Barriers to Cancer Patient Information.

BACKGROUND: Covid-19 vaccination has started in the majority of the countries at the global level. Cancer patients are at high risk for infection, serious illness, and death from COVID-19 and need vaccination guidance and support. Guidance availability in the English language only is a major limit for recommendations' delivery and their application in the world's population and generates information inequalities across the different populations. METHODS: Most of the available COVID-19 vaccination guidance for cancer patients was screened and scrutinized by the European Cancer Patients Coalition (ECPC) and an international oncology panel of 52 physicians from 33 countries. RESULTS: A summary guidance was developed and provided in 28 languages in order to reach more than 70 percent of the global population. CONCLUSION: Language barrier and e-guidance availability in the native language are the most important barriers when communicating with patients. E-guidance availability in various native languages should be considered a major priority by international medical and health organizations that are communicating with patients at the global level.