Biotinylation of reducing and non-reducing termini to create plug-and-play polysaccharides.

Single-molecule studies continue to grow in popularity. In cases where biopolymer samples of interest exhibit variations in fine-structure between individual chains such single-molecule studies uniquely offer the promise of revealing deep structure-function relationships. Polysaccharides are typically studied in bulk and, as such, their study could greatly benefit from the application of single-molecule techniques. However, while for example single-molecule optical tweezers (OT) studies have become commonplace for DNA, studies of polysaccharides have lagged behind somewhat, complicated by the difficulty of studying molecules that amongst other things have more complex end-group chemistry. Recently, divalent streptavidin linkers have been shown to be capable of concatenating two pieces of biotin-terminated DNA to produce robust composite strings that run intact through conventional gels, and can be used in single-molecule OT experiments (Mohandas, Kent, Raudsepp, Jameson, & Williams, 2022). By using two such streptavidin linkers, biotin-terminated polymers could be inserted between two sections of DNA in order to facilitate single-molecule experiments on biopolymers that are currently difficult to address by other means. Here, we describe a generic approach for placing the required biotin moieties at both ends of polysaccharide chains, producing plug-and-play polysaccharide inserts that can be incorporated into composite polymer strings using streptavidin linking hubs.

Allied health transdisciplinary models of care in hospital settings: A scoping review  .

Improving the productivity of the allied health workforce is a global priority in response to the increasing incidence of chronic disease, associated healthcare costs, and insufficient workforce volume. Team-based healthcare, specifically allied health transdisciplinary teams, might be a solution to improve the utilization of workforce while maintaining high-quality and value-based healthcare. Allied health transdisciplinary teams can be a valuable solution in settings where care is delivered by different allied health professionals. Transdisciplinary teams embrace overlapping skills and blur traditional professional boundaries, allowing one professional to deliver certain aspects of care without eroding the skills and knowledge that each profession offers. The objective of this scoping review is to systematically examine and map the characteristics, outcomes, facilitators, and barriers of contemporary allied health transdisciplinary models of care that have been implemented in hospital settings. The scoping review was guided by the Joanna Briggs Institute methodology and reported in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR). Three screening rounds were completed by two independent reviewers. Included sources were synthesized using descriptive and tabular analysis. Nine studies that evaluated hospital-based allied health transdisciplinary teams were included. One study was a randomized controlled trial, five were experimental quantitative studies, two utilized qualitative analyses, and one was a conference abstract. Most studies reported improvements in time-efficiency, quality of care, and positive stakeholder perceptions. One study reported labor and capital cost savings. Barriers and facilitators of transdisciplinary teams were categorized by the authors as person/interpersonal, workflow, organizational or implementation factors. This review presents some evidence that demonstrates the potential of hospital-based allied health transdisciplinary teams, however high-quality evidence is scarce. Further primary research should focus on stakeholder perceptions, and labor and capital cost outcomes.

Modeling multiple duplex DNA attachments in a force-extension experiment.

Optical tweezers-based DNA stretching often relies on tethering a single end-activated DNA molecule between optically manipulated end-binding beads. Measurement success can depend on DNA concentration. At lower DNA concentrations tethering is less common, and many trials may be required to observe a single-molecule stretch. At higher DNA concentrations tethering is more common; however, the resulting force-extensions observed are more complex and may vary from measurement to measurement. Typically these more complex results are attributed to the formation of multiple tethers between the beads; however, to date there does not appear to have been a critical examination of this hypothesis or the potential usefulness of such data. Here we examine stretches at a higher DNA concentration and use analysis and simulation to show how the more complex force-extensions observed can be understood in terms of multiple DNA attachments.

Depressive traits are associated with a reduced effect of choice on intentional binding.

A sense of agency (SoA) over wilful actions is thought to be dependent on the level of choice and the nature of the outcome. In a preregistered study, we manipulated choice and valence of outcome to assess the relationship between SoA across the depression and psychosis continuum. Participants (N = 151) completed a Libet Clock task, in which they had either a free or forced choice to press one of two buttons and received either a rewarding or punishing outcome. Participants also completed questionnaires on depressive and psychosis-like traits. Rewarding outcomes increased intentional binding. The evidence favoured no effect of choice on average, but this was influenced by inter-individual differences. Individuals reporting more depressive traits had less of a difference in intentional binding between free and forced choice conditions. We show that implicit SoA is sensitive to outcome valence and the effect of choice differs across the depression continuum.

Social Inference from Middle to Older Adulthood.

OBJECTIVE: Inferring the emotional state or the true meaning of another person's utterance is a key aspect of social cognition and critical for successful social interactions. In this study, we assess age-related differences in emotion recognition and sincere and sarcastic social exchanges in the context of cognitive and demographic factors. METHOD: One hundred and eighty-seven adults ranging from middle to older adulthood completed the Mini-SEA Emotion Recognition test and Part B of The Awareness of Social Inference Test - Short Form (TASIT-S). Fluid intelligence and executive abilities were also assessed. Sex differences and the relationship with education level were also investigated. Regression models were used to assess age-related differences controlling for baseline cognitive and demographic factors. RESULTS: Age was negatively associated with accuracy for inferring sincere social exchanges. No differences were identified for accuracy for inferring sarcastic exchanges. Likewise, no age differences were identified for emotion recognition (Mini-SEA). Fluid intelligence was associated with accuracy for inferring sincere exchanges, but this was independent of age-related effects. A female advantage was identified for emotion recognition. CONCLUSION: Age is associated with difficulty in inferring sincere exchanges, which is not explained by fluid intelligence, verbal abstract reasoning, or auditory verbal attention. A female advantage in emotion recognition is consistent with findings reported in younger adults. Both age and sex should be considered in clinical assessments using the Mini-SEA and the TASIT-S.

Interfacial colloidal assembly guided by optical tweezers and tuned via surface charge.

HYPOTHESIS: The size, shape and dynamics of assemblies of colloidal particles optically-trapped at an air-water interface can be tuned by controlling the optical potential, particle concentration, surface charge density and wettability of the particles and the surface tension of the solution. EXPERIMENTS: The assembly dynamics of different colloidal particle types (silica, polystyrene and carboxyl coated polystyrene particles) at an air-water interface in an optical potential were systematically explored allowing the effect of surface charge on assembly dynamics to be investigated. Additionally, the pH of the solutions were varied in order to modulate surface charge in a controllable fashion. The effect of surface tension on these assemblies was also explored by reducing the surface tension of the supporting solution by mixing ethanol with water. FINDINGS: Silica, polystyrene and carboxyl coated polystyrene particles showed distinct assembly behaviours at the air-water interface that could be rationalised taking into account changes in surface charge (which in addition to being different between the particles could be modified systematically by changing the solution pH). Additionally, this is the first report showing that wettability of the colloidal particles and the surface tension of the solution are critical in determining the resulting assembly at the solution surface.

Progress toward Plug-and-Play Polymer Strings for Optical Tweezers Experiments: Concatenation of DNA Using Streptavidin Linkers.

Streptavidin is a tetrameric protein that is renowned for its strong binding to biotin. The robustness and strength of this noncovalent coupling has led to multitudinous applications of the pairing. Within the streptavidin tetramer, each protein monomer has the potential to specifically bind one biotin-bearing moiety. Herein, by separating various streptavidin species that have had differing numbers of their four potential binding sites blocked, several different types of "linking hub" were obtained, each with a different valency. The identification of these species and the study of the plugging process used to block sites during their preparation were carried out using capillary electrophoresis. Subsequently, a specific species, namely, a trans-divalent linker, in which the two open biotin-binding pockets are approximately opposite one another, was used to concatenate two ∼5 kb pieces of biotin-terminated double-stranded DNA. Following the incubation of this DNA with the prepared linker, a fraction of ∼10 kb strings was identified using gel electrophoresis. Finally, these concatenated DNA strings were stretched in an optical tweezer experiment, demonstrating the potential of the methodology for coupling and extending molecules for use in single-molecule biophysical experiments.

Estimating orientation of optically trapped, near vertical, microsphere dimers using central moments and off-focus imaging.

Near vertical optically trapped dimers, composed of pairs of microspheres, and constructed in situ, were imaged in bright-field in flow and at rest, and with displacement Δz from the transverse xy imaging plane of an inverted microscope. Image first central moments µ01 were measured, and their dependence on the imposed flow velocity of the surrounding fluid was calculated. This dependence was related to the at-rest restricted diffusion statistics. It was assumed that, for small perturbations, the torque T on the dimer was proportional to the velocity of flow v and resulting angular deflection Δθ so that T∝v∝Δθ. Displacements Δz at which vâˆΔµ01∝Δθ, which are typically off focus, were examined in more detail; in this range, Δθ=hΔµ01. The hydrodynamics of the dimer were modeled as that of a prolate ellipsoid, and the constant of proportionality h was determined by comparing the short-time mean-squared variation measured during diffusion to that predicted by the model calculation: h2⟨Δµ012(t)⟩=⟨Δθ2(t)⟩. With h determined, the optical trap stiffness kθ was determined from the long-time restricted diffusion of the dimer. The measured kθ and Δθ can then be used compute torque: T=kθΔθ, potentially enabling the near vertical optically trapped dimer to be used as a torque probe.

