Merging Modular Molecular Design with High Throughput Screening of Cell Adhesion on Antimicrobial Supramolecular Biomaterials.

A polymer microarray based on the supramolecular ureido-pyrimidinone (UPy) moiety is fabricated to screen antimicrobial materials for their ability to support cell adhesion. UPy-functionalized additives, either cell-adhesive, antimicrobial or control peptides, are used, and investigated in different combinations at different concentrations, resulting in a library of 194 spots. These are characterized on composition and morphology to evaluate the microarray fabrication. Normal human dermal fibroblasts are cultured on the microarrays and cell adhesion to the spots is systematically analyzed. Results demonstrate enhanced cell adhesion on spots with combinations including the antimicrobial peptides. This study clearly proves the power of the high throughput approach in combination with supramolecular molecules, to screen additive libraries for desired biological response.

Spatially Resolved Molecular Analysis of Host Response to Medical Device Implantation Using the 3D OrbiSIMS Highlights a Critical Role for Lipids.

A key goal for implanted medical devices is that they do not elicit a detrimental immune response. Macrophages play critical roles in the modulation of the host immune response and are the cells responsible for persistent inflammatory reactions to implanted biomaterials. Two novel immune-instructive polymers that stimulate pro- or anti-inflammatory responses from macrophages in vitro are investigated. These also modulate in vivo foreign body responses (FBR) when implanted subcutaneously in mice. Immunofluorescent staining of tissue abutting the polymer reveals responses consistent with pro- or anti-inflammatory responses previously described for these polymers. Three Dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) analysis to spatially characterize the metabolites in the tissue surrounding the implant, providing molecular histology insight into the metabolite response in the host is applied. For the pro-inflammatory polymer, monoacylglycerols (MG) and diacylglycerols (DG) are observed at increased intensity, while for the anti-inflammatory coating, the number of phospholipid species detected decreased, and pyridine and pyrimidine levels are elevated. Small molecule signatures from single-cell studies of M2 macrophages in vitro correlate with the in vivo observations, suggesting potential for prediction. Metabolite characterization by the 3D OrbiSIMS is shown to provide insight into the mechanism of bio-instructive materials as medical devices and to inform on the FBR to biomaterials.

Unlocking Bio-Instructive Polymers: A Novel Multi-Well Screening Platform Based on Secretome Sampling.

Biomaterials are designed to interact with biological systems to replace, support, enhance, or monitor their function. However, there are challenges associated with traditional biomaterials' development due to the lack of underlying theory governing cell response to materials' chemistry. This leads to the time-consuming process of testing different materials plus the adverse reactions in the body such as cytotoxicity and foreign body response. High-throughput screening (HTS) offers a solution to these challenges by enabling rapid and simultaneous testing of a large number of materials to determine their bio-interactions and biocompatibility. Secreted proteins regulate many physiological functions and determine the success of implanted biomaterials through directing cell behaviour. However, the majority of biomaterials' HTS platforms are suitable for microscopic analyses of cell behaviour and not for investigating non-adherent cells or measuring cell secretions. Here, we describe a multi-well platform adaptable to robotic printing of polymers and suitable for secretome profiling of both adherent and non-adherent cells. We detail the platform's development steps, encompassing the preparation of individual cell culture chambers, polymer printing, and the culture environment, as well as examples to demonstrate surface chemical characterisation and biological assessments of secreted mediators. Such platforms will no doubt facilitate the discovery of novel biomaterials and broaden their scope by adapting wider arrays of cell types and incorporating assessments of both secretome and cell-bound interactions. Key features • Detailed protocols for preparation of substrate for contact printing of acrylate-based polymers including O2 plasma etching, functionalisation process, and Poly(2-hydroxyethyl methacrylate) (pHEMA) dip coating. • Preparations of 7 mm × 7 mm polymers employing pin printing system. • Provision of confined area for each polymer using ProPlate® multi-well chambers. • Compatibility of this platform was validated using adherent cells [primary human monocyte-derived macrophages (MDMs)) and non-adherent cells (primary human monocyte-derived dendritic cells (moDCs)]. • Examples of the adaptability of the platform for secretome analysis including five different cytokines using enzyme-linked immunosorbent assay (ELISA, DuoSet®). Graphical overview.

Toward Comprehensive Analysis of the 3D Chemistry of Pseudomonas aeruginosa Biofilms.

