Theory for nonlinear dynamic force spectroscopy.

Dynamic force spectroscopy (DFS) is an experimental technique that is commonly used to assess information on the strength, energy landscape, and lifetime of noncovalent bio-molecular interactions. DFS traditionally requires an applied force that increases linearly with time so that the bio-complex under investigation is exposed to a constant loading rate. However, tethers or polymers can modulate the applied force in a nonlinear manner. For example, bacterial adhesion pili and polymers with worm-like chain properties are structures that show nonlinear force responses. In these situations, the theory for traditional DFS cannot be readily applied. In this work, we expand the theory for DFS to also include nonlinear external forces while still maintaining compatibility with the linear DFS theory. To validate the theory, we modeled a bio-complex expressed on a stiff, an elastic, and a worm-like chain polymer, using Monte Carlo methods, and assessed the corresponding rupture force spectra. It was found that the nonlinear DFS (NLDFS) theory correctly predicted the numerical results. We also present a protocol suggesting an experimental approach and analysis method of the data to estimate the bond length and the thermal off-rate.

Assessing bacterial adhesion on an individual adhesin and single pili level using optical tweezers.

Optical tweezers (OT) are a technique that, by focused laser light, can both manipulate micrometer sized objects and measure minute forces (in the pN range) in biological systems. The technique is therefore suitable for assessment of bacterial adhesion on an individual adhesin-receptor and single attachment organelle (pili) level. This chapter summarizes the use of OT for assessment of adhesion mechanisms of both non-piliated and piliated bacteria. The latter include the important helix-like pili expressed by uropathogenic Escherichia coli (UPEC), which have shown to have unique and intricate biomechanical properties. It is conjectured that the large flexibility of this type of pili allows for a redistribution of an external shear force among several pili, thereby extending the adhesion lifetime of bacteria. Systems with helix-like adhesion organelles may therefore act as dynamic biomechanical machineries, enhancing the ability of bacteria to withstand high shear forces originating from rinsing flows such as in the urinary tract. This implies that pili constitute an important virulence factor and a possible target for future anti-microbial drugs.

Quantitative Evaluation of Single-Use Particle Filtering Half Masks for SARS-CoV-2 Protection.

Background: The SARS-CoV-2 pandemic put the entire healthcare sector under severe strain due to shortages of personal protection equipment. A large number of new filtering mask models were introduced on the market, claiming effectiveness that had undergone little or no objective and reliable verifications. Methods and Materials: Filter materials were tested against sodium chloride particles according to the EN149 §7.9.2 standard for particle penetration. Particle counters were used to measure the particle penetration of the filtering mask models, resolved over sizes in the range of 27-1000 nm. Results: We report on the results for 86 different filtering mask models. The majority of the tested models showed <3% penetration, whereas almost one third (i.e., 27 of 86) of the models performed poorly. Discussion: Interestingly, the poorest performing masks showed a tendency to have worse filtering effectiveness for larger particles than for smaller sized particles, following the opposite tendency of the best filtering masks. Conclusion: Almost one third of the filtering mask models tested failed the specified pass criteria as specified in the temporary EU COVID-19 standard. This fact, and the high health risks of COVID-19, highlights the need for independent testing.

Catch-bond behavior of bacteria binding by slip bonds.

It is shown that multipili-adhering bacteria expressing helix-like pili binding by slip bonds can show catch-bond behavior. When exposed to an external force, such bacteria can mediate adhesion to their hosts by either of two limiting means: sequential or simultaneous pili force exposure (referring to when the pili mediate force in a sequential or simultaneous manner, respectively). As the force is increased, the pili can transition from sequential to simultaneous pili force exposure. Since the latter mode of adhesion gives rise to a significantly longer bacterial adhesion lifetime than the former, this results in a prolongation of the lifetime, which shows up as a catch-bond behavior. The properties and conditions of this effect were theoretically investigated and assessed in some detail for dual-pili-adhering bacteria, by both analytical means and simulations. The results indicate that the adhesion lifetime of such bacteria can be prolonged by more than an order of magnitude. This implies that the adhesion properties of multibinding systems cannot be directly conveyed to the individual adhesion-receptor bonds.

Correction: Multilevel model for airborne transmission of foot-and-mouth disease applied to Swedish livestock.

[This corrects the article DOI: 10.1371/journal.pone.0232489.].

Multilevel model for airborne transmission of foot-and-mouth disease applied to Swedish livestock.