Changes in the properties of membrane tethers in response to HP1α depletion in MCF7 cells.

Plasma membrane tension is known to regulate many cell functions, such as motility and membrane trafficking. Membrane tether pulling is an effective method for measuring the apparent membrane tension of cells and exploring membrane-cytoskeleton interactions. In this article, the mechanical properties of HP1α-depleted MCF7 breast cancer cells are explored in comparison to controls, by pulling membrane tethers using optical tweezers. These studies were inspired by previous findings that a loss of HP1α correlates with an increase in the invasive potential of malignant cancer cells. Specifically, the membrane tension and force relaxation curves for tethers pulled from MCF7 breast cancer cells with HP1α knockdown and their matched controls were measured, and shown to be significantly different.

Inside the ensemble: unlocking the potential of one-at-a-time experiments with lab-on-a-chip automation.

The advent of technologies that allow the interactions of individual microscopic particles to be probed "one-at-a-time" has paved the way for new experimental avenues of enquiry in colloidal systems. For example, investigating whether a particular pair of colloidal particles isolated from a macroscopic sample might adhere to each other when brought into close proximity is certainly possible. However, given the probabilistic nature of the process (different particles within the ensemble may have slightly different surface charge distributions and asperities, and interaction energies involved can be close to thermal values), it is important that many hundreds or thousands of pairs of particles are tested under each set of experimental conditions of interest. Currently it is still an arduous task to perform such an experiment a sufficient number of times in order to acquire a data-set that truly represents the ensemble. Herein an automated particle collider for measuring particle-particle interactions has been realized by combining elements of microfluidics, holographic optical tweezers and image processing. Each individual measurement consists of confining two particles within a predetermined chemical micro-environment, and observing whether their interactions lead to aggregation. To automate the measurements, computer software consisting of LabVIEW and Red Tweezers with a custom plugin was used. Preliminary experiments carried out using 1 µm diameter polystyrene particles demonstrated that many hundreds of pairwise-interaction measurements could be carried out autonomously within a matter of hours. Further exemplar real-world experiments, designed to examine the stickiness of emulsion drops as a function of bulk measurements of the ζ-potential (zeta potential) of the sample, were then performed. It is envisaged that such robust approaches to the automation of "one-at-a-time" experiments will find applications in a large number of areas, and enable previously unthinkable experiments to be carried out in a timely fashion, thus allowing the focus to shift away from tedious experimental frustrations to more profound scientific questions.

Optical microlever assisted DNA stretching.

Optical microrobotics is an emerging field that has the potential to improve upon current optical tweezer studies through avenues such as limiting the exposure of biological molecules of interest to laser radiation and overcoming the current limitations of low forces and unwanted interactions between nearby optical traps. However, optical microrobotics has been historically limited to rigid, single-body end-effectors rather than even simple machines, limiting the tasks that can be performed. Additionally, while multi-body machines such as microlevers exist in the literature, they have not yet been successfully demonstrated as tools for biological studies, such as molecule stretching. In this work we have taken a step towards moving the field forward by developing two types of microlever, produced using two-photon absorption polymerisation, to perform the first lever-assisted stretches of double-stranded DNA. The aim of the work is to provide a proof of concept for using optical micromachines for single molecule studies. Both styles of microlevers were successfully used to stretch single duplexes of DNA, and the results were analysed with the worm-like chain model to show that they were in good agreement.

Complexes of ß-lactoglobulin and high methyl-esterified pectin as a one-shot delivery system for reinforcing oil/water interfaces.

Electrostatic complexation of negatively charged polysaccharides with ß-lactoglobulin (ß-lg) has been shown to bolster the protein films at oil/water interfaces thereby improving emulsion stability. However, recent sub-phase exchange experiments demonstrated that highly charged polysaccharides such as low methyl-esterified pectin are complementary only if sequentially introduced to a pre-formed interfacial ß-lg film. In this study, results of transient interfacial shear rheology show that, by using high-methylesterified pectins instead, complexes can be formed in pre-mixed solutions with ß-lg at pH 4 that can lead to reinforced protein films at dodecane/water interfaces. Using this one-shot adsorption of such complexes, pectins as well as short chain polysaccharides like homogalacturonan nearly doubled the steady state shear elastic moduli as compared to that of a pure ß-lg film. The lag times of film formation were established to be primarily decided by the charge density and pattern on the polysaccharide. Based on the results from mixed solutions of ß-lg monomers, it is proposed that the polysaccharide at pH 4 strengthens the resulting interfacial layer by concatenating adsorbed ß-lg molecules thereby establishing cross-links in the aqueous phase.

Stimulation of the Social Brain Improves Perspective Selection in Older Adults: A HD-tDCS Study.

There is evidence for dissociable, causal roles for two key social brain regions in young adults. Specifically, the right temporoparietal junction (rTPJ) is associated with embodied perspective taking, whereas the dorsomedial prefrontal cortex (dmPFC) is associated with the integration of social information. However, it is unknown whether these causal brain-behaviour associations are evident in older adults. Fifty-two healthy older adults were stratified to receive either rTPJ or dmPFC anodal high-definition transcranial direct current stimulation in a sham-controlled, double-blinded, repeated-measures design. Self-other processing was assessed across implicit and explicit level one (line-of-sight) and level two (embodied rotation) visual perspective taking (VPT) tasks, and self-other encoding effects on episodic memory. Both rTPJ and dmPFC stimulation reduced the influence of the alternate perspective during level one VPT, indexed by a reduced congruency effect (difference between congruent and incongruent perspectives). There were no stimulation effects on level two perspective taking nor self-other encoding effects on episodic memory. Stimulation to the rTPJ and dmPFC improved perspective selection during level one perspective taking. However, dissociable effects on self-other processing, previously observed in young adults, were not identified in older adults. The results provide causal evidence for age-related changes in social brain function that requires further scrutinization.

Depletion of HP1α alters the mechanical properties of MCF7 nuclei.

Within the nucleus of the eukaryotic cell, DNA is partitioned into domains of highly condensed, transcriptionally silent heterochromatin and less condensed, transcriptionally active euchromatin. Heterochromatin protein 1α (HP1α) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. Although the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin-modifying enzymes have been studied previously, here we measure how the material properties of the nuclei are modified after the knockdown of HP1α. These studies were inspired by the observation that poorly invasive MCF7 breast cancer cells become more invasive after knockdown of HP1α expression and that, indeed, in many solid tumors the loss of HP1α correlates with the onset of tumor cell invasion. Atomic force microscopy (AFM), optical tweezers (OT), and techniques based on micropipette aspiration (MA) were each used to characterize the mechanical properties of nuclei extracted from HP1α knockdown or matched control MCF7 cells. Using AFM or OT to locally indent nuclei, those extracted from MCF7 HP1α knockdown cells were found to have apparent Young's moduli that were significantly lower than nuclei from MCF7 control cells, consistent with previous studies that assert heterochromatin plays a major role in governing the mechanical response in such experiments. In contrast, results from pipette-based techniques in the spirit of MA, in which the whole nuclei were deformed and aspirated into a conical pipette, showed considerably less variation between HP1α knockdown and control, consistent with previous studies reporting that it is predominantly the lamins in the nuclear envelope that determine the mechanical response to large whole-cell deformations. The differences in chromatin organization observed by various microscopy techniques between the MCF7 control and HP1α knockdown nuclei correlate well with the results of our measured mechanical responses and our hypotheses regarding their origin.

Improving Cross-cultural "Mind-reading" with Electrical Brain Stimulation.

A cross-cultural disadvantage exists when inferring the mental state of others, which may be detrimental for individuals acting in an increasingly globalized world. The dorsomedial prefrontal cortex (dmPFC) is a key hub of the social brain involved in ToM. We explored whether facilitation of dmPFC function by focal high-definition tDCS can improve cross-cultural mind-reading. 52 (26 F/M) Singaporeans performed the Caucasian version of the Reading the Mind in the Eyes Test (RMET) and received HD-tDCS to either the dmPFC or a control site (right temporoparietal junction, rTPJ) in sham-controlled, double-blinded, crossover studies. Contact with Caucasians was determined for the Singaporean cohort as a potential mediator of RMET performance and HD-tDCS response. 52 Caucasians completed the RMET during sham-tDCS and served as a comparison group. A cross-cultural disadvantage on the RMET was confirmed in the Singaporean cohort and this disadvantage was more pronounced in those participants who had less contact with Caucasians. Importantly, HD-tDCS to the dmPFC improved RMET performance in those with less contact. No effect was identified for rTPJ HD-tDCS or for the age/sex control task demonstrating task and site specificity of the stimulation effects. Electrical stimulation of the dmPFC selectively improves the rate of cross-cultural ToM inference from facial cues, effectively removing cross-cultural disadvantage that was found in individuals with lower cross-cultural exposure.

Response initiation and inhibition and the relationship with fluid intelligence across the adult lifespan.