Bacterial biofilms are structured communities consisting of cells enmeshed in a self-generated extracellular matrix usually attached to a surface. They contain diverse classes of molecules including polysaccharides, lipids, proteins, nucleic acids, and diverse small organic molecules (primary and secondary metabolites) which are organized to optimize survival and facilitate dispersal to new colonization sites. In situ characterization of the chemical composition and structure of bacterial biofilms is necessary to fully understand their development on surfaces relevant to biofouling in health, industry, and the environment. Biofilm development has been extensively studied using confocal microscopy using targeted fluorescent labels providing important insights into the architecture of biofilms. Recently, cryopreparation has been used to undertake targeted in situ chemical characterization using Orbitrap secondary ion mass spectrometry (OrbiSIMS), providing a label-free method for imaging biofilms in their native state. Although the high mass resolution of OrbiSIMS enables more confident peak assignments, it is still very challenging to assign most of the peaks in the spectra due to complexity of SIMS spectra and lack of automatic peak assignment methods. Here, we analyze the same OrbiSIMS depth profile data generated from the frozen-hydrated biofilm, but employ a new untargeted chemical filtering process utilizing mass spectral databases to assign secondary ions to decipher the large number of fragments present in the SIMS spectra. To move towards comprehensive analysis of different chemistries in the sample, we apply a molecular formula prediction approach which putatively assigns 81% of peaks in the 3D OrbiSIMS depth profile analysis. This enables us to catalog over 1000 lipids and their fragments, 3500 protein fragments, 71 quorum sensing-related molecules (2-alkyl-4-quinolones and N-acylhomoserine lactones), 150 polysaccharide fragments, and glycolipids simultaneously from one data set and map these separated molecular classes spatially through a Pseudomonas aeruginosa biofilm. Assignment of different chemistries in this sample facilitates identification of differences between biofilms grown on biofilm-promoting and biofilm-resistant polymers.

A potential alternative to fungicides using actives-free (meth)acrylate polymers for protection of wheat crops from fungal attachment and infection.

Fungicidal compounds are actives widely used for crop protection from fungal infection, but they can also kill beneficial organisms, enter the food chain and promote resistant pathogen strains from overuse. Here we report the first field crop trial of homopolymer materials that prevent fungal attachment, showing successful crop protection via an actives-free approach. In the trial, formulations containing two candidate polymers were applied to young wheat plants that were subject to natural infection with the wheat pathogen Zymoseptoria tritici. A formulation containing one of the candidate polymers, poly(di(ethylene glycol) ethyl ether acrylate) (abbreviated DEGEEA), produced a significant reduction (26%) in infection of the crop by Z. tritici, delivering protection against fungal infection that compared favourably with three different commercially established fungicide programmes tested in parallel. Furthermore, the sprayed polymers did not negatively affect wheat growth. The two lead polymer candidates were initially identified by bio-performance testing using in vitro microplate- and leaf-based assays and were taken forward successfully into a programme to optimize and scale-up their synthesis and compound them into a spray formulation. Therefore, the positive field trial outcome has also established the validity of the smaller-scale, laboratory-based bioassay data and scale-up methodologies used. Because fungal attachment to plant surfaces is a first step in many crop infections, this non-eluting polymer: (i) now offers significant potential to deliver protection against fungal attack, while (ii) addressing the fourth and aligning with the eleventh principles of green chemistry by using chemical products designed to preserve efficacy of function while reducing toxicity. A future focus should be to develop the material properties for this and other applications including other fungal pathogens.

High-Throughput Screening of Thiol-ene Click Chemistries for Bone Adhesive Polymers.

Metal surgical pins and screws are employed in millions of orthopedic surgical procedures every year worldwide, but their usability is limited in the case of complex, comminuted fractures or in surgeries on smaller bones. Therefore, replacing such implants with a bone adhesive material has long been considered an attractive option. However, synthesizing a biocompatible bone adhesive with a high bond strength that is simple to apply presents many challenges. To rapidly identify candidate polymers for a biocompatible bone adhesive, we employed a high-throughput screening strategy to assess human mesenchymal stromal cell (hMSC) adhesion toward a library of polymers synthesized via thiol-ene click chemistry. We chose thiol-ene click chemistry because multifunctional monomers can be rapidly cured via ultraviolet (UV) light while minimizing residual monomer, and it provides a scalable manufacturing process for candidate polymers identified from a high-throughput screen. This screening methodology identified a copolymer (1-S2-FT01) composed of the monomers 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione (TATATO) and pentaerythritol tetrakis (3-mercaptopropionate) (PETMP), which supported highest hMSC adhesion across a library of 90 polymers. The identified copolymer (1-S2-FT01) exhibited favorable compressive and tensile properties compared to existing commercial bone adhesives and adhered to bone with adhesion strengths similar to commercially available bone glues such as Histoacryl. Furthermore, this cytocompatible polymer supported osteogenic differentiation of hMSCs and could adhere 3D porous polymer scaffolds to the bone tissue, making this polymer an ideal candidate as an alternative bone adhesive with broad utility in orthopedic surgery.

