The spatial organization of sphingofungin biosynthesis in Aspergillus fumigatus and its cross-interaction with sphingolipid metabolism.

Sphingofungins are sphinganine analog mycotoxins acting as inhibitors of serine palmitoyl transferases, enzymes responsible for the first step in the sphingolipid biosynthesis. Eukaryotic cells are highly organized with various structures and organelles to facilitate cellular processes and chemical reactions, including the ones occurring as part of the secondary metabolism. We studied how sphingofungin biosynthesis is compartmentalized in the human-pathogenic fungus Aspergillus fumigatus, and we observed that it takes place in the endoplasmic reticulum (ER), ER-derived vesicles, and the cytosol. This implies that sphingofungin and sphingolipid biosynthesis colocalize to some extent. Automated analysis of confocal microscopy images confirmed the colocalization of the fluorescent proteins. Moreover, we demonstrated that the cluster-associated aminotransferase (SphA) and 3-ketoreductase (SphF) play a bifunctional role, supporting sphingolipid biosynthesis, and thereby antagonizing the toxic effects caused by sphingofungin production.IMPORTANCEA balanced sphingolipid homeostasis is critical for the proper functioning of eukaryotic cells. To this end, sphingolipid inhibitors have therapeutic potential against diseases related to the deregulation of sphingolipid balance. In addition, some of them have significant antifungal activity, suggesting that sphingolipid inhibitors-producing fungi have evolved mechanisms to escape self-poisoning. Here, we propose a novel self-defense mechanism, with cluster-associated genes coding for enzymes that play a dual role, being involved in both sphingofungin and sphingolipid production.

Engineering stringent genetic biocontainment of yeast with a protein stability switch.

Synthetic biology holds immense promise to tackle key problems in resource use, environmental remediation, and human health care. However, comprehensive safety measures are lacking to employ engineered microorganisms in open-environment applications. Genetically encoded biocontainment systems may solve this issue. Here, we describe such a system based on conditional stability of essential proteins. We used a destabilizing domain degron stabilized by estradiol addition (ERdd). We ERdd-tagged 775 essential genes and screened for strains with estradiol dependent growth. Three genes, SPC110, DIS3 and RRP46, were found to be particularly suitable targets. Respective strains showed no growth defect in the presence of estradiol and strong growth inhibition in its absence. SPC110-ERdd offered the most stringent containment, with an escape frequency of <5×10-7. Removal of its C-terminal domain decreased the escape frequency further to <10-8. Being based on conditional protein stability, the presented approach is mechanistically orthogonal to previously reported genetic biocontainment systems.

Heterologous pulcherrimin production in Saccharomyces cerevisiae confers inhibitory activity on Botrytis conidiation.

Pulcherrimin is an iron (III) chelate of pulcherriminic acid that plays a role in antagonistic microbial interactions, iron metabolism, and stress responses. Some bacteria and yeasts produce pulcherriminic acid, but so far, pulcherrimin could not be produced in Saccharomyces cerevisiae. Here, multiple integrations of the Metschnikowia pulcherrima PUL1 and PUL2 genes in the S. cerevisiae genome resulted in red colonies, which indicated pulcherrimin formation. The coloration correlated positively and significantly with the number of PUL1 and PUL2 genes. The presence of pulcherriminic acid was confirmed by mass spectrometry. In vitro competition assays with the plant pathogenic fungus Botrytis caroliana revealed inhibitory activity on conidiation by an engineered, strong pulcherrimin-producing S. cerevisiae strain. We demonstrate that the PUL1 and PUL2 genes from M. pulcherrima, in multiple copies, are sufficient to transfer pulcherrimin production to S. cerevisiae and represent the starting point for engineering and optimizing this biosynthetic pathway in the future.

Safety by design: Biosafety and biosecurity in the age of synthetic genomics.