The foot-and-mouth disease is an ever-present hazard to the livestock industry due to the huge economic consequences following an outbreak that necessitates culling of possibly infected animals in vast numbers. The disease is highly contagious and previous epizootics have shown that it spreads by many routes. One such route is airborne transmission, which has been investigated in this study by means of a detailed multilevel model that includes all scales of an outbreak. Local spread within an infected farm is described by a stochastic compartment model while the spread between farms is quantified by atmospheric dispersion simulations using a network representation of the set of farms. The model was applied to the Swedish livestock industry and the risk for an epizootic outbreak in Sweden was estimated using the basic reproduction number of each individual livestock-holding farm as the endpoint metric. The study was based on comprehensive official data sets for both the current livestock holdings and regional meteorological conditions. Three species of farm animals are susceptible to the disease and are present in large numbers: cattle, pigs and sheep. These species are all included in this study using their individual responses and consequences to the disease. It was concluded that some parts of southern Sweden are indeed preconditioned to harbor an airborne epizootic, while the sparse farm population of the north renders such events unlikely to occur there. The distribution of the basic reproduction number spans over several orders of magnitudes with low risk of disease spread from the majority of the farms while some farms may act as very strong disease transmitters. The results may serve as basic data in the planning of the national preparedness for this type of events.

Characterization of the biomechanical properties of T4 pili expressed by Streptococcus pneumoniae--a comparison between helix-like and open coil-like pili.

Bacterial adhesion organelles, known as fimbria or pili, are expressed by gram-positive as well as gram-negative bacteria families. These appendages play a key role in the first steps of the invasion and infection processes, and they therefore provide bacteria with pathogenic abilities. To improve the knowledge of pili-mediated bacterial adhesion to host cells and how these pili behave under the presence of an external force, we first characterize, using force measuring optical tweezers, open coil-like T4 pili expressed by gram-positive Streptococcus pneumoniae with respect to their biomechanical properties. It is shown that their elongation behavior can be well described by the worm-like chain model and that they possess a large degree of flexibility. Their properties are then compared with those of helix-like pili expressed by gram-negative uropathogenic Escherichia coli (UPEC), which have different pili architecture. The differences suggest that these two types of pili have distinctly dissimilar mechanisms to adhere and sustain external forces. Helix-like pili expressed by UPEC bacteria adhere to host cells by single adhesins located at the distal end of the pili while their helix-like structures act as shock absorbers to dampen the irregularly shear forces induced by urine flow and to increase the cooperativity of the pili ensemble, whereas open coil-like pili expressed by S. pneumoniae adhere to cells by a multitude of adhesins distributed along the pili. It is hypothesized that these two types of pili represent different strategies of adhering to host cells in the presence of external forces. When exposed to significant forces, bacteria expressing helix-like pili remain attached by distributing the external force among a multitude of pili, whereas bacteria expressing open coil-like pili sustain large forces primarily by their multitude of binding adhesins which presumably detach sequentially.

Methods and estimations of uncertainties in single-molecule dynamic force spectroscopy.

In dynamic force spectroscopy, access to the characteristic parameters of single molecular bonds requires nontrivial measurements and data processing as the rupture forces are found not only to be distributed over a wide range, but are also dependent on the loading rate. The choice of measurement procedure and data processing methods has a considerable impact on the accuracy and precision of the final results. We analyze, by means of numerical simulations, methods to minimize and assess the magnitude of the expected errors for different combinations of experimental and evaluation methods. It was found that the choice of fitting function is crucial to extract correct parameter values. Applying a Gaussian function, which is a common practice, is equivalent to introducing a systematic error, and leads to a consequent overestimation of the thermal off-rate by more than 30%. We found that the precision of the bond length and the thermal off-rate, in presence of unbiased noise, were improved by reducing the number of loading rates for a given number of measurements. Finally, the results suggest that the minimum number of measurements needed to obtain the bond strength, with acceptable precision, exceeds the common number of approximately 100 reported in literature.

Physical properties of the specific PapG-galabiose binding in E. coli P pili-mediated adhesion.

Detailed analyses of the mechanisms that mediate binding of the uropathogenic Escherichia coli to host cells are essential, as attachment is a prerequisite for the subsequent infection process. We explore, by means of force measuring optical tweezers, the interaction between the galabiose receptor and the adhesin PapG expressed by P pili on single bacterial cells. Two variants of dynamic force spectroscopy were applied based on constant and non-linear loading force. The specific PapG-galabiose binding showed typical slip-bond behaviour in the force interval (30-100 pN) set by the pilus intrinsic biomechanical properties. Moreover, it was found that the bond has a thermodynamic off-rate and a bond length of 2.6 x 10(-3) s(-1) and 5.0 A, respectively. Consequently, the PapG-galabiose complex is significantly stronger than the internal bonds in the P pilus structure that stabilizes the helical chain-like macromolecule. This finding suggests that the specific binding is strong enough to enable the P pili rod to unfold when subjected to strong shear forces in the urinary tract. The unfolding process of the P pili rod promotes the formation of strong multipili interaction, which is important for the bacterium to maintain attachment to the host cells.