Label-free Chemical Characterization of Polarized Immune Cells in vitro and Host Response to Implanted Bio-instructive Polymers in vivo Using 3D OrbiSIMS.

The Three-dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) is a secondary ion mass spectrometry instrument, a combination of a Time of Flight (ToF) instrument with an Orbitrap analyzer. The 3D OrbiSIMS technique is a powerful tool for metabolic profiling in biological samples. This can be achieved at subcellular spatial resolution, high sensitivity, and high mass-resolving power coupled with MS/MS analysis. Characterizing the metabolic signature of macrophage subsets within tissue sections offers great potential to understand the response of the human immune system to implanted biomaterials. Here, we describe a protocol for direct analysis of individual cells after in vitro differentiation of naïve monocytes into M1 and M2 phenotypes using cytokines. As a first step in vivo, we investigate explanted silicon catheter sections as a medical device in a rodent model of foreign body response. Protocols are presented to allow the host response to different immune instructive materials to be compared. The first demonstration of this capability illustrates the great potential of direct cell and tissue section analysis for in situ metabolite profiling to probe functional phenotypes using molecular signatures. Details of the in vitro cell approach, materials, sample preparation, and explant handling are presented, in addition to the data acquisition approaches and the data analysis pipelines required to achieve useful interpretation of these complex spectra. This method is useful for in situ characterization of both in vitro single cells and ex vivo tissue sections. This will aid the understanding of the immune response to medical implants by informing the design of immune-instructive biomaterials with positive interactions. It can also be used to investigate a broad range of other clinically relevant therapeutics and immune dysregulations. Graphical overview.

Identification of Pseudomonas aeruginosa exopolysaccharide Psl in biofilms using 3D OrbiSIMS.

Secondary ion mass spectrometry (SIMS) offers advantages over both liquid extraction mass spectrometry and matrix assisted laser desorption mass spectrometry in that it provides the direct in situ analysis of molecules and has the potential to preserve the 3D location of an analyte in a sample. Polysaccharides are recognized as challenging analytes in the mass spectrometry of liquids and are also difficult to identify and assign using SIMS. Psl is an exopolysaccharide produced by Pseudomonas aeruginosa, which plays a key role in biofilm formation and maturation. In this Letter, we describe the use of the OrbiTrap analyzer with SIMS (3D OrbiSIMS) for the label-free mass spectrometry of Psl, taking advantage of its high mass resolving power for accurate secondary ion assignment. We study a P. aeruginosa biofilm and compare it with purified Psl to enable the assignment of secondary ions specific to the Psl structure. This resulted in the identification of 17 peaks that could confidently be ascribed to Psl fragments within the biofilm matrix. The complementary approach of the following neutral loss sequences is also shown to identify multiple oligosaccharide fragments without the requirement of a biological reference sample.

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.

Identifying new biomarkers of aggressive Group 3 and SHH medulloblastoma using 3D hydrogel models, single cell RNA sequencing and 3D OrbiSIMS imaging.

The most common malignant brain tumour in children, medulloblastoma (MB), is subdivided into four clinically relevant molecular subgroups, although targeted therapy options informed by understanding of different cellular features are lacking. Here, by comparing the most aggressive subgroup (Group 3) with the intermediate (SHH) subgroup, we identify crucial differences in tumour heterogeneity, including unique metabolism-driven subpopulations in Group 3 and matrix-producing subpopulations in SHH. To analyse tumour heterogeneity, we profiled individual tumour nodules at the cellular level in 3D MB hydrogel models, which recapitulate subgroup specific phenotypes, by single cell RNA sequencing (scRNAseq) and 3D OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) imaging. In addition to identifying known metabolites characteristic of MB, we observed intra- and internodular heterogeneity and identified subgroup-specific tumour subpopulations. We showed that extracellular matrix factors and adhesion pathways defined unique SHH subpopulations, and made up a distinct shell-like structure of sulphur-containing species, comprising a combination of small leucine-rich proteoglycans (SLRPs) including the collagen organiser lumican. In contrast, the Group 3 tumour model was characterized by multiple subpopulations with greatly enhanced oxidative phosphorylation and tricarboxylic acid (TCA) cycle activity. Extensive TCA cycle metabolite measurements revealed very high levels of succinate and fumarate with malate levels almost undetectable particularly in Group 3 tumour models. In patients, high fumarate levels (NMR spectroscopy) alongside activated stress response pathways and high Nuclear Factor Erythroid 2-Related Factor 2 (NRF2; gene expression analyses) were associated with poorer survival. Based on these findings we predicted and confirmed that NRF2 inhibition increased sensitivity to vincristine in a long-term 3D drug treatment assay of Group 3 MB. Thus, by combining scRNAseq and 3D OrbiSIMS in a relevant model system we were able to define MB subgroup heterogeneity at the single cell level and elucidate new druggable biomarkers for aggressive Group 3 and low-risk SHH MB.

Predictive Molecular Design and Structure-Property Validation of Novel Terpene-Based, Sustainably Sourced Bacterial Biofilm-Resistant Materials.

Presented in this work is the use of a molecular descriptor, termed the α parameter, to aid in the design of a series of novel, terpene-based, and sustainable polymers that were resistant to biofilm formation by the model bacterial pathogen Pseudomonas aeruginosa. To achieve this, the potential of a range of recently reported, terpene-derived monomers to deliver biofilm resistance when polymerized was both predicted and ranked by the application of the α parameter to key features in their molecular structures. These monomers were derived from commercially available terpenes (i.e., α-pinene, ß-pinene, and carvone), and the prediction of the biofilm resistance properties of the resultant novel (meth)acrylate polymers was confirmed using a combination of high-throughput polymerization screening (in a microarray format) and in vitro testing. Furthermore, monomers, which both exhibited the highest predicted biofilm anti-biofilm behavior and required less than two synthetic stages to be generated, were scaled-up and successfully printed using an inkjet "valve-based" 3D printer. Also, these materials were used to produce polymeric surfactants that were successfully used in microfluidic processing to create microparticles that possessed bio-instructive surfaces. As part of the up-scaling process, a novel rearrangement was observed in a proposed single-step synthesis of α-terpinyl methacrylate via methacryloxylation, which resulted in isolation of an isobornyl-bornyl methacrylate monomer mixture, and the resultant copolymer was also shown to be bacterial attachment-resistant. As there has been great interest in the current literature upon the adoption of these novel terpene-based polymers as green replacements for petrochemical-derived plastics, these observations have significant potential to produce new bio-resistant coatings, packaging materials, fibers, medical devices, etc.

Discovery of a polymer resistant to bacterial biofilm, swarming, and encrustation.

Innovative approaches to prevent catheter-associated urinary tract infections (CAUTIs) are urgently required. Here, we describe the discovery of an acrylate copolymer capable of resisting single- and multispecies bacterial biofilm formation, swarming, encrustation, and host protein deposition, which are major challenges associated with preventing CAUTIs. After screening ~400 acrylate polymers, poly(tert-butyl cyclohexyl acrylate) was selected for its biofilm- and encrustation-resistant properties. When combined with the swarming inhibitory poly(2-hydroxy-3-phenoxypropyl acrylate), the copolymer retained the bioinstructive properties of the respective homopolymers when challenged with Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. Urinary tract catheterization causes the release of host proteins that are exploited by pathogens to colonize catheters. After preconditioning the copolymer with urine collected from patients before and after catheterization, reduced host fibrinogen deposition was observed, and resistance to diverse uropathogens was maintained. These data highlight the potential of the copolymer as a urinary catheter coating for preventing CAUTIs.

Optimization of diffuse Raman spectroscopy for in-vivo quantification of foreign body response in a small animal model.