Technologies to profoundly engineer biology are becoming increasingly affordable, powerful, and accessible to a widening group of actors. While offering tremendous potential to fuel biological research and the bioeconomy, this development also increases the risk of inadvertent or deliberate creation and dissemination of pathogens. Effective regulatory and technological frameworks need to be developed and deployed to manage these emerging biosafety and biosecurity risks. Here, we review digital and biological approaches of a range of technology readiness levels suited to address these challenges. Digital sequence screening technologies already are used to control access to synthetic DNA of concern. We examine the current state of the art of sequence screening, challenges and future directions, and environmental surveillance for the presence of engineered organisms. As biosafety layer on the organism level, we discuss genetic biocontainment systems that can be used to created host organisms with an intrinsic barrier against unchecked environmental proliferation.

Vanishing Boycott Impetus: Why and How Consumer Participation in a Boycott Decreases Over Time.

Media reports that a company behaves in a socially nonresponsible manner frequently result in consumer participation in a boycott. As time goes by, however, the number of consumers participating in the boycott starts dwindling. Yet, little is known on why individual participation in a boycott declines and what type of consumer is more likely to stop boycotting earlier rather than later. Integrating research on drivers of individual boycott participation with multi-stage models and the hot/cool cognition system, suggests a "heat-up" phase in which boycott participation is fueled by expressive drivers, and a "cool-down" phase in which instrumental drivers become more influential. Using a diverse set of real contexts, four empirical studies provide evidence supporting a set of hypotheses on promotors and inhibitors of boycott participation over time. Study 1 provides initial evidence for the influence of expressive and instrumental drivers in a food services context. Extending the context to video streaming services, e-tailing, and peer-to-peer ridesharing, Study 2, Study 3, and Study 4 show that the reasons consumers stop/continue boycotting vary systematically across four distinct groups. Taken together, the findings help activists sustain boycott momentum and assist firms in dealing more effectively with boycotts. Supplementary Information: The online version contains supplementary material available at 10.1007/s10551-021-04997-9.

Moral-psychological mechanisms of rebound effects from a consumer-centered perspective: A conceptualization and research directions.

Rebound effects on the consumer level occur when consumers' realized greenhouse gas emission savings caused by behaviors that might be beneficial to the environment are lower than their potential greenhouse gas emission savings because the savings are offset by behavioral adjustments. While previous literature mainly studied the economic mechanisms of such rebound effects, research has largely neglected the moral-psychological mechanisms. A comprehensive conceptualization of rebound effects on the consumer level can help fill this void and stimulate more empirical research in this relevant area. To this end, the paper introduces three focal dimensions of rebound effects on the consumer level: mechanism of rebound effects, product category, and consumption context. Based on this conceptualization, and integrating assumptions from the theory of moral licensing, the theory of categorization, and the construal level theory, this paper further refines the conceptualization of the moral component as an explanatory factor for rebound effects and highlights that the moral-psychological mechanisms of indirect rebound effects (i.e., rebound effects that occur across different product categories or consumption contexts) are more complex and diverse than the economic mechanisms. The paper outlines promising directions for future studies considering the different quantification and characteristics of economic and moral currencies that explain rebound effects on the consumer level and the strategic categorization of products and consumption contexts.

Monitoring orthodontic tooth movement in rats after piezocision by bone scintigraphy.

