Co-designing complex therapy interventions with parents as partners in the care of children with cerebral palsy: An Experience-based Co-design study in England.

BACKGROUND: Parents of children with hemiplegic cerebral palsy are increasingly involved in therapy intervention delivery. Enhancing the ways that parents are supported in delivery is key to optimising outcomes. This study aimed to refine an existing programme in England to better support parents partnering in their child's intervention delivery. METHODS AND PROCEDURES: Experience-based Co-design (EBCD) fostered collaboration between parents and therapists to identify shared improvement priorities and develop solutions. The study included eighteen interviews and sixteen co-design meetings involving twenty parents and eight therapists in total. Intervention development followed the MRC Framework for developing and evaluating complex interventions. OUTCOMES AND RESULTS: Themes from parent and therapist interviews informed priority setting for the co-design work. Three key shared priorities emerged a) accessing rehabilitation; b) fostering partnership and c) parent learning. Aligned with these priorities, three mixed parent and therapist co-design teams produced a) a parent booklet; an education outline for healthcare professionals; b) partnership principles; adaptations to intervention logbooks c) an online parent education session. CONCLUSIONS AND IMPLICATIONS: Engaging parents and therapists in a structured co-design process using EBCD yielded innovative interventions supporting parents in delivering therapy for children with hemiplegia. This collaborative approach is anticipated to enhance programme implementation and effectiveness.

PTM-Psi: A python package to facilitate the computational investigation of post-translational modification on protein structures and their impacts on dynamics and functions.

Post-translational modification (PTM) of a protein occurs after it has been synthesized from its genetic template, and involves chemical modifications of the protein's specific amino acid residues. Despite of the central role played by PTM in regulating molecular interactions, particularly those driven by reversible redox reactions, it remains challenging to interpret PTMs in terms of protein dynamics and function because there are numerous combinatorially enormous means for modifying amino acids in response to changes in the protein environment. In this study, we provide a workflow that allows users to interpret how perturbations caused by PTMs affect a protein's properties, dynamics, and interactions with its binding partners based on inferred or experimentally determined protein structure. This Python-based workflow, called PTM-Psi, integrates several established open-source software packages, thereby enabling the user to infer protein structure from sequence, develop force fields for non-standard amino acids using quantum mechanics, calculate free energy perturbations through molecular dynamics simulations, and score the bound complexes via docking algorithms. Using the S-nitrosylation of several cysteines on the GAP2 protein as an example, we demonstrated the utility of PTM-Psi for interpreting sequence-structure-function relationships derived from thiol redox proteomics data. We demonstrate that the S-nitrosylated cysteine that is exposed to the solvent indirectly affects the catalytic reaction of another buried cysteine over a distance in GAP2 protein through the movement of the two ligands. Our workflow tracks the PTMs on residues that are responsive to changes in the redox environment and lays the foundation for the automation of molecular and systems biology modeling.

Activity-based protein profiling identifies alternating activation of enzymes involved in the bifidobacterium shunt pathway or mucin degradation in the gut microbiome response to soluble dietary fiber.

While deprivation of dietary fiber has been associated with adverse health outcomes, investigations concerning the effect of dietary fiber on the gut microbiome have been largely limited to compositional sequence-based analyses or utilize a defined microbiota not native to the host. To extend understanding of the microbiome's functional response to dietary fiber deprivation beyond correlative evidence from sequence-based analyses, approaches capable of measuring functional enzymatic activity are needed. In this study, we use an activity-based protein profiling (ABPP) approach to identify sugar metabolizing and transport proteins in native mouse gut microbiomes that respond with differential activity to the deprivation or supplementation of the soluble dietary fibers inulin and pectin. We found that the microbiome of mice subjected to a high fiber diet high in soluble fiber had increased functional activity of multiple proteins, including glycoside hydrolases, polysaccharide lyases, and sugar transport proteins from diverse taxa. The results point to an increase in activity of the Bifidobacterium shunt metabolic pathway in the microbiome of mice fed high fiber diets. In those subjected to a low fiber diet, we identified a shift from the degradation of dietary fibers to that of gut mucins, in particular by the recently isolated taxon "Musculibacterium intestinale", which experienced dramatic growth in response to fiber deprivation. When combined with metabolomics and shotgun metagenomics analyses, our findings provide a functional investigation of dietary fiber metabolism in the gut microbiome and demonstrates the power of a combined ABPP-multiomics approach for characterizing the response of the gut microbiome to perturbations.