Diffuse Raman spectroscopy (DRS) allows subsurface molecular analysis of optically turbid samples. Numerical modeling of light propagation was used as a method for improving the design of an DRS instrument to maximize the signal to noise ratio (SNR) while ensuring safe laser exposure parameters required for in-vivo measurements. Experimental validation of the model was performed on both phantom samples and disks implanted postmortem to mimic the typical response to foreign bodies (formation of a fibrotic capsule around an implant). A reduction of laser exposure of over 1500-fold was achieved over previous studies whilst maintaining the same Raman collection rates and reaching the safe power density of 3 mW/mm2. The validation of this approach in a subcutaneous implant in a mouse cadaver showed a further improvement of 1.5-fold SNR, with a thickness limit of detection for the fibrotic layer of 23 µm, under the same acquisition times. In the animal body, a thickness limit of detection of 16 µm was achieved. These results demonstrate the feasibility of numerical model-based optimization for DRS, and that the technique can be improved sufficiently to be used for in-vivo measurement of collagenous capsule formation as a result of the foreign body response in murine models.

Microparticles Decorated with Cell-Instructive Surface Chemistries Actively Promote Wound Healing.

Wound healing is a complex biological process involving close crosstalk between various cell types. Dysregulation in any of these processes, such as in diabetic wounds, results in chronic nonhealing wounds. Fibroblasts are a critical cell type involved in the formation of granulation tissue, essential for effective wound healing. 315 different polymer surfaces are screened to identify candidates which actively drive fibroblasts toward either pro- or antiproliferative functional phenotypes. Fibroblast-instructive chemistries are identified, which are synthesized into surfactants to fabricate easy to administer microparticles for direct application to diabetic wounds. The pro-proliferative microfluidic derived particles are able to successfully promote neovascularization, granulation tissue formation, and wound closure after a single application to the wound bed. These active novel bio-instructive microparticles show great potential as a route to reducing the burden of chronic wounds.

Evaluation of the relative potential for contact and doffing transmission of SARS-CoV-2 by a range of personal protective equipment materials.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-has caused a global public health emergency. Personal protective equipment (PPE) is the primary defence against viral exposure in healthcare and community settings. However, the surfaces of PPE materials may trap virus for contact transmission or through laden aerosols generated during removal of PPE, through cleaning or during movement. In this study, the relative efficacy of current PPE materials in terms of virion adsorption to materials and their antiviral potency, has been evaluated on a wide range of PPE for the first time, including four polymer glove types, two types of scrubs, apron material, a mask, visor and a selection of other commercial polymers and products. Although differences in virion adsorption to the test materials were observed, none of the existing polymer-based PPE resulted in more than tenfold reduction in the SARS-CoV-2 titre within either 10 min or 30 min contact period. The wettability and surface chemistry of the test materials were analysed to investigate any correlations with their surface physicochemical properties. While no correlation was found between wettability and viral retention under air flow challenge, one secondary ion of m/z 101.03 (+) and three secondary ions of m/z 31.98 (-), 196.93 (-) and 394.33 (+) in ToF-SIMS data of the test materials showed positive and negative correlations with the viral retention, respectively, which was identified by PLS regression model, suggesting that the surface chemistry plays a role in determining the extent of virion adsorption. Our findings outline the material aspects that influence the efficacy of current PPE against SARS-CoV-2 transmission and give suggestions on the development of novel simple polymer-based PPE for better infection protection.

Time resolved growth of (N)-polycyclic aromatic hydrocarbons in engine deposits uncovered with OrbiSIMS depth profiling.

Carbonaceous deposits are ubiquitous, being formed on surfaces in engines, fuel systems and on catalysts operating at high temperatures for hydrocarbon transformations. In internal combustion engines, their formation negatively affects worldwide vehicle emissions and fuel economy, leading to premature deaths and environmental damage. Deposit composition and formation pathways are poorly understood due to their insolubility and the intrinsic complexity of their layered carbonaceous matrix. Here, we apply the in situ high mass resolving power capabilities of 3D Orbitrap secondary ion mass spectrometry (3D OrbiSIMS) argon cluster depth profiling on 16 lab grown deposits and evidence common molecular distributions in deposit depth and in positions relative to the combustion chamber. We observe the products of the growth of both planar and curved polycyclic aromatic hydrocarbons to form small fullerenes over time in the engine and propose possible formation pathways which explain the molecular distributions observed. These include alkyl scission, cyclisation of aliphatic side chains and hydrogen abstraction C2H2 addition to form larger aromatic structures. We apply this pathway to previously unidentified nitrogen containing structures in deposits including quinolines and carbazoles. For the first time, 3D OrbiSIMS results were compared and validated with data from atmospheric pressure matrix assisted laser desorption ionization MS. The comprehensive characterization provided will help the development of a new generation of chemical additives to reduce deposits, and thus improve vehicle emissions and global air quality.