AIM: Piezocision, corticocision of mineralized tissue by ultrasound showed promising results in accelerating tooth movement induced by orthodontic appliances although the biologic effects of this procedure are not well-understood so far. The aim of this study was to investigate the impact of piezocision on bone remodeling in rats by bone SPECT imaging. MATERIAL AND METHODS: Ten male Wistar rats underwent surgical placement of orthodontic appliances on each side of the maxilla followed by piezocision on one side only. Each rat underwent 99mTc-MDP bone SPECT/CT imaging before surgery (T0), and 2 (T1) and 4 weeks (T2) after surgery. Bone uptake is expressed as median [IQR] min-max in percentage of the injected activity per ml computed from the 10 voxels with the highest uptake (%IAmax10/ml). RESULTS: Pooled data regardless of the piezocision showed a significant increase in bone uptake from T0 (3.2 [2.8-3.9] 2.6-4.9) to T1 (4.4 [3.8-4.6] 3.4-4.8; p = 0.001). Thereafter, the uptake decreased to T2 (3.8 [3.1-4.4] 2.8-4.8; p = 0.116). No significant differences in bone uptake were found between the maxilla sides without and with piezocision: T1: without (4.3 [3.8-4.5] 3.4-4.8) vs. with (4.5 [3.7-4.6] 3.5-4.7; p=0.285), T2: without (4.0 [3.1-4.5] 2.8-4.8) vs. with (3.7 [3.0-4.4] 2.8-4.8; p=0.062). CONCLUSION: 99mTc-MDP bone SPECT imaging in rats was able to reproduce changes in bone uptake in the maxilla after placement of orthodontic appliances inducing measurable tooth movement. An additional effect of piezocision on bone remodeling in terms of bone uptake was not detectable which is probably due to the pronounced and significant effects induced by the orthodontic appliances per se, which may mask the potential effects of additional piezocision.

Augmented reality-delivered product information at the point of sale: when information controllability backfires.

Augmented reality-delivered product information (ARPI) can overcome the limited space at the point of sale to inform shoppers on demand and will therefore become more widespread in brick-and-mortar stores. To fill the void of academic research, this paper develops a model of how consumers process ARPI and how ARPI can shape brand image and purchase intentions. Making use of the cues-filtered-out theory, this paper suggests that the effect of ARPI controllability depends on information detailedness. An unintended backfire effect of controllability occurs when the accessible information is detailed, which is explained by the mediating process via perceived comprehensiveness. This backfire effect is a risk primarily in busy shopping times. The main experiment conducted in a hypermarket and four follow-up studies (using field, lab, and video settings) empirically confirm the proposed model on the basis of different data sources, including usage tracking, questionnaires, and scanner data. The paper derives managerial implications and outlines directions for future research. Supplementary Information: The online version contains supplementary material available at 10.1007/s11747-022-00855-w.

Ethical Principles in Digital Palliative Care for Children: The MyPal Project and Experiences Made in Designing a Trustworthy Approach.

This paper explores the ethical dimension of the opportunity to offer improved electronic patient-reported outcome (ePRO) systems addressing personal needs of pediatric cancer patients, their parents and caregivers, with regard to technological advance of digital health. This opportunity has been explored in the MyPal research project, which aims to assess a patient-centered service for palliative care relying on the adaptation and extension of digital health tools and concepts available from previous projects. Development and implementation of ePROs need to take place in a safe, secure and responsible manner, preventing any possible harm and safeguarding the integrity of humans. To that end, although the final results will be published at the end of the project, this paper aims to increase awareness of the ethical ramifications we had to address in the design and testing of new technologies and to show the essentiality of protection and promotion of privacy, safety and ethical standards. We have thus reached a final design complying with the following principles: (a) respect for the autonomy of participants, especially children, (b) data protection and transparency, (c) fairness and non-discrimination, (d) individual wellbeing of participants in relation to their physical and psychological health status and e) accessibility and acceptability of digital health technologies for better user-engagement. These principles are adapted from the Ethics Guidelines for a trustworthy Artificial Intelligence (AI) which provide the framework for similar interventions to be lawful, complying with all applicable laws and regulations, ethical, ensuring compliance to ethical principles and values and robust, both from a technical and social perspective.

New opportunities for genetic code expansion in synthetic yeast.

The synthetic yeast, Sc2.0, is nearing completion as consolidation of all 17 synthetic chromosomes into a single cell advances. This organism will be the first synthetic eukaryote and provides a highly plastic biological chassis built from the bottom-up using principles of biological design. This synthetic approach to genome construction has allowed the genetic code to be re-wired in this background to liberate the amber stop codon as a dedicated triplet for encoding non-canonical amino acids. The availability of an expanded set of amino acid building blocks allows precise control of protein structure and function, providing new opportunities to develop protein-based therapeutics, materials and catalysts. In this article, we review the challenges facing genetic code expansion research in yeast and highlight how the development of Sc2.0 provides new and exciting opportunities to address existing limitations.

AquaScouts: ePROs Implemented as a Serious Game for Children With Cancer to Support Palliative Care.

MyPal is a European initiative focusing on the use of the electronic patient reported outcome (ePRO) measures to enhance patient engagement in palliative cancer care via digital self-reporting palliative care for patients with cancer. As a part of its approach, MyPal also focuses on pediatric patients, implementing a specific digital health platform including a serious game to facilitate the reporting of the symptoms and overall status regarding their quality of life (QoL). To this end, the reduction of psychological burden related to frequent reporting, a.k.a. as "reporting fatigue" has been identified as a priority. In this study, we present the MyPal-CHILD platform, emphasizing on the serious game named AquaScouts and its key design decisions, while also emphasizing on the respective challenges. More specifically, we provide insights on the participatory design approach applied during the design of the platform and the high-level goals defined based on end-user input. In addition, the validation process applied before the use of the platform under real-world conditions is also presented. Finally, we discuss a number of challenges and the prospects of deploying eHealth interventions to support palliative care.

Impact of piezocision on orthodontic tooth movement.

OBJECTIVE: This study investigated the impact of a single piezocision in the maxillary alveolar process on the speed of tooth movement. The null hypothesis was that the speed of tooth movement will be equal with and without piezocision. METHODS: All maxillary molars on one side were moved against the combined incisors in 10 ten-week-old male Wistar rats. Under general anesthesia, a force of 25 cN was applied on either side using a Sentalloy closed coil spring. After placing the orthodontic appliance, vertical corticision was performed using a piezotome under local anesthesia, 2 mm mesial from the mesial root of the first molar on a randomly selected side; the other side served as the control. At the beginning of the treatment, and 2 and 4 weeks later, skull micro-computed tomography was performed. After image reconstruction, the distance between the mesial root of the first molar and the incisive canal, and the length of the mesial root of the first maxillary molar were measured. Moreover, the root resorption score was determined as described by Lu et al. RESULTS: Significantly higher speed of tooth movement was observed on the corticision side; thus, the null hypothesis was rejected. The loss of root length and root resorption score were significantly more pronounced after piezocision than before. A strong correlation was observed between the speed of tooth movement and root resorption on the surgical side, but the control side only showed a weak correlation. CONCLUSIONS: Piezocision accelerates orthodontic tooth movement and causes increased root resorption.

Thermomechanical properties of zero thermal expansion materials from theory and experiments.

Origin and composition dependence of the anisotropic thermomechanical properties are elucidated for Ba1-xSrxZn2Si2O7 (BZS) solid solutions. The high-temperature phase of BZS shows negative thermal expansion (NTE) along one crystallographic axis and highly anisotropic elastic properties characterized by X-ray diffraction experiments and simulations at the density functional theory level. Ab initio molecular dynamics simulations provide accurate predictions of the anisotropic thermal expansion in excellent agreement with experimental observations. The NTE considerably decreases with increasing Sr content x. This is connected with the composition dependence of the vibrational density of states (VDOS) and the anisotropic Grüneisen parameters. The VDOS shifts to higher frequencies between 0-5 THz due to substitution of Ba with Sr. In the same frequency range, vibrational modes contributing most to the NTE are found. In addition, phonon calculations using the quasi-harmonic approximation revealed that the NTE is mainly connected with deformation of four-membered rings formed by SiO4 and ZnO4 tetrahedra. The thermomechanical and vibrational properties obtained in this work provide the basis for future studies facilitating the targeted design of BZS solid solutions as zero or negative thermal expansion material.

Computer-Aided Whole-Cell Design: Taking a Holistic Approach by Integrating Synthetic With Systems Biology.