Synthetic Soil Aggregates: Bioprinted Habitats for High-Throughput Microbial Metaphenomics.

The dynamics of microbial processes are difficult to study in natural soil, owing to the small spatial scales on which microorganisms operate and to the opacity and chemical complexity of the soil habitat. To circumvent these challenges, we have created a 3D-bioprinted habitat that mimics aspects of natural soil aggregates while providing a chemically defined and translucent alternative culturing method for soil microorganisms. Our Synthetic Soil Aggregates (SSAs) retain the porosity, permeability, and patchy resource distribution of natural soil aggregates-parameters that are expected to influence emergent microbial community interactions. We demonstrate the printability and viability of several different microorganisms within SSAs and show how the SSAs can be integrated into a multi-omics workflow for single SSA resolution genomics, metabolomics, proteomics, lipidomics, and biogeochemical assays. We study the impact of the structured habitat on the distribution of a model co-culture microbial community and find that it is significantly different from the spatial organization of the same community in liquid culture, indicating a potential for SSAs to reproduce naturally occurring emergent community phenotypes. The SSAs have the potential as a tool to help researchers quantify microbial scale processes in situ and achieve high-resolution data from the interplay between environmental properties and microbial ecology.

Activity-Based Protein Profiling of Chitin Catabolism.

The microbial catabolism of chitin, an abundant and ubiquitous environmental organic polymer, is a fundamental cog in terrestrial and aquatic carbon and nitrogen cycles. Despite the importance of this critical bio-geochemical function, there is a limited understanding of the synergy between the various hydrolytic and accessory enzymes involved in chitin catabolism. To address this deficit, we synthesized activity-based probes (ABPs) designed to target active chitinolytic enzymes by modifying the chitin subunits N-acetyl glucosamine and chitotriose. The ABPs were used to determine the active complement of chitinolytic enzymes produced over time by the soil bacterium Cellvibrio japonicus treated with various C substrates. We demonstrate the utility of these ABPs in determining the synergy between various enzymes involved in chitin catabolism. The strategy can be used to gain molecular-level insights that can be used to better understand microbial roles in soil bio-geochemical cycling in the face of a changing climate.

Deep learning predicts microbial interactions from self-organized spatiotemporal patterns.

Microbial communities organize into spatial patterns that are largely governed by interspecies interactions. This phenomenon is an important metric for understanding community functional dynamics, yet the use of spatial patterns for predicting microbial interactions is currently lacking. Here we propose supervised deep learning as a new tool for network inference. An agent-based model was used to simulate the spatiotemporal evolution of two interacting organisms under diverse growth and interaction scenarios, the data of which was subsequently used to train deep neural networks. For small-size domains (100 µm × 100 µm) over which interaction coefficients are assumed to be invariant, we obtained fairly accurate predictions, as indicated by an average R2 value of 0.84. In application to relatively larger domains (450 µm × 450 µm) where interaction coefficients are varying in space, deep learning models correctly predicted spatial distributions of interaction coefficients without any additional training. Lastly, we evaluated our model against real biological data obtained using Pseudomonas fluorescens and Escherichia coli co-cultures treated with polymeric chitin or N-acetylglucosamine, the hydrolysis product of chitin. While P. fluorescens can utilize both substrates for growth, E. coli lacked the ability to degrade chitin. Consistent with our expectations, our model predicted context-dependent interactions across two substrates, i.e., degrader-cheater relationship on chitin polymers and competition on monomers. The combined use of the agent-based model and machine learning algorithm successfully demonstrates how to infer microbial interactions from spatially distributed data, presenting itself as a useful tool for the analysis of more complex microbial community interactions.