Multipili attachment of bacteria with helixlike pili exposed to stress.

A number of biomechanical properties of various types of pili expressed by Escherichia coli, predominantly their force-versus-elongation behavior, have previously been assessed in detail on a single pilus level. In vivo, however, bacteria bind in general to host cells by a multitude of pili, which presumably provides them with adhesion properties that differs from those of single pili. Based upon the previously assessed biomechanical properties of individual pili, this work presents a theoretical analysis of the adhesion properties of multipili-attaching bacteria expressing helixlike pili exposed to an external force. Expressions for the adhesion lifetime of dual- and multipili-attaching bacteria are derived and their validity is verified by Monte Carlo simulations. It is demonstrated that the adhesion lifetime of a multipili-binding bacterium depends to a large degree on the cooperativity of the attaching pili, which, in turn, depends strongly on their internal biomechanical properties, in particular their helixlike structure and its ability to elongate, which, in turn, depends on the intrinsic properties of the bonds, e.g., their lengths and activation energies. It is shown, for example, that a decrease in the length of a layer-to-layer bond in the rod of P pili, expressed by E. coli, by 50% leads to a decrease in the adhesion lifetime of a bacterium attaching by ten pili and exposed to a force of 500 pN by three orders of magnitude. The results indicate moreover that the intrinsic properties of the rod for this particular type of pili are optimized for multipili attachment under a broad range of external forces and presumably also to its in vivo environment. For example, P pili seems to be optimized to withstand a force exposure during approximately 3 s, which correspond to the time it takes for a bolus to pass a bacterium attached to the ureteral wall. Even though the results presented in this work apply quantitatively to one type of pilus, they are assumed to apply qualitatively to all helixlike pili systems expressing slip bonds.

Time optimization of 90Sr determinations: sequential measurement of multiple samples during decay of 90Y.

This work presents an optimized method for the determination of multiple samples containing 90Sr when its daughter 90Y is measured after chemical separation and in sequence, i.e. during its decay. Consequently the measurement times will increase for each subsequent sample, since there has been a longer time for decay before measurement. Compared to a previously published approach, when 90Y is measured during its ingrowth, the gain in total analysis time (time for ingrowth+ summation of measurement times) is not that large, particularly not for low background instruments. However, results for a large part of the samples can be delivered earlier.

Helicobacter pylori Adapts to Chronic Infection and Gastric Disease via pH-Responsive BabA-Mediated Adherence.

The BabA adhesin mediates high-affinity binding of Helicobacter pylori to the ABO blood group antigen-glycosylated gastric mucosa. Here we show that BabA is acid responsive-binding is reduced at low pH and restored by acid neutralization. Acid responsiveness differs among strains; often correlates with different intragastric regions and evolves during chronic infection and disease progression; and depends on pH sensor sequences in BabA and on pH reversible formation of high-affinity binding BabA multimers. We propose that BabA's extraordinary reversible acid responsiveness enables tight mucosal bacterial adherence while also allowing an effective escape from epithelial cells and mucus that are shed into the acidic bactericidal lumen and that bio-selection and changes in BabA binding properties through mutation and recombination with babA-related genes are selected by differences among individuals and by changes in gastric acidity over time. These processes generate diverse H. pylori subpopulations, in which BabA's adaptive evolution contributes to H. pylori persistence and overt gastric disease.

Time optimization of (90)Sr measurements: Sequential measurement of multiple samples during ingrowth of (90)Y.

The aim of this paper is to contribute to a more rapid determination of a series of samples containing (90)Sr by making the Cherenkov measurement of the daughter nuclide (90)Y more time efficient. There are many instances when an optimization of the measurement method might be favorable, such as; situations requiring rapid results in order to make urgent decisions or, on the other hand, to maximize the throughput of samples in a limited available time span. In order to minimize the total analysis time, a mathematical model was developed which calculates the time of ingrowth as well as individual measurement times for n samples in a series. This work is focused on the measurement of (90)Y during ingrowth, after an initial chemical separation of strontium, in which it is assumed that no other radioactive strontium isotopes are present. By using a fixed minimum detectable activity (MDA) and iterating the measurement time for each consecutive sample the total analysis time will be less, compared to using the same measurement time for all samples. It was found that by optimization, the total analysis time for 10 samples can be decreased greatly, from 21h to 6.5h, when assuming a MDA of 1Bq/L and at a background count rate of approximately 0.8cpm.

