Stress relaxation analysis facilitates a quantitative approach towards antimicrobial penetration into biofilms

Biofilm-related infections can develop everywhere in the human body and are rarely cleared by the host immune system. Moreover, biofilms are often tolerant to antimicrobials, due to a combination of inherent properties of bacteria in their adhering, biofilm mode of growth and poor physical penetration of antimicrobials through biofilms. Current understanding of biofilm recalcitrance toward antimicrobial penetration is based on qualitative descriptions of biofilms. Here we hypothesize that stress relaxation of biofilms will relate with antimicrobial penetration. Stress relaxation analysis of single-species oral biofilms grown in vitro identified a fast, intermediate and slow response to an induced deformation, corresponding with outflow of water and extracellular polymeric substances, and bacterial re-arrangement, respectively. Penetration of chlorhexidine into these biofilms increased with increasing relative importance of the slow and decreasing importance of the fast relaxation element. Involvement of slow relaxation elements suggests that biofilm structures allowing extensive bacterial re-arrangement after deformation are more open, allowing better antimicrobial penetration. Involvement of fast relaxation elements suggests that water dilutes the antimicrobial upon penetration to an ineffective concentration in deeper layers of the biofilm. Next, we collected biofilms formed in intra-oral collection devices bonded to the buccal surfaces of the maxillary first molars of human volunteers. Ex situ chlorhexidine penetration into two weeks old in vivo formed biofilms followed a similar dependence on the importance of the fast and slow relaxation elements as observed for in vitro formed biofilms. This study demonstrates that biofilm properties can be derived that quantitatively explain antimicrobial penetration into a biofilm

How digital health affects the patient-physician relationship: An empirical-ethics study into the perspectives and experiences in obstetric care

Background: Digital health technologies are believed to change the patient-physician relationship. Such changes are still speculative, as there are no studies in which both patients and health care professionals are asked for their experiences and perspectives on how digital health affects the patient-physician relationship. Methods: We performed a qualitative interview study (n = 25) to identify relevant aspects of the patient-physician relationship as perceived by both health care professionals (n = 14) and patients (n = 11) of a digital monitoring platform for hypertensive disorders related to pregnancy. We focus on roles, responsibilities and medical decision-making. Results: Digital monitoring helps patients to better understand their own condition and contributes to shared decision-making in terms of information exchange. Yet for clinical decision-making both patients and health care professionals argue that health care professionals should stay in the lead. The collected data is by some health care professionals considered hard data that allows objective and more standardized decision-making, while others believe digital monitoring requires further interpretation in order to personalize the clinical care to the patient. Conclusion: Digital technologies have subtle, yet double-edged, effects on the patient-physician relationship in terms of roles and responsibilities and the value addressed to the digital data. These insights let to 6 ethical recommendations for the implementation of digital health technologies to replace and support clinical care

A Broad Set of Different Llama Antibodies Specific for a 16 kDa Heat Shock Protein of Mycobacterium tuberculosis

Background Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. Methodology/Principal Findings Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4×10-10 M. Conclusions/Significance A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria

Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors

Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative method using a sphingosine-1-phosphate (S1P) receptor as a model. Because of a lack of suitable binding studies, it has been difficult to study S1P receptor internalisation. Using a N-terminal HisG-tag, S1P1 receptors on the cell membrane can be visualised via immunocytochemistry with a specific anti-HisG antibody. S1P-induced internalisation was concentration dependent and was quantified using a microplate reader, detecting either absorbance, a fluorescent or luminescent signal, depending on the antibodies used. Among those, the fluorescence detection method was the most convenient to use. The relative ease of this method makes it suitable to measure a large number of data points, e.g. to compare the potency and efficacy of receptor ligands

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to 'rescue' a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.

The ethical significance of user-control in AI-driven speech-BCIs: a narrative review

AI-driven brain-computed interfaces aimed at restoring speech for individuals living with locked-in-syndrome are paired with ethical implications for user’s autonomy, privacy and responsibility. Embedding options for sufficient levels of user-control in speech-BCI design has been proposed to mitigate these ethical challenges. However, how user-control in speech-BCIs is conceptualized and how it relates to these ethical challenges is underdetermined. In this narrative literature review, we aim to clarify and explicate the notion of user-control in speech-BCIs, to better understand in what way user-control could operationalize user’s autonomy, privacy and responsibility and explore how such suggestions for increasing user-control can be translated to recommendations for the design or use of speech-BCIs. First, we identified types of user control, including executory control that can protect voluntariness of speech, and guidance control that can contribute to semantic accuracy. Second, we identified potential causes for a loss of user-control, including contributions of predictive language models, a lack of ability for neural control, or signal interference and external control. Such a loss of user control may have implications for semantic accuracy and mental privacy. Third we explored ways to design for user-control. While embedding initiation signals for users may increase executory control, they may conflict with other aims such as speed and continuity of speech. Design mechanisms for guidance control remain largely conceptual, similar trade-offs in design may be expected. We argue that preceding these trade-offs, the overarching aim of speech-BCIs needs to be defined, requiring input from current and potential users. Additionally, conceptual clarification of user-control and other (ethical) concepts in this debate has practical relevance for BCI researchers. For instance, different concepts of inner speech may have distinct ethical implications. Increased clarity of such concepts can improve anticipation of ethical implications of speech-BCIs and may help to steer design decisions

Meaningful public engagement in the context of open science: reflections from early and mid-career academics

How is public engagement perceived to contribute to open science? This commentary highlights common reflections on this question from interviews with 12 public engagement fellows in Utrecht University’s Open Science Programme in the Netherlands. We identify four reasons why public engagement is an essential enabler of open science. Interaction between academics and society can: (1) better align science with the needs of society; (2) secure a relationship of trust between science and society; (3) increase the quality and impact of science; and (4) support the impact of open access and FAIR data practices (data which meet principles of findability, accessibility, interoperability and reusability). To be successful and sustainable, such public engagement requires support in skills training and a form of institutionalisation in a university-wide system, but, most of all, the fellows express the importance of a formal and informal recognition and rewards system. Our findings suggest that in order to make public engagement an integral part of open science, universities should invest in institutional support, create awareness, and stimulate dialogue among staff members on how to ‘do’ good public engagement

Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient