A Label-Free Approach for Relative Spatial Quantitation of c-di-GMP in Microbial Biofilms.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three-dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable toward understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest from a single sample.

Seeing is Believing: Developing Multimodal Metabolic Insights at the Molecular Level.

This outlook explores how two different molecular imaging approaches might be combined to gain insight into dynamic, subcellular metabolic processes. Specifically, we discuss how matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and stimulated Raman scattering (SRS) microscopy, which have significantly pushed the boundaries of imaging metabolic and metabolomic analyses in their own right, could be combined to create comprehensive molecular images. We first briefly summarize the recent advances for each technique. We then explore how one might overcome the inherent limitations of each individual method, by envisioning orthogonal and interchangeable workflows. Additionally, we delve into the potential benefits of adopting a complementary approach that combines both MSI and SRS spectro-microscopy for informing on specific chemical structures through functional-group-specific targets. Ultimately, by integrating the strengths of both imaging modalities, researchers can achieve a more comprehensive understanding of biological and chemical systems, enabling precise metabolic investigations. This synergistic approach holds substantial promise to expand our toolkit for studying metabolites in complex environments.

Revalorization of Yerba Mate Residues: Biopolymers-Based Films of Dual Wettability as Potential Mulching Materials.

Biodegradable mulching films are a very attractive solution to agronomical practices intended to achieve more successful crop results. And, in this context, the employment of agricultural and industrial food residues as starting material for their production is an alternative with economic and environmental advantages. This work reports the preparation of bilayer films having two different wettability characteristics from three bio-derived biopolymers: TEMPO-oxidized cellulose nanofibers isolated from infused Yerba Mate residues, Chitosan and Polylactic acid. The infused Yerba Mate residues, the isolated and oxidized cellulose nanofibers, and the films were characterized. Nanofibrillation yield, optical transmittance, cationic demand, carboxyl content, intrinsic viscosity, degree of polymerization, specific surface area and length were studied for the (ligno)cellulose nanofibers. Textural and chemical analysis, thermal and mechanical properties studies, as well as water and light interactions were included in the characterization of the films. The bilayer films are promising materials to be used as mulching films.

Norepinephrine induces anoikis resistance in high-grade serous ovarian cancer precursor cells.

High-grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to stess-induced sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines were able to survive in ultra-low-attachment (ULA) culture in a ß-adrenergic receptor-dependent (ß-AR-dependent) manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE were abrogated using the ß-AR blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony-stimulating factor 2. These findings provide mechanistic insight and identify targets that may be regulated by ovary-derived NE in early HGSC.

Unveiling a CAAX Protease-Like Protein Involved in Didemnin Drug Maturation and Secretion.

The assembly line biosynthesis of the powerful anticancer-antiviral didemnin cyclic peptides is proposed to follow a prodrug release mechanism in Tristella bacteria. This strategy commences with the formation of N-terminal prodrug scaffolds and culminates in their cleavage during the cellular export of the mature products. In this study, a comprehensive exploration of the genetic and biochemical aspects of the enzymes responsible for both the assembly and cleavage of the acylated peptide prodrug scaffolds is provided. This process involves the assembly of N-acyl-polyglutamine moieties orchestrated by the nonribosomal peptide synthetase DidA and the cleavage of these components at the post-assembly stage by DidK, a transmembrane CAAX hydrolase homolog. The findings not only shed light on the complex prodrug mechanism that underlies the synthesis and secretion of didemnin compounds but also offer novel insights into the expanded role of CAAX hydrolases in microbes. Furthermore, this knowledge can be leveraged for the strategic design of genome mining approaches aimed at discovering new bioactive natural products that employ similar prodrug biochemical strategies.

Reducing Mass Confusion over the Microbiome.

As genetic tools continue to emerge and mature, more information is revealed about the identity and diversity of microbial community members. Genetic tools can also be used to make predictions about the chemistry that bacteria and fungi produce to function and communicate with one another and the host. Ongoing efforts to identify these products and link genetic information to microbiome chemistry rely on analytical tools. This tutorial highlights recent advancements in microbiome studies driven by techniques in mass spectrometry.

A label-free approach for relative spatial quantitation of c-di-GMP in microbial biofilms.

Microbial biofilms represent an important lifestyle for bacteria and are dynamic three dimensional structures. Cyclic dimeric guanosine monophosphate (c-di-GMP) is a ubiquitous signaling molecule that is known to be tightly regulated with biofilm processes. While measurements of global levels of c-di-GMP have proven valuable towards understanding the genetic control of c-di-GMP production, there is a need for tools to observe the local changes of c-di-GMP production in biofilm processes. We have developed a label-free method for the direct detection of c-di-GMP in microbial colony biofilms using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). We applied this method to the enteric pathogen Vibrio cholerae, the marine symbiont V. fischeri, and the opportunistic pathogen Pseudomonas aeruginosa PA14 and detected spatial and temporal changes in c-di-GMP signal that accompanied genetic alterations in factors that synthesize and degrade the compound. We further demonstrated how this method can be simultaneously applied to detect additional metabolites of interest in a single experiment.

An Integrated Approach to Protein Discovery and Detection From Complex Biofluids.

Ovarian cancer, a leading cause of cancer-related deaths among women, has been notoriously difficult to screen for and diagnose early, as early detection significantly improves survival. Researchers and clinicians seek routinely usable and noninvasive screening methods; however, available methods (i.e., biomarker screening) lack desirable sensitivity/specificity. The most fatal form, high-grade serous ovarian cancer, often originate in the fallopian tube; therefore, sampling from the vaginal environment provides more proximal sources for tumor detection. To address these shortcomings and leverage proximal sampling, we developed an untargeted mass spectrometry microprotein profiling method and identified cystatin A, which was validated in an animal model. To overcome the limits of detection inherent to mass spectrometry, we demonstrated that cystatin A is present at 100 pM concentrations using a label-free microtoroid resonator and translated our workflow to patient-derived clinical samples, highlighting the potential utility of early stage detection where biomarker levels would be low.

Metabolomics of bacterial-fungal pairwise interactions reveal conserved molecular mechanisms.

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecules in BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis sp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and that elucidating their role in complex communities should continue to be a priority.

Metabolomics of bacterial-fungal pairwise interactions reveal conserved molecular mechanisms.

Bacterial-fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial-fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecule mediated BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis spp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles (i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and their role in complex communities should continue to be a priority.

Fallopian tube secreted protein affects ovarian metabolites in high grade serous ovarian cancer.

High grade serous ovarian cancer (HGSOC), the most lethal histotype of ovarian cancer, frequently arises from fallopian tube epithelial cells (FTE). Once transformed, tumorigenic FTE often migrate specifically to the ovary, completing the crucial primary metastatic step and allowing the formation of the ovarian tumors after which HGSOC was originally named. As only the fimbriated distal ends of the fallopian tube that reside in close proximity to the ovary develop precursor lesions such as serous tubal intraepithelial carcinomas, this suggests that the process of transformation and primary metastasis to the ovary is impacted by the local microenvironment. We hypothesize that chemical cues, including small molecules and proteins, may help stimulate the migration of tumorigenic FTE to the ovary. However, the specific mediators of this process are still poorly understood, despite a recent growth in interest in the tumor microenvironment. Our previous work utilized imaging mass spectrometry (IMS) to identify the release of norepinephrine (NE) from the ovary in co-cultures of tumorigenic FTE cells with an ovarian explant. We predicted that tumorigenic FTE cells secreted a biomolecule, not produced or produced with low expression by non-tumorigenic cells, that stimulated the ovary to release NE. As such, we utilized an IMS mass-guided bioassay, using NE release as our biological marker, and bottom-up proteomics to demonstrate that a secreted protein, SPARC, is a factor produced by tumorigenic FTE responsible for enhancing release of ovarian NE and influencing primary metastasis of HGSOC. This discovery highlights the bidirectional interplay between different types of biomolecules in the fallopian tube and ovarian microenvironment and their combined roles in primary metastasis and disease progression.

TIMSCONVERT: a workflow to convert trapped ion mobility data to open data formats.