A Probe-Enabled Approach for the Selective Isolation and Characterization of Functionally Active Subpopulations in the Gut Microbiome.

Commensal microorganisms in the mammalian gut play important roles in host health and physiology, but a central challenge remains in achieving a detailed mechanistic understanding of specific microbial contributions to host biochemistry. New function-based approaches are needed that analyze gut microbial function at the molecular level by coupling detection and measurements of in situ biochemical activity with identification of the responsible microbes and enzymes. We developed a platform employing ß-glucuronidase selective activity-based probes to detect, isolate, and identify microbial subpopulations in the gut responsible for this xenobiotic metabolism. We find that metabolic activity of gut microbiota can be plastic and that between individuals and during perturbation, phylogenetically disparate populations can provide ß-glucuronidase activity. Our work links biochemical activity with molecular-scale resolution without relying on genomic inference.

Multifunctional Activity-Based Protein Profiling of the Developing Lung.

Lung diseases and disorders are a leading cause of death among infants. Many of these diseases and disorders are caused by premature birth and underdeveloped lungs. In addition to developmentally related disorders, the lungs are exposed to a variety of environmental contaminants and xenobiotics upon birth that can cause breathing issues and are progenitors of cancer. In order to gain a deeper understanding of the developing lung, we applied an activity-based chemoproteomics approach for the functional characterization of the xenometabolizing cytochrome P450 enzymes, active ATP and nucleotide binding enzymes, and serine hydrolases using a suite of activity-based probes (ABPs). We detected P450 activity primarily in the postnatal lung; using our ATP-ABP, we characterized a wide range of ATPases and other active nucleotide- and nucleic acid-binding enzymes involved in multiple facets of cellular metabolism throughout development. ATP-ABP targets include kinases, phosphatases, NAD- and FAD-dependent enzymes, RNA/DNA helicases, and others. The serine hydrolase-targeting probe detected changes in the activities of several proteases during the course of lung development, yielding insights into protein turnover at different stages of development. Select activity-based probe targets were then correlated with RNA in situ hybridization analyses of lung tissue sections.

High-Fat Diets Alter the Modulatory Effects of Xenobiotics on Cytochrome P450 Activities.

Cytochrome P450 monooxygenase (P450) enzymes metabolize critical endogenous chemicals and oxidize nearly all xenobiotics. Dysregulated P450 activities lead to altered capacity for drug metabolism and cellular stress. The effects of mixed exposures on P450 expression and activity are variable and elusive. A high-fat diet (HFD) is a common exposure that results in obesity and associated pathologies including hepatotoxicity. Herein, we report the effects of cigarette smoke on P450 activities of normal weight and HFD induced obese mice. Activity-based protein profiling results indicate that HFD mice had significantly decreased P450 activity, likely instigated by proinflammatory chemicals, and that P450 enzymes involved in detoxification, xenobiotic metabolism, and bile acid synthesis were effected by HFD and smoke interaction. Smoking increased activity of all lung P450 and coexposure to diet effected P450 2s1. We need to expand our understanding of common exposures coupled to altered P450 metabolism to enhance the safety and efficacy of therapeutic drug dosing.

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases.

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding "G site" and a substrate binding "H site". Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the "G site", and (2) an ABP designed to mimic a substrate molecule and have "H site" activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and "G" and "H" site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.

Indirect Interspecies Regulation: Transcriptional and Physiological Responses of a Cyanobacterium to Heterotrophic Partnership.

