Modelled-Microgravity Reduces Virulence Factor Production in Staphylococcus aureus through Downregulation of agr-Dependent Quorum Sensing.

Bacterial contamination during space missions is problematic for human health and damages filters and other vital support systems. Staphylococcus aureus is both a human commensal and an opportunistic pathogen that colonizes human tissues and causes acute and chronic infections. Virulence and colonization factors are positively and negatively regulated, respectively, by bacterial cell-to-cell communication (quorum sensing) via the agr (accessory gene regulator) system. When cultured under low-shear modelled microgravity conditions (LSMMG), S. aureus has been reported to maintain a colonization rather than a pathogenic phenotype. Here, we show that the modulation of agr expression via reduced production of autoinducing peptide (AIP) signal molecules was responsible for this behavior. In an LSMMG environment, the S. aureus strains JE2 (methicillin-resistant) and SH1000 (methicillin-sensitive) both exhibited reduced cytotoxicity towards the human leukemia monocytic cell line (THP-1) and increased fibronectin binding. Using S. aureus agrP3::lux reporter gene fusions and mass spectrometry to quantify the AIP concentrations, the activation of agr, which depends on the binding of AIP to the transcriptional regulator AgrC, was delayed in the strains with an intact autoinducible agr system. This was because AIP production was reduced under these growth conditions compared with the ground controls. Under LSMMG, S. aureus agrP3::lux reporter strains that cannot produce endogenous AIPs still responded to exogenous AIPs. Provision of exogenous AIPs to S. aureus USA300 during microgravity culture restored the cytotoxicity of culture supernatants for the THP-1 cells. These data suggest that microgravity does not affect AgrC-AIP interactions but more likely the generation of AIPs.

Spaceflight Induces Strength Decline in Caenorhabditis elegans.

Background: Understanding and countering the well-established negative health consequences of spaceflight remains a primary challenge preventing safe deep space exploration. Targeted/personalized therapeutics are at the forefront of space medicine strategies, and cross-species molecular signatures now define the 'typical' spaceflight response. However, a lack of direct genotype-phenotype associations currently limits the robustness and, therefore, the therapeutic utility of putative mechanisms underpinning pathological changes in flight. Methods: We employed the worm Caenorhabditis elegans as a validated model of space biology, combined with 'NemaFlex-S' microfluidic devices for assessing animal strength production as one of the most reproducible physiological responses to spaceflight. Wild-type and dys-1 (BZ33) strains (a Duchenne muscular dystrophy (DMD) model for comparing predisposed muscle weak animals) were cultured on the International Space Station in chemically defined media before loading second-generation gravid adults into NemaFlex-S devices to assess individual animal strength. These same cultures were then frozen on orbit before returning to Earth for next-generation sequencing transcriptomic analysis. Results: Neuromuscular strength was lower in flight versus ground controls (16.6% decline, p < 0.05), with dys-1 significantly more (23% less strength, p < 0.01) affected than wild types. The transcriptional gene ontology signatures characterizing both strains of weaker animals in flight strongly corroborate previous results across species, enriched for upregulated stress response pathways and downregulated mitochondrial and cytoskeletal processes. Functional gene cluster analysis extended this to implicate decreased neuronal function, including abnormal calcium handling and acetylcholine signaling, in space-induced strength declines under the predicted control of UNC-89 and DAF-19 transcription factors. Finally, gene modules specifically altered in dys-1 animals in flight again cluster to neuronal/neuromuscular pathways, suggesting strength loss in DMD comprises a strong neuronal component that predisposes these animals to exacerbated strength loss in space. Conclusions: Highly reproducible gene signatures are strongly associated with space-induced neuromuscular strength loss across species and neuronal changes in calcium/acetylcholine signaling require further study. These results promote targeted medical efforts towards and provide an in vivo model for safely sending animals and people into deep space in the near future.

Linear Binary Classifier to Predict Bacterial Biofilm Formation on Polyacrylates.