MOTIVATION: Advances in mass spectrometry have led to the development of mass spectrometers with ion mobility spectrometry capabilities and dual-source instrumentation; however, the current software ecosystem lacks interoperability with downstream data analysis using open-source software and pipelines. RESULTS: Here, we present TIMSCONVERT, a data conversion high-throughput workflow from timsTOF Pro/fleX mass spectrometer raw data files to mzML and imzML formats that incorporates ion mobility data while maintaining compatibility with data analysis tools. We showcase several examples using data acquired across different experiments and acquisition modalities on the timsTOF fleX MS. AVAILABILITY AND IMPLEMENTATION: TIMSCONVERT and its documentation can be found at https://github.com/gtluu/timsconvert and is available as a standalone command-line interface tool for Windows and Linux, NextFlow workflow and online in the Global Natural Products Social (GNPS) platform. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Home-Built Spinning Apparatus for Drying Agarose-Based Imaging Mass Spectrometry Samples.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a useful technique for mapping the spatial distribution of molecules across biological samples. Sample preparation is crucial for MALDI-IMS; samples must be flat, dry, and cocrystallized with a matrix prior to analysis. Agarose-based samples can be difficult to consistently prepare as they are susceptible to environmental changes, which can lead to inconsistent drying and wrinkling on the sample surface. Small height differences may cause low ionization of target analytes or introduce artifacts in imaging data depending on the instrument used for analysis. To overcome the variations, a home-built robotic spinner was constructed and applied to agarose-based samples. This robotic spinner is inexpensive and easy to assemble, and when it was applied to agarose-based samples, accelerated the drying process and reduced wrinkles, improving the overall quality of the resulting IMS data.

Cellulose nanofibers/PVA blend polymeric beads containing in-situ prepared magnetic nanorods as dye pollutants adsorbents.

Magnetic beads were developed from polyvinyl alcohol and different amounts of cellulose nanofibers (CNF) by in-situ preparation of iron oxide nanoparticles in an alkaline aqueous medium at room temperature. The CNF were isolated from wheat straw, whereas the magnetic nanoparticles (MNPs) precursors were simple iron salts. The complete characterization of all the obtained materials was conducted, and among some other outstanding results it showed that all the components were strongly interacting via hydrogen bonding, while the nano-rods and husks like MNPs were effectively acting as crosslinking dots. All the prepared materials had good magnetic responses, and they were able to remove not only cationic, but also anionic dye pollutants from aqueous model solutions.

Utilizing imaging mass spectrometry to analyze microbial biofilm chemical responses to exogenous compounds.

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an appealing label-free method for imaging biological samples which focuses on the spatial distribution of chemical signals. This approach has been used to study the chemical ecology of microbes and can be applied to study the chemical responses of microbes to treatment with exogenous compounds. Specific conjugated cholic acids such as taurocholic acid (TCA), have been shown to inhibit biofilm formation in the enteric pathogen Vibrio cholerae and MALDI-IMS can be used to directly observe the chemical responses of V. cholerae biofilm colonies to treatment with TCA. A major challenge of MALDI-IMS is optimizing the sample preparation and drying for a particular growth condition and microbial strain. Here we demonstrate how V. cholerae is cultured and prepared for MALDI-IMS analysis and highlight critical steps to ensure proper sample adherence to a MALDI target plate and maintain spatial distributions when applying this technique to any microbial strain. We additionally show how to use both manual interrogation and statistical analyses of MALDI-IMS data to establish the adequacy of the sample preparation protocol. This protocol can serve as a guideline for the development of sample preparation techniques and the acquisition of high quality MALDI-IMS data.

Automated Microbial Library Generation Using the Bioinformatics Platform IDBac.

Libraries of microorganisms have served as a cornerstone of therapeutic drug discovery, though the continued re-isolation of known natural product chemical entities has remained a significant obstacle to discovery efforts. A major contributing factor to this redundancy is the duplication of bacterial taxa in a library, which can be mitigated through the use of a variety of DNA sequencing strategies and/or mass spectrometry-informed bioinformatics platforms so that the library is created with minimal phylogenetic, and thus minimal natural product overlap. IDBac is a MALDI-TOF mass spectrometry-based bioinformatics platform used to assess overlap within collections of environmental bacterial isolates. It allows environmental isolate redundancy to be reduced while considering both phylogeny and natural product production. However, manually selecting isolates for addition to a library during this process was time intensive and left to the researcher's discretion. Here, we developed an algorithm that automates the prioritization of hundreds to thousands of environmental microorganisms in IDBac. The algorithm performs iterative reduction of natural product mass feature overlap within groups of isolates that share high homology of protein mass features. Employing this automation serves to minimize human bias and greatly increase efficiency in the microbial strain prioritization process.

Cellulose Nanofiber-Based Aerogels from Wheat Straw: Influence of Surface Load and Lignin Content on Their Properties and Dye Removal Capacity.

Water pollution is one of the most serious problems worldwide. Nanocellulose-based aerogels usually show excellent adsorption capacities due to their high aspect ratio, specific surface area and surface charge, making them ideal for water purification. In this work, (ligno)cellulose nanofibers (LCNFs/CNFs) from wheat straw residues were obtained using two types of pre-treatments: mechanical (Mec) and TEMPO-mediated oxidization (TO), to obtain different consistency (0.2, 0.4, 0.6 and 0.8) bioaerogels, and their adsorption capacities as dye removers were further studied. The materials were characterized in terms of density, porosity and mechanical properties. An inversely proportional relationship was observed between the consistencies of the aerogels and their achieved densities. Despite the increase in density, all samples showed porosities above 99%. In terms of mechanical properties, the best results were obtained for the 0.8% consistency LCNF and CNF-Mec aerogels, reaching 67.87 kPa and 64.6 kPa for tensile strength and Young's modulus, respectively. In contrast, the adsorption capacity of the aerogels was better for TEMPO-oxidized aerogels, reaching removal rates of almost 100% for the CNF-TO5 samples. Furthermore, the residual lignin content in LCNF-Mec aerogels showed a great improvement in the removal capacity, reaching rates higher than 80%, further improving the cost efficiency of the samples due to the reduction in chemical treatments.

Relationship between bacterial phylotype and specialized metabolite production in the culturable microbiome of two freshwater sponges.

Microbial drug discovery programs rely heavily on accessing bacterial diversity from the environment to acquire new specialized metabolite (SM) lead compounds for the therapeutic pipeline. Therefore, knowledge of how commonly culturable bacterial taxa are distributed in nature, in addition to the degree of variation of SM production within those taxa, is critical to informing these front-end discovery efforts and making the overall sample collection and bacterial library creation process more efficient. In the current study, we employed MALDI-TOF mass spectrometry and the bioinformatics pipeline IDBac to analyze diversity within phylotype groupings and SM profiles of hundreds of bacterial isolates from two Eunapius fragilis freshwater sponges, collected 1.5 km apart. We demonstrated that within two sponge samples of the same species, the culturable bacterial populations contained significant overlap in approximate genus-level phylotypes but mostly nonoverlapping populations of isolates when grouped lower than the level of genus. Further, correlations between bacterial phylotype and SM production varied at the species level and below, suggesting SM distribution within bacterial taxa must be analyzed on a case-by-case basis. Our results suggest that two E. fragilis freshwater sponges collected in similar environments can exhibit large culturable diversity on a species-level scale, thus researchers should scrutinize the isolates with analyses that take both phylogeny and SM production into account to optimize the chemical space entering into a downstream bacterial library.

Toward improvement of screening through mass spectrometry-based proteomics: ovarian cancer as a case study.

Ovarian cancer is one of the leading causes of cancer related deaths affecting United States women. Early-stage detection of ovarian cancer has been linked to increased survival, however, current screening methods, such as biomarker testing, have proven to be ineffective in doing so. Therefore, further developments are necessary to be able to achieve positive patient prognosis. Ongoing efforts are being made in biomarker discovery towards clinical applications in screening for early-stage ovarian cancer. In this perspective, we discuss and provide examples for several workflows employing mass spectrometry-based proteomics towards protein biomarker discovery and characterization in the context of ovarian cancer; workflows include protein identification and characterization as well as intact protein profiling. We also discuss the opportunities to merge these workflows for a multiplexed approach for biomarkers. Lastly, we provide our insight as to future developments that may serve to enhance biomarker discovery workflows while also considering translational potential.