Computer-aided design (CAD) for synthetic biology promises to accelerate the rational and robust engineering of biological systems. It requires both detailed and quantitative mathematical and experimental models of the processes to (re)design biology, and software and tools for genetic engineering and DNA assembly. Ultimately, the increased precision in the design phase will have a dramatic impact on the production of designer cells and organisms with bespoke functions and increased modularity. CAD strategies require quantitative models of cells that can capture multiscale processes and link genotypes to phenotypes. Here, we present a perspective on how whole-cell, multiscale models could transform design-build-test-learn cycles in synthetic biology. We show how these models could significantly aid in the design and learn phases while reducing experimental testing by presenting case studies spanning from genome minimization to cell-free systems. We also discuss several challenges for the realization of our vision. The possibility to describe and build whole-cells in silico offers an opportunity to develop increasingly automatized, precise and accessible CAD tools and strategies.

Probing eukaryotic genome functions with synthetic chromosomes.

The ability to redesign and reconstruct a cell at whole-genome level provides new platforms for biological study. The international synthetic yeast genome project-Sc2.0, designed by interrogating knowledge amassed by the yeast community to date, exemplifies how a classical synthetic biology "design-build-test-learn" engineering cycle can effectively test hypotheses about various genome fundamentals. The genome reshuffling SCRaMbLE system implemented in synthetic yeast strains also provides unprecedented diversified resources for genotype-phenotype study and yeast metabolic engineering. Further development of genome synthesis technology will shed new lights on complex biological processes in higher eukaryotes.

Five-Year Results of the Bioflow-III Registry: Real-World Experience with a Biodegradable Polymer Sirolimus-Eluting Stent.

PURPOSE: We aimed to assess long-term safety and performance of the Orsiro sirolimus-eluting coronary stent with biodegradable polymer in a large unselected population and in pre-specified subgroups. METHODS: BIOFLOW-III is a prospective, multicenter, international, observational registry with follow-up visits scheduled at 6 and 12 months, and at 3 and 5 years (NCT01553526). RESULTS: 1356 patients with 1738 lesions were enrolled. Of those, 392 (28.9%) declined to participate in the study extension from 18 months to 5 years, 37 (2.7%) withdrew consent, and 89 (6.6%) were lost to follow-up. At 5-years, Kaplan-Meier estimates of target lesion failure, defined as a composite of cardiac death, target-vessel myocardial infarction, coronary artery bypass grafting and clinically driven target lesion revascularization was 10.0% [95% confidence interval (CI): 8.4; 12.0] in the overall population, and 14.0% [95% CI: 10.5; 18.6], 10.3% [95% CI: 7.8; 13.5], 1.8% [95% CI: 0.3; 12.0], and 11.3% [95% CI: 8.5; 15.1] in the pre-defined risk groups of patients with diabetes mellitus, small vessels ≤2.75 mm, chronic total occlusion, and acute myocardial infarction. Definite stent thrombosis was observed in 0.3% [95% CI: 0.1; 0.9] of patients. CONCLUSION: These long-term outcomes provide further evidence on the safety and performance of a sirolimus-eluting biodegradable polymer stent within daily clinical practice. The very low definite stent thrombosis rate affirms biodegradable polymer safety and performance.

Differences of the tumour cell glycocalyx affect binding of capsaicin-loaded chitosan nanocapsules.

The glycocalyx regulates the interaction of mammalian cells with extracellular molecules, such as cytokines. However, it is unknown to which extend the glycocalyx of distinct cancer cells control the binding and uptake of nanoparticles. In the present study, exome sequencing data of cancer patients and analysis of distinct melanoma and bladder cancer cell lines suggested differences in cancer cell-exposed glycocalyx components such as heparan sulphate. Our data indicate that glycocalyx differences affected the binding of cationic chitosan nanocapsules (Chi-NCs). The pronounced glycocalyx of bladder cancer cells enhanced the internalisation of nanoencapsulated capsaicin. Consequently, capsaicin induced apoptosis in the cancer cells, but not in the less glycosylated benign urothelial cells. Moreover, we measured counterion condensation on highly negatively charged heparan sulphate chains. Counterion condensation triggered a cooperative binding of Chi-NCs, characterised by a weak binding rate at low Chi-NC doses and a strongly increased binding rate at high Chi-NC concentrations. Our results indicate that the glycocalyx of tumour cells controls the binding and biological activity of nanoparticles. This has to be considered for the design of tumour cell directed nanocarriers to improve the delivery of cytotoxic drugs. Differential nanoparticle binding may also be useful to discriminate tumour cells from healthy cells.

