Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population.

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes. Most prominently, Dhtkd1 was identified as a primary regulator of 2-aminoadipate, explaining variance in fasted glucose and diabetes status in both mice and humans. These integrated molecular profiles also allowed further characterization of complex pathways, particularly the mitochondrial unfolded protein response (UPR(mt)). UPR(mt) shows strikingly variant responses at the transcript and protein level that are remarkably conserved among C. elegans, mice, and humans. Overall, these examples demonstrate the value of an integrated multilayered omics approach to characterize complex metabolic phenotypes.

Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.

The Hippo pathway is crucial in organ size control, and its dysregulation contributes to tumorigenesis. However, upstream signals that regulate the mammalian Hippo pathway have remained elusive. Here, we report that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling. Serum-borne lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P) act through G12/13-coupled receptors to inhibit the Hippo pathway kinases Lats1/2, thereby activating YAP and TAZ transcription coactivators, which are oncoproteins repressed by Lats1/2. YAP and TAZ are involved in LPA-induced gene expression, cell migration, and proliferation. In contrast, stimulation of Gs-coupled receptors by glucagon or epinephrine activates Lats1/2 kinase activity, thereby inhibiting YAP function. Thus, GPCR signaling can either activate or inhibit the Hippo-YAP pathway depending on the coupled G protein. Our study identifies extracellular diffusible signals that modulate the Hippo pathway and also establishes the Hippo-YAP pathway as a critical signaling branch downstream of GPCR.

Field-resolved infrared spectroscopy of biological systems.

The proper functioning of living systems and physiological phenotypes depends on molecular composition. Yet simultaneous quantitative detection of a wide variety of molecules remains a challenge1-8. Here we show how broadband optical coherence opens up opportunities for fingerprinting complex molecular ensembles in their natural environment. Vibrationally excited molecules emit a coherent electric field following few-cycle infrared laser excitation9-12, and this field is specific to the sample's molecular composition. Employing electro-optic sampling10,12-15, we directly measure this global molecular fingerprint down to field strengths 107 times weaker than that of the excitation. This enables transillumination of intact living systems with thicknesses of the order of 0.1 millimetres, permitting broadband infrared spectroscopic probing of human cells and plant leaves. In a proof-of-concept analysis of human blood serum, temporal isolation of the infrared electric-field fingerprint from its excitation along with its sampling with attosecond timing precision results in detection sensitivity of submicrograms per millilitre of blood serum and a detectable dynamic range of molecular concentration exceeding 105. This technique promises improved molecular sensitivity and molecular coverage for probing complex, real-world biological and medical settings.

Plasma Instead of Serum Avoids Critical Confounding of Clinical Metabolomics Studies by Platelets.

Metabolomics is an emerging and powerful bioanalytical method supporting clinical investigations. Serum and plasma are commonly used without rational prioritization. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may affect the metabolome and increase the variance, as platelet counts and function may vary substantially in individuals. A multiomics approach systematically investigating the suitability of serum and plasma for clinical studies demonstrated that metabolites correlated well (n = 461, R2 = 0.991), whereas lipid mediators (n = 83, R2 = 0.906) and proteins (n = 322, R2 = 0.860) differed substantially between specimen. Independently, analysis of platelet releasates identified most biomolecules significantly enriched in serum compared to plasma. A prospective, randomized, controlled parallel group metabolomics trial with acetylsalicylic acid administered for 7 days demonstrated that the apparent drug effects significantly differ depending on the analyzed specimen. Only serum analyses of healthy individuals suggested a significant downregulation of TXB2 and 12-HETE, which were specifically formed during coagulation in vitro. Plasma analyses reliably identified acetylsalicylic acid effects on metabolites and lipids occurring in vivo such as an increase in serotonin, 15-deoxy-PGJ2 and sphingosine-1-phosphate and a decrease in polyunsaturated fatty acids. The present data suggest that plasma should be preferred above serum for clinical metabolomics studies as the serum metabolome may be substantially confounded by platelets.

Serum Ammonia Levels Do Not Correlate With Overt Hepatic Encephalopathy Severity in Hospitalized Patients With Cirrhosis.

Although ammonia is involved in the pathophysiology of hepatic encephalopathy (HE), the use of ammonia levels in clinical practice is problematic.1-3 For example, in a study of 551 patients with overt HE (OHE) receiving lactulose who had ammonia levels tested, only 60% had an increased ammonia level (defined as >72 µmol/L).2 Overall, there was no correlation