Bacterial infections are increasingly problematic due to the rise of antimicrobial resistance. Consequently, the rational design of materials naturally resistant to biofilm formation is an important strategy for preventing medical device-associated infections. Machine learning (ML) is a powerful method to find useful patterns in complex data from a wide range of fields. Recent reports showed how ML can reveal strong relationships between bacterial adhesion and the physicochemical properties of polyacrylate libraries. These studies used robust and predictive nonlinear regression methods that had better quantitative prediction power than linear models. However, as nonlinear models' feature importance is a local rather than global property, these models were hard to interpret and provided limited insight into the molecular details of material-bacteria interactions. Here, we show that the use of interpretable mass spectral molecular ions and chemoinformatic descriptors and a linear binary classification model of attachment of three common nosocomial pathogens to a library of polyacrylates can provide improved guidance for the design of more effective pathogen-resistant coatings. Relevant features from each model were analyzed and correlated with easily interpretable chemoinformatic descriptors to derive a small set of rules that give model features tangible meaning that elucidate relationships between the structure and function. The results show that the attachment of Pseudomonas aeruginosa and Staphylococcus aureus can be robustly predicted by chemoinformatic descriptors, suggesting that the obtained models can predict the attachment response to polyacrylates to identify anti-attachment materials to synthesize and test in the future.

Routine omics collection is a golden opportunity for European human research in space and analog environments.

Widespread generation and analysis of omics data have revolutionized molecular medicine on Earth, yet its power to yield new mechanistic insights and improve occupational health during spaceflight is still to be fully realized in humans. Nevertheless, rapid technological advancements and ever-regular spaceflight programs mean that longitudinal, standardized, and cost-effective collection of human space omics data are firmly within reach. Here, we consider the practicality and scientific return of different sampling methods and omic types in the context of human spaceflight. We also appraise ethical and legal considerations pertinent to omics data derived from European astronauts and spaceflight participants (SFPs). Ultimately, we propose that a routine omics collection program in spaceflight and analog environments presents a golden opportunity. Unlocking this bright future of artificial intelligence (AI)-driven analyses and personalized medicine approaches will require further investigation into best practices, including policy design and standardization of omics data, metadata, and sampling methods.

Space Medicines for Space Health.

Scientists from around the world are studying the effects of microgravity and cosmic radiation via the "off-Earth" International Space Station (ISS) laboratory platform. The ISS has helped scientists make discoveries that go beyond the basic understanding of Earth. Over 300 medical experiments have been performed to date, with the goal of extending the knowledge gained for the benefit of humanity. This paper gives an overview of these numerous space medical findings, critically identifies challenges and gaps, and puts the achievements into perspective toward long-term space traveling and also adding benefits to our home planet. The medical contents are trifold structured, starting with the well-being of space travelers (astronaut health studies), followed by medical formulation research under space conditions, and then concluding with a blueprint for space pharmaceutical manufacturing. The review covers essential elements of our Earth-based pharmaceutical research such as drug discovery, drug and formulation stability, drug-organ interaction, drug disintegration/bioavailability/pharmacokinetics, pathogen virulence, genome mutation, and body's resistance. The information compiles clinical, medicinal, biological, and chemical research as well as fundamentals and practical applications.

Astropharmacy: Pushing the boundaries of the pharmacists' role for sustainable space exploration.

BACKGROUND: Human physiology undergoes extensive changes in space potentially leading to alterations in the way a medication functions. Understanding the efficacy behind Pharmacological Countermeasures (PCMs) and deliverable pharmacy services is imperative for the future presence of humans in space. However, while the pharmacist plays an integral role for human health terrestrially, pharmacist input has been minimal for human health in the space sector. Here, we explore the pharmacist's potential role in larger medical teams for future missions. OBJECTIVE: To explore pharmacy and space sector stakeholder perspectives regarding the pharmacist's role in the space sector. METHODS: Semi-structured interviews and focus groups were conducted with pharmacy (n = 31) and human health-related space sector stakeholders (n = 26) across the globe from governmental, commercial, industry and academic sectors. Purposive and snowball sampling were used to identify stakeholders. Interviews and focus groups were audio recorded, transcribed verbatim and thematically analysed. RESULTS: Three themes - medication management, medication-related research and medication and health information - were generated. The importance of medication optimisation within commercial and federal spaceflight participant medication regimens was cited as necessary for sustainable space exploration. Both groups advocated for pharmacists' involvement with in-situ medication manufacturing and medication-related research, particularly regarding space-based pharmacokinetic and pharmacodynamic drug profiling. Other essential roles included the pharmacist's role in providing medication information to spaceflight participants and other healthcare professionals on their health status and medication use risk in the context of space. CONCLUSIONS: With the advancement of accessible, commercial space travel and humans becoming an inter-planetary species, the opportunity to tackle PCM needs via a more extensive and comprehensive collaborative effort between the space, medical and pharmacy sectors is essential for sustainable space exploration.