Single-Cell Metabolic Profiling of Macrophages Using 3D OrbiSIMS: Correlations with Phenotype.

Macrophages are important immune cells that respond to environmental cues acquiring a range of activation statuses represented by pro-inflammatory (M1) and anti-inflammatory (M2) phenotypes at each end of their spectrum. Characterizing the metabolic signature (metabolic profiling) of different macrophage subsets is a powerful tool to understand the response of the human immune system to different stimuli. Here, the recently developed 3D OrbiSIMS instrument is applied to yield useful insight into the metabolome from individual cells after in vitro differentiation of macrophages into naïve, M1, and M2 phenotypes using different cytokines. This analysis strategy not only requires more than 6 orders of magnitude less sample than traditional mass spectrometry approaches but also allows the study of cell-to-cell variance. Characteristic metabolites in macrophage subsets are identified using a targeted lipid and data-driven multivariate approach highlighting amino acids and other small molecules. The diamino acids alanylasparagine and lipid sphingomyelin SM(d18/16:0) are uniquely found in M1 macrophages, while pyridine and pyrimidine are observed at increased intensity in M2 macrophages, findings which link to known biological pathways. The first demonstration of this capability illustrates the great potential of direct cell analysis for in situ metabolite profiling with the 3D OrbiSIMS to probe functional phenotype at the single-cell level using molecular signatures and to understand the response of the human body to implanted devices and immune diseases.

Developing Novel Biointerfaces: Using Chlorhexidine Surface Attachment as a Method for Creating Anti-Fungal Surfaces.

There is an increasing focus in healthcare environments on combatting antimicrobial resistant infections. While bacterial infections are well reported, infections caused by fungi receive less attention, yet have a broad impact on society and can be deadly. Fungi are eukaryotes with considerable shared biology with humans, therefore limited technologies exist to combat fungal infections and hospital infrastructure is rarely designed for reducing microbial load. In this study, a novel antimicrobial surface (AMS) that is modified with the broad-spectrum biocide chlorhexidine is reported. The surfaces are shown to kill the opportunistic fungal pathogens Candida albicans and Cryptococcus neoformans very rapidly (<15 min) and are significantly more effective than current technologies available on the commercial market, such as silver and copper.

Elucidating the molecular landscape of the stratum corneum.

SignificanceSkin is recognized as an intricate assembly of molecular components, which facilitate cell signaling, metabolism, and protein synthesis mechanisms in order to offer protection, regulation, and sensation to the body. Our study takes significant steps to characterize in more detail the complex chemistry of the skin, in particular by generating a better understanding of the uppermost layer, the stratum corneum. Using a state-of-the-art 3D OrbiSIMS technique, we were able to observe the depth distribution, in situ, for a wide range of molecular species. This unprecedented molecular characterization of skin provides information that has the potential to benefit research into fundamental processes, such as those associated with skin aging and disease, and the development and delivery of effective topical formulations.

Molecular Formula Prediction for Chemical Filtering of 3D OrbiSIMS Datasets.

Modern mass spectrometry techniques produce a wealth of spectral data, and although this is an advantage in terms of the richness of the information available, the volume and complexity of data can prevent a thorough interpretation to reach useful conclusions. Application of molecular formula prediction (MFP) to produce annotated lists of ions that have been filtered by their elemental composition and considering structural double bond equivalence are widely used on high resolving power mass spectrometry datasets. However, this has not been applied to secondary ion mass spectrometry data. Here, we apply this data interpretation approach to 3D OrbiSIMS datasets, testing it for a series of increasingly complex samples. In an organic on inorganic sample, we successfully annotated the organic contaminant overlayer separately from the substrate. In a more challenging purely organic human serum sample we filtered out both proteins and lipids based on elemental compositions, 226 different lipids were identified and validated using existing databases, and we assigned amino acid sequences of abundant serum proteins including albumin, fibronectin, and transferrin. Finally, we tested the approach on depth profile data from layered carbonaceous engine deposits and annotated previously unidentified lubricating oil species. Application of an unsupervised machine learning method on filtered ions after performing MFP from this sample uniquely separated depth profiles of species, which were not observed when performing the method on the entire dataset. Overall, the chemical filtering approach using MFP has great potential in enabling full interpretation of complex 3D OrbiSIMS datasets from a plethora of material types.