Measurements of the binding force between the Helicobacter pylori adhesin BabA and the Lewis b blood group antigen using optical tweezers.

Helicobacter pylori is a world-wide spread bacterium that causes persistent infections and chronic inflammations that can develop into gastritis and peptic ulcer disease. It expresses several adhesin proteins on its surface that bind to specific receptors in the gastric epithelium. The most well-known adhesin is BabA, which has previously been shown to bind specifically to the fucosylated blood group antigen Lewis b (Leb). The adhesion forces between BabA and the Leb antigen are investigated in this work and assessed by means of optical tweezers. A model system for in situ measurements of the interaction forces between individual bacteria and beads coated with Leb is developed. It is found that the de-adhesion force in this model system, measured with a loading rate of approximately 100 pNs, ranges from 20 to 200 pN. The de-adhesion force appears predominantly as multiples of an elementary force, which is determined to 25+/-1.5 pN and identified as the unbinding force of an individual BabA-Leb binding. It is concluded that adhesion in general is mediated by a small number of bindings (most often 1 to 4) despite that the contact surface between the bacterium and the bead encompassed significantly more binding sites.

A structural basis for sustained bacterial adhesion: biomechanical properties of CFA/I pili.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease worldwide. Adhesion pili (or fimbriae), such as the CFA/I (colonization factor antigen I) organelles that enable ETEC to attach efficiently to the host intestinal tract epithelium, are critical virulence factors for initiation of infection. We characterized the intrinsic biomechanical properties and kinetics of individual CFA/I pili at the single-organelle level, demonstrating that weak external forces (7.5 pN) are sufficient to unwind the intact helical filament of this prototypical ETEC pilus and that it quickly regains its original structure when the force is removed. While the general relationship between exertion of force and an increase in the filament length for CFA/I pili associated with diarrheal disease is analogous to that of P pili and type 1 pili, associated with urinary tract and other infections, the biomechanical properties of these different pili differ in key quantitative details. Unique features of CFA/I pili, including the significantly lower force required for unwinding, the higher extension speed at which the pili enter a dynamic range of unwinding, and the appearance of sudden force drops during unwinding, can be attributed to morphological features of CFA/I pili including weak layer-to-layer interactions between subunits on adjacent turns of the helix and the approximately horizontal orientation of pilin subunits with respect to the filament axis. Our results indicate that ETEC CFA/I pili are flexible organelles optimized to withstand harsh motion without breaking, resulting in continued attachment to the intestinal epithelium by the pathogenic bacteria that express these pili.

Dynamic force spectroscopy of the Helicobacter pylori BabA-Lewis b binding.

The binding strength of the Helicobacter pylori adhesin-receptor complex BabA-ABO/Lewis b has been analyzed by means of dynamic force spectroscopy. High-resolution measurements of rupture forces were performed in situ on single bacterial cells, expressing the high-affinity binding BabA adhesin, by the use of force measuring optical tweezers. The resulting force spectra revealed the mechanical properties of a single BabA-Leb bond. It was found that the bond is dominated by one single energy barrier and that it is a slip-bond. The bond length and thermal off-rate were assessed to be 0.86+/-0.07 nm and 0.015+/-0.006 s(-1), respectively.

Modeling of the elongation and retraction of Escherichia coli P pili under strain by Monte Carlo simulations.

P pili are fimbrial adhesion organelles expressed by uropathogenic Escherichia coli in the upper urinary tract. They constitute a stiff helix-like polymer consisting of a number of subunits joined by head-to-tail bonds. The elongation and retraction properties of individual P pili exposed to strain have been modeled by Monte Carlo (MC) simulations. The simulation model is based upon a three-state energy landscape that deforms under an applied force. Bond opening and closure are modeled by Bells theory while the elongation of the linearized part of the pilus is described by a worm-like chain model. The simulations are compared with measurements made by force measuring optical tweezers. It was found that the simulations can reproduce pili elongation as well as retraction, under both equilibrium and dynamic conditions, including entropic effects. It is shown that the simulations allow for an assessment of various model parameters, e.g. the unfolding force, energy barrier heights, and various distances in the energy landscape, including their stochastic spread that analytical models are unable to do. The results demonstrate that MC simulations are useful to model elongation and retraction properties of P pili, and therefore presumably also other types of pili, exposed to strain and/or stress. MC simulations are particularly suited for description of helix-like pili since these have an intricate self-regulating mechanical elongation behavior that makes analytical descriptions non-trivial when dynamic processes are studied, or if additional interactions in the rod or the behavior of the adhesion tip needs to be modeled.