Characterizing Transcriptional Interference between Converging Genes in Bacteria.

Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two.

Chitosan-based nanodelivery systems applied to the development of novel triclabendazole formulations.

Triclabendazole is a poorly-water soluble (0.24 µg/mL) compound classified into the Class II/IV of the Biopharmaceutical Classification System. It is the drug of choice to treat fascioliasis, a neglected parasitic disease worldwide disseminated. Triclabendazole is registered as veterinary medicine and it is only available for human treatment as 250 mg tablets. Thus, the aim of this work was to develop novel drug delivery systems based on nanotechnology approaches. The chitosan-based nanocapsules and nanoemulsions of triclabendazole were fully characterized regarding their particle size distribution, polydispersity index and zeta potential, in-vitro release and stability in biological media. Cytotoxicity evaluation and cellular uptake studies using CaCo-2 cell line were also investigated. The results indicated an average hydrodynamic size around ~160 nm were found for unloaded nanoemulsions which were slightly increased up to ~190 nm for loaded one. In contrast, the average hydrodynamic size of the nanocapsules increased from ~160 nm up to ~400 nm when loaded with triclabendazole. The stability studies upon 30 days storage at 4, 25 and 37°C showed that average size of nanoemulsions was not modified with varying amounts of loaded TCBZ while an opposite result was seen in case of loaded nanocapsules. In addition, a slight reduction of zeta potential values over time was observed in both triclabendazole nanosystems. Release of TCBZ from nanoformulations over 6 h in simulated gastric fluid was 9 to 16-fold higher than with untreated TCBZ dispersion. In phosphate buffer saline solution there was no drug release for neither nanocapsules nor nanoemulsions. Cell viabilities studies indicated that at certain concentrations, drug encapsulation can lower its cytotoxic effects when compared to untreated drug. Confocal laser scanning microscopy study has shown that nanocapsules strongly interacted with Caco-2 cells in vitro which could increase the passage time of triclabendazole after oral administration. The results of this study constitute the first step towards the development of nanoformulations intended for the oral delivery of anti-parasitic drugs of enhanced bioavailability.

Dietary Behavior: An Interdisciplinary Conceptual Analysis and Taxonomy.

Background: Dietary behavior encompasses many aspects, terms for which are used inconsistently across different disciplines and research traditions. This hampers communication and comparison across disciplines and impedes the development of a cumulative science. We describe the conceptual analysis of the fuzzy umbrella concept "dietary behavior" and present the development of an interdisciplinary taxonomy of dietary behavior. Methods: A four-phase multi-method approach was employed. Input was provided by 76 scholars involved in an international research project focusing on the determinants of dietary behavior. Input was collected from the scholars via an online mind mapping procedure. After structuring, condensing, and categorizing this input into a compact taxonomy, the result was presented to all scholars, discussed extensively, and adapted. A second revision round was then conducted among a core working group. Results: A total of 145 distinct entries were made in the original mind mapping procedure. The subsequent steps allowed us to reduce and condense the taxonomy into a final product consisting of 34 terms organized into three main categories: Food Choice, Eating Behavior, and Dietary Intake/Nutrition. In a live discussion session attended by 50 of the scholars involved in the development of the taxonomy, it was judged to adequately reflect their input and to be a valid and useful starting point for interdisciplinary understanding and collaboration. Conclusion: The current taxonomy can be used as a tool to facilitate understanding and cooperation between different disciplines investigating dietary behavior, which may contribute to a more successful approach to tackling the complex public health challenges faced by the field. The taxonomy need not be viewed as a final product, but can continue to grow in depth and width as additional experts provide their input.