The mechanisms by which microbes interact in communities remain poorly understood. Here, we interrogated specific interactions between photoautotrophic and heterotrophic members of a model consortium to infer mechanisms that mediate metabolic coupling and acclimation to partnership. This binary consortium was composed of a cyanobacterium, Thermosynechococcus elongatus BP-1, which supported growth of an obligate aerobic heterotroph, Meiothermus ruber strain A, by providing organic carbon, O2, and reduced nitrogen. Species-resolved transcriptomic analyses were used in combination with growth and photosynthesis kinetics to infer interactions and the environmental context under which they occur. We found that the efficiency of biomass production and resistance to stress induced by high levels of dissolved O2 increased, beyond axenic performance, as a result of heterotrophic partnership. Coordinated transcriptional responses transcending both species were observed and used to infer specific interactions resulting from the synthesis and exchange of resources. The cyanobacterium responded to heterotrophic partnership by altering expression of core genes involved with photosynthesis, carbon uptake/fixation, vitamin synthesis, and scavenging of reactive oxygen species (ROS). IMPORTANCE This study elucidates how a cyanobacterial primary producer acclimates to heterotrophic partnership by modulating the expression levels of key metabolic genes. Heterotrophic bacteria can indirectly regulate the physiology of the photoautotrophic primary producers, resulting in physiological changes identified here, such as increased intracellular ROS. Some of the interactions inferred from this model system represent putative principles of metabolic coupling in phototrophic-heterotrophic partnerships.

Dinitrogenase-Driven Photobiological Hydrogen Production Combats Oxidative Stress in Cyanothece sp. Strain ATCC 51142.

Photobiologically synthesized hydrogen (H2) gas is carbon neutral to produce and clean to combust, making it an ideal biofuel. Cyanothece sp. strain ATCC 51142 is a cyanobacterium capable of performing simultaneous oxygenic photosynthesis and H2 production, a highly perplexing phenomenon because H2 evolving enzymes are O2 sensitive. We employed a system-level in vivo chemoproteomic profiling approach to explore the cellular dynamics of protein thiol redox and how thiol redox mediates the function of the dinitrogenase NifHDK, an enzyme complex capable of aerobic hydrogenase activity. We found that NifHDK responds to intracellular redox conditions and may act as an emergency electron valve to prevent harmful reactive oxygen species formation in concert with other cell strategies for maintaining redox homeostasis. These results provide new insight into cellular redox dynamics useful for advancing photolytic bioenergy technology and reveal a new understanding for the biological function of NifHDK. IMPORTANCE: Here, we demonstrate that high levels of hydrogen synthesis can be induced as a protection mechanism against oxidative stress via the dinitrogenase enzyme complex in Cyanothece sp. strain ATCC 51142. This is a previously unknown feature of cyanobacterial dinitrogenase, and we anticipate that it may represent a strategy to exploit cyanobacteria for efficient and scalable hydrogen production. We utilized a chemoproteomic approach to capture the in situ dynamics of reductant partitioning within the cell, revealing proteins and reactive thiols that may be involved in redox sensing and signaling. Additionally, this method is widely applicable across biological systems to achieve a greater understanding of how cells navigate their environment and how redox chemistry can be utilized to alter metabolism and achieve homeostasis.

Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development.

Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography-mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.

Systematic Survey of Serine Hydrolase Activity in Mycobacterium tuberculosis Defines Changes Associated with Persistence.

The transition from replication to non-replication underlies much of Mycobacterium tuberculosis (Mtb) pathogenesis, as non- or slowly replicating Mtb are responsible for persistence and poor treatment outcomes. Therapeutic targeting of non-replicating populations is a priority for tuberculosis treatment, but few drug targets in non-replicating Mtb are currently known. Here, we directly measured the activity of the highly diverse and druggable serine hydrolases (SHs) during active replication and non-replication using activity-based proteomics. We predict SH activity for 78 proteins, including 27 proteins with unknown function, and identify 37 SHs that remain active in the absence of replication, providing a set of candidate persistence targets. Non-replication was associated with major shifts in SH activity. These activity changes were largely independent of SH abundance, indicating extensive post-translational regulation of SHs. By probing a large cross-section of druggable Mtb enzyme space during replication and non-replication, we identify new SHs and suggest new persistence targets.

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.

Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2 (-)) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4 (+)-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with ß-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA.

Role of Cytochrome P450 Hydroxylase in the Decreased Accumulation of Vitamin E in Muscle from Turkeys Compared to that from Chickens.