Impact of cold plasma on the biomolecules and organoleptic properties of foods: A review.

Cold plasma is formed by the nonthermal ionization of gas into free electrons, ions, reactive atomic and molecular species, and ultraviolet (UV) radiation. This cold plasma can be used to alter the surface of solid and liquid foods, and it offers multiple advantages over traditional thermal treatments, such as no thermal damage and increased output variation (due to the various input parameters gas, power, plasma type, etc.). Cold plasma appears to have limited impact on the sensory and color properties, at lower power and treatment times, but there has been a statistically significant reduction in pH for most of the cold plasma treatments reviewed (p < 0.05). Carbohydrates (cross linking and glycosylation), lipids (oxidation), and proteins (secondary structure) are more significantly impacted due to cold plasma at higher intensities and longer treatment times. Although cold plasma treatments and food matrices can vary considerably, this review has identified the literary evidence of some of the influences and impacts of the vast array of cold plasma treatment parameters on the biomolecular and organoleptic properties of these foods. Due to the rapidly evolving nature of the field, we have also identified that authors prioritize the presentation of different information when publishing from different research areas. Therefore, we have proposed a number of key physical and chemical cold plasma parameters that should be considered for inclusion in all future publications in the field.

Generation and Characterization of a Library of Novel Biologically Active Functional Surfactants (Surfmers) Using Combined High-Throughput Methods.

We report the first successful combination of three distinct high-throughput techniques to deliver the accelerated design, synthesis, and property screening of a library of novel, bio-instructive, polymeric, comb-graft surfactants. These three-dimensional, surface-active materials were successfully used to control the surface properties of particles by forming a unimolecular deep layer on the surface of the particles via microfluidic processing. This strategy deliberately utilizes the surfactant to both create the stable particles and deliver a desired cell-instructive behavior. Therefore, these specifically designed, highly functional surfactants are critical to promoting a desired cell response. This library contained surfactants constructed from 20 molecularly distinct (meth)acrylic monomers, which had been pre-identified by HT screening to exhibit specific, varied, and desirable bacterial biofilm inhibitory responses. The surfactant's self-assembly properties in water were assessed by developing a novel, fully automated, HT method to determine the critical aggregation concentration. These values were used as the input data to a computational-based evaluation of the key molecular descriptors that dictated aggregation behavior. Thus, this combination of HT techniques facilitated the rapid design, generation, and evaluation of further novel, highly functional, cell-instructive surfaces by application of designed surfactants possessing complex molecular architectures.

Droplet Microfluidic Optimisation Using Micropipette Characterisation of Bio-Instructive Polymeric Surfactants.

Droplet microfluidics can produce highly tailored microparticles whilst retaining monodispersity. However, these systems often require lengthy optimisation, commonly based on a trial-and-error approach, particularly when using bio-instructive, polymeric surfactants. Here, micropipette manipulation methods were used to optimise the concentration of bespoke polymeric surfactants to produce biodegradable (poly(d,l-lactic acid) (PDLLA)) microparticles with unique, bio-instructive surface chemistries. The effect of these three-dimensional surfactants on the interfacial tension of the system was analysed. It was determined that to provide adequate stabilisation, a low level (0.1% (w/v)) of poly(vinyl acetate-co-alcohol) (PVA) was required. Optimisation of the PVA concentration was informed by micropipette manipulation. As a result, successful, monodisperse particles were produced that maintained the desired bio-instructive surface chemistry.

Immunity in Space: Prokaryote Adaptations and Immune Response in Microgravity.