BACKGROUND: Cognitive processes associated with frontal lobe functioning are often termed "executive functions." Two such processes are initiation and inhibition or the starting and stopping of responses. It has recently been claimed dysfunction of executive abilities can be explained by a single measure of fluid intelligence. Here, we test this claim, specifically for the executive abilities of response initiation and inhibition, across the healthy lifespan. METHOD: In a cohort of 336 healthy adults (18-89 years), initiation and inhibition were assessed with the Hayling test, Stroop test, and phonemic and semantic verbal fluency. All participants also completed a measure of fluid intelligence. The relationship between fluid intelligence and executive measures was explored across the lifespan using a continuous approach. Mediation models were computed to assess whether age-related decline across the four initiation/inhibition tasks could be fully explained by a single measure of fluid intelligence. RESULTS: Age was negatively correlated with response initiation/inhibition and fluid intelligence. The mediation analyses identified only partial mediation of fluid intelligence for age and Hayling performance. By contrast, fluid intelligence did not mediate performance on the Stroop test or phonemic and semantic verbal fluency. CONCLUSIONS: Response initiation/inhibition are not able to be explained by fluid intelligence. The results support a multifactorial theory of executive functions and provide evidence for the inclusion of multiple specific executive measures in a thorough neuropsychological assessment of age-related cognitive decline.

Optical Micromachines for Biological Studies.

Optical tweezers have been used for biological studies since shortly after their inception. However, over the years research has suggested that the intense laser light used to create optical traps may damage the specimens being studied. This review aims to provide a brief overview of optical tweezers and the possible mechanisms for damage, and more importantly examines the role of optical micromachines as tools for biological studies. This review covers the achievements to date in the field of optical micromachines: improvements in the ability to produce micromachines, including multi-body microrobots; and design considerations for both optical microrobots and the optical trapping set-up used for controlling them are all discussed. The review focuses especially on the role of micromachines in biological research, and explores some of the potential that the technology has in this area.

Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM.

Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 µm long was observed.

Common and unique effects of HD-tDCS to the social brain across cultural groups.

Cultural background influences social cognition, however no study has examined brain stimulation differences attributable to cultural background. 104 young adults [52 South-East Asian Singaporeans (SEA); 52 Caucasian Australians (CA)] received anodal high-definition transcranial direct current stimulation (HD-tDCS) to the dorsomedial prefrontal cortex (dmPFC) or the right temporoparietal junction (rTPJ). Participants completed tasks with varying demands on self-other processing including visual perspective taking (VPT)and episodic memory with self and other encoding. At baseline, SEA showed greater self-other integration than CA in the level one (line-of-sight) VPT task as indexed by greater interference from the alternate perspective. Anodal HD-tDCS to the dmPFC resulted in the CA performing closer to the SEA during egocentric perspective judgements. Baseline performance on level two (embodied rotation) VPT task and the self-reference effect (SRE) in episodic memory was comparable between the two groups. In the combined sample, HD-tDCS to the rTPJ decreased the interference from the egocentric perspective during level two VPT and dmPFC HD-tDCS removed the SRE in episodic memory. Stimulation effects were comparable when baseline performance was comparable. When baseline performance differed, stimulation differences were identified. Therefore, social cognitive differences due to cultural background are an important consideration in social brain stimulation studies.

Aggregation and coalescence of partially crystalline emulsion drops investigated using optical tweezers.

The solid content of viscoelastic emulsion drops is known to affect their propensity for aggregation and their subsequent coalescence behaviour, where the balance between the drive to reduce surface tension and the straining of an internal viscoelastic network is able to create a plethora of stable partially-coalesced states. The latter has previously been elegantly demonstrated in synthetic systems, generated using oil containing different phase volumes of added solids, with micro-pipette experiments carried out on emulsion drops of several tens of microns in size. Herein we carry out experiments in the same spirit but aided by optical tweezers (OT) and using smaller micron-sized emulsion drops generated from milk fat. Given the size dependence of Brownian fluctuations and Laplace pressure the experimental investigation of these smaller drops is not necessarily a trivial extension of the previous work. The solid content of initially separated drops is controlled using a temperature-cycling regime in the sample preparation protocol, and subsequently the propensity for drops to remain joined or not after being brought into contact was examined. Aggregated pairs of drops were then subjected to an increase in temperature, either locally using a high-powered laser, or more globally using a custom-made Peltier temperature-controller. By heating to different degrees, the amount of fat crystals in the drops was able to be controlled, with progressively more compact partially-coalesced states, and eventually complete coalescence generated as the solid content was reduced. While in contrast to previous studies, the emulsion studied here was quite different in size and nature, and the solid content was controlled using temperature, the same underlying physics was nevertheless observed.