Turkeys and chickens reared to 5 weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). Alpha tocopherol hydroxylase activity was greater in liver microsomes prepared from turkeys compared to that from chickens (p < 0.01). Alpha and gamma tocopherol metabolites were higher in turkey bile than in chicken when assessing the RRR AcT diet and the all-racemic AcT diet at 58 mg/kg feed (p < 0.01). Turkey cytochrome P450 2C29 was increased relative to its chicken ortholog on the basis of RNA-Seq transcript abundance (p < 0.001) and activity-based protein profiling (p < 0.01) of liver tissue. Alpha tocopherol concentrations in plasma, liver, and muscle from turkey were lower than the respective tissues from chicken (p < 0.05). Lipid oxidation was greater in turkey thigh than in chicken (p < 0.05). These results suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylase(s) in turkeys contributes to the decreased accumulation of alpha tocopherol in turkey tissues compared to that of chickens.

Multi-Omic Dynamics Associate Oxygenic Photosynthesis with Nitrogenase-Mediated H2 Production in Cyanothece sp. ATCC 51142.

To date, the proposed mechanisms of nitrogenase-driven photosynthetic H2 production by the diazotrophic unicellular cyanobacterium Cyanothece sp. ATCC 51142 have assumed that reductant and ATP requirements are derived solely from glycogen oxidation and cyclic-electron flow around photosystem I. Through genome-scale transcript and protein profiling, this study presents and tests a new hypothesis on the metabolic relationship between oxygenic photosynthesis and nitrogenase-mediated H2 production in Cyanothece 51142. Our results show that net-positive rates of oxygenic photosynthesis and increased expression of photosystem II reaction centers correspond and are synchronized with nitrogenase expression and H2 production. These findings provide a new and more complete view on the metabolic processes contributing to the energy budget of photosynthetic H2 production and highlight the role of concurrent photocatalytic H2O oxidation as a participating process.

Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling.

The development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosic bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.

Activity-based protein profiling of microbes.

Activity-based protein profiling (ABPP) in conjunction with multimodal characterization techniques has yielded impactful findings in microbiology, particularly in pathogen, bioenergy, drug discovery, and environmental research. Using small molecule chemical probes that react irreversibly with specific proteins or protein families in complex systems has provided insights in enzyme functions in central metabolic pathways, drug-protein interactions, and regulatory protein redox, for systems ranging from photoautotrophic cyanobacteria to mycobacteria, and combining live cell or cell extract ABPP with proteomics, molecular biology, modeling, and other techniques has greatly expanded our understanding of these systems. New opportunities for application of ABPP to microbial systems can enhance protein annotation, characterize protein activities in myriad environments, and reveal signal transduction and regulatory mechanisms in microbial systems.

Deficient expression of aldehyde dehydrogenase 1A1 is consistent with increased sensitivity of Gorlin syndrome patients to radiation carcinogenesis.

Human phenotypes that are highly susceptible to radiation carcinogenesis have been identified. Sensitive phenotypes often display robust regulation of molecular features that modify biological response, which can facilitate identification of the pathways/networks that contribute to pathophysiological outcomes. Here we interrogate primary dermal fibroblasts isolated from Gorlin syndrome patients (GDFs), who display a pronounced inducible tumorigenic response to radiation, in comparison to normal human dermal fibroblasts (NHDFs). Our approach exploits newly developed thiol reactive probes to define changes in protein thiol profiles in live cell studies, which minimizes artifacts associated with cell lysis. Redox probes revealed deficient expression of an apparent 55 kDa protein thiol in GDFs from independent Gorlin syndrome patients, compared with NHDFs. Proteomics tentatively identified this protein as aldehyde dehydrogenase 1A1 (ALDH1A1), a key enzyme regulating retinoic acid synthesis, and ALDH1A1 protein deficiency in GDFs was confirmed by Western blot. A number of additional protein thiol differences in GDFs were identified, including radiation responsive annexin family members and lamin A/C. Collectively, candidates identified in our study have plausible implications for radiation health effects and cancer susceptibility.