Immune dysfunction has long been reported by medical professionals regarding astronauts suffering from opportunistic infections both during their time in space and a short period afterwards once back on Earth. Various species of prokaryotes onboard these space missions or cultured in a microgravity analogue exhibit increased virulence, enhanced formation of biofilms, and in some cases develop specific resistance for specific antibiotics. This poses a substantial health hazard to the astronauts confined in constant proximity to any present bacterial pathogens on long space missions with a finite number of resources including antibiotics. Furthermore, some bacteria cultured in microgravity develop phenotypes not seen in Earth gravity conditions, providing novel insights into bacterial evolution and avenues for research. Immune dysfunction caused by exposure to microgravity may increase the chance of bacterial infection. Immune cell stimulation, toll-like receptors and pathogen-associated molecular patterns can all be altered in microgravity and affect immunological crosstalk and response. Production of interleukins and other cytokines can also be altered leading to immune dysfunction when responding to bacterial infection. Stem cell differentiation and immune cell activation and proliferation can also be impaired and altered by the microgravity environment once more adding to immune dysfunction in microgravity. This review elaborates on and contextualises these findings relating to how bacteria can adapt to microgravity and how the immune system subsequently responds to infection.

Protein identification by 3D OrbiSIMS to facilitate in situ imaging and depth profiling.

Label-free protein characterization at surfaces is commonly achieved using digestion and/or matrix application prior to mass spectrometry. We report the assignment of undigested proteins at surfaces in situ using secondary ion mass spectrometry (SIMS). Ballistic fragmentation of proteins induced by a gas cluster ion beam (GCIB) leads to peptide cleavage producing fragments for subsequent OrbitrapTM analysis. In this work we annotate 16 example proteins (up to 272 kDa) by de novo peptide sequencing and illustrate the advantages of this approach by characterizing a protein monolayer biochip and the depth distribution of proteins in human skin.

Evolutionary drivers of protein shape.

Diffusional motion within the crowded environment of the cell is known to be crucial to cellular function as it drives the interactions of proteins. However, the relationships between protein diffusion, shape and interaction, and the evolutionary selection mechanisms that arise as a consequence, have not been investigated. Here, we study the dynamics of triaxial ellipsoids of equivalent steric volume to proteins at different aspect ratios and volume fractions using a combination of Brownian molecular dynamics and geometric packing. In general, proteins are found to have a shape, approximately Golden in aspect ratio, that give rise to the highest critical volume fraction resisting gelation, corresponding to the fastest long-time self-diffusion in the cell. The ellipsoidal shape also directs random collisions between proteins away from sites that would promote aggregation and loss of function to more rapidly evolving nonsticky regions on the surface, and further provides a greater tolerance to mutation.

Nanofibrous Scaffolds Support a 3D in vitro Permeability Model of the Human Intestinal Epithelium.

Advances in drug research not only depend on high throughput screening to evaluate large numbers of lead compounds but also on the development of in vitro models which can simulate human tissues in terms of drug permeability and functions. Potential failures, such as poor permeability or interaction with efflux drug transporters, can be identified in epithelial Caco-2 monolayer models and can impact a drug candidate's progression onto the next stages of the drug development process. Whilst monolayer models demonstrate reasonably good prediction of in vivo permeability for some compounds, more developed in vitro tools are needed to assess new entities that enable closer in vivo in vitro correlation. In this study, an in vitro model of the human intestinal epithelium was developed by utilizing nanofibers, fabricated using electrospinning, to mimic the structure of the basement membrane. We assessed Caco-2 cell response to these materials and investigated the physiological properties of these cells cultured on the fibrous supports, focusing on barrier integrity and drug-permeability properties. The obtained data illustrate that 2D Caco-2 Transwell® cultures exhibit artificially high trans-epithelial electrical resistance (TEER) compared to cells cultured on the 3D nanofibrous scaffolds which show TEER values similar to ex vivo porcine tissue (also measured in this study). Furthermore, our results demonstrate that the 3D nanofibrous scaffolds influence the barrier integrity of the Caco-2 monolayer to confer drug-absorption properties that more closely mimic native gut tissue particularly for studying passive epithelial transport. We propose that this 3D model is a suitable in vitro model for investigating drug absorption and intestinal metabolism.

CRISPR/Cas9 editing in human pluripotent stem cell-cardiomyocytes highlights arrhythmias, hypocontractility, and energy depletion as potential therapeutic targets for hypertrophic cardiomyopathy.

Aims: Sarcomeric gene mutations frequently underlie hypertrophic cardiomyopathy (HCM), a prevalent and complex condition leading to left ventricle thickening and heart dysfunction. We evaluated isogenic genome-edited human pluripotent stem cell-cardiomyocytes (hPSC-CM) for their validity to model, and add clarity to, HCM. Methods and results: CRISPR/Cas9 editing produced 11 variants of the HCM-causing mutation c.C9123T-MYH7 [(p.R453C-ß-myosin heavy chain (MHC)] in 3 independent hPSC lines. Isogenic sets were differentiated to hPSC-CMs for high-throughput, non-subjective molecular and functional assessment using 12 approaches in 2D monolayers and/or 3D engineered heart tissues. Although immature, edited hPSC-CMs exhibited the main hallmarks of HCM (hypertrophy, multi-nucleation, hypertrophic marker expression, sarcomeric disarray). Functional evaluation supported the energy depletion model due to higher metabolic respiration activity, accompanied by abnormalities in calcium handling, arrhythmias, and contraction force. Partial phenotypic rescue was achieved with ranolazine but not omecamtiv mecarbil, while RNAseq highlighted potentially novel molecular targets. Conclusion: Our holistic and comprehensive approach showed that energy depletion affected core cardiomyocyte functionality. The engineered R453C-ßMHC-mutation triggered compensatory responses in hPSC-CMs, causing increased ATP production and αMHC to energy-efficient ßMHC switching. We showed that pharmacological rescue of arrhythmias was possible, while MHY7: MYH6 and mutant: wild-type MYH7 ratios may be diagnostic, and previously undescribed lncRNAs and gene modifiers are suggestive of new mechanisms.

A novel DFP tripeptide motif interacts with the coagulation factor XI apple 2 domain.

Factor XI (FXI) is the zymogen of FXIa, which cleaves FIX in the intrinsic pathway of coagulation. FXI is known to exist as a dimer and interacts with multiple proteins via its 4 apple domains in the "saucer section" of the enzyme; however, to date, no complex crystal structure has been described. To investigate protein interactions of FXI, a large random peptide library consisting of 10(6) to 10(7) peptides was screened for FXI binding, which identified a series of FXI binding motifs containing the signature Asp-Phe-Pro (DFP) tripeptide. Motifs containing this core tripeptide were found in diverse proteins, including the known ligand high-molecular-weight kininogen (HK), as well as the extracellular matrix proteins laminin and collagen V. To define the binding site on FXI, we determined the crystal structure of FXI in complex with the HK-derived peptide NPISDFPDT. This revealed the location of the DFP peptide bound to the FXI apple 2 domain, and central to the interaction, the DFP phenylalanine side-chain inserts into a major hydrophobic pocket in the apple 2 domain and the isoleucine occupies a flanking minor pocket. Two further structures of FXI in complex with the laminin-derived peptide EFPDFP and a DFP peptide from the random screen demonstrated binding in the same pocket, although in a slightly different conformation, thus revealing some flexibility in the molecular interactions of the FXI apple 2 domain.

Monitoring the Dissolution Mechanisms of Amorphous Bicalutamide Solid Dispersions via Real-Time Raman Mapping.

Real-time in situ Raman mapping has been employed to monitor, during dissolution, the crystallization transitions of amorphous bicalutamide formulated as a molecular dispersion in a copovidone VA64 matrix. The dissolution performance was also investigated using the rotating disc dissolution rate methodology, which allows simultaneous determination of the dissolution rate of both active ingredient and polymer. The dissolution behavior of two bicalutamide:copovidone VA64 dispersion formulations, containing 5% (w/w) and 50% (w/w) bicalutamide, respectively, was investigated, with the aim of exploring the effect of increasing the bicalutamide loading on the dissolution performance. Spatially time-resolved Raman maps generated using multivariate curve resolution indicated the simultaneous transformation of amorphous bicalutamide present in the 50% drug-loaded extrudate into metastable polymorphic form II and low-energy polymorphic form I. Fitting a kinetic model and spatially correlating the data extracted from the Raman maps also allowed us to understand the re-crystallization mechanisms by which the low-energy form I appears. Form I was shown to crystallize mainly directly from the amorphous solid dispersion, with crystallization from the metastable form II being a minor contribution.

Multivariate ToF-SIMS image analysis of polymer microarrays and protein adsorption.

The complexity of hyperspectral time of flight secondary ion mass spectrometry (ToF-SIMS) datasets makes their subsequent analysis and interpretation challenging, and is often an impasse to the identification of trends and differences within large sample-sets. The application of multivariate data analysis has become a routine method to successfully deconvolute and analyze objectively these datasets. The advent of high-resolution large area ToF-SIMS imaging capability has enlarged further the data handling challenges. In this work, a modified multivariate curve resolution image analysis of a polymer microarray containing 70 different poly(meth)acrylate type spots (over a 9.2 × 9.2 mm area) is presented. This analysis distinguished key differences within the polymer library such as the differentiation between acrylate and methacrylate polymers and variance specific to side groups. Partial least squares (PLS) regression analysis was performed to identify correlations between the ToF-SIMS surface chemistry and the protein adsorption. PLS analysis identified a number of chemical moieties correlating with high or low protein adsorption, including ions derived from the polymer backbone and polyethylene glycol side-groups. The retrospective validation of the findings from the PLS analysis was also performed using the secondary ion images for those ions found to significantly contribute to high or low protein adsorption.

Bacterial attachment to polymeric materials correlates with molecular flexibility and hydrophilicity.

A new class of material resistant to bacterial attachment has been discovered that is formed from polyacrylates with hydrocarbon pendant groups. In this study, the relationship between the nature of the hydrocarbon moiety and resistance to bacteria is explored, comparing cyclic, aromatic, and linear chemical groups. A correlation is shown between bacterial attachment and a parameter derived from the partition coefficient and the number of rotatable bonds of the materials' pendant groups. This correlation is applicable to 86% of the hydrocarbon pendant moieties surveyed, quantitatively supporting the previous qualitative observation that bacteria are repelled from poly(meth)acrylates containing a hydrophilic ester group when the pendant group is both rigid and hydrophobic. This insight will help inform and predict the further development of polymers resistant to bacterial attachment.

Strategies for MCR image analysis of large hyperspectral data-sets.

Polymer microarrays are a key enabling technology for high throughput materials discovery. In this study, multivariate image analysis, specifically multivariate curve resolution (MCR), is applied to the hyperspectral time of flight secondary ion mass spectroscopy (ToF-SIMS) data from eight individual microarray spots. Rather than analysing the data individually, the data-sets are collated and analysed as a single large data-set. Desktop computing is not a practical method for undertaking MCR analysis of such large data-sets due to the constraints of memory and computational overhead. Here, a distributed memory High-Performance Computing facility (HPC) is used. Similar to what is achieved using MCR analysis of individual samples, the results from this consolidated data-set allow clear identification of the substrate material; furthermore, specific chemistries common to different spots are also identified. The application of the HPC facility to the MCR analysis of ToF-SIMS hyperspectral data-sets demonstrates a potential methodology for the analysis of macro-scale data without compromising spatial resolution (data 'binning'). Copyright © 2012 John Wiley & Sons, Ltd.

Protein unfolding under force: crack propagation in a network.

The mechanical unfolding of a set of 12 proteins with diverse topologies is investigated using an all-atom constraint-based model. Proteins are represented as polypeptides cross-linked by hydrogen bonds, salt bridges, and hydrophobic contacts, each modeled as a harmonic inequality constraint capable of supporting a finite load before breaking. Stereochemically acceptable unfolding pathways are generated by minimally overloading the network in an iterative fashion, analogous to crack propagation in solids. By comparing the pathways to those from molecular dynamics simulations and intermediates identified from experiment, it is demonstrated that the dominant unfolding pathways for 9 of the 12 proteins studied are well described by crack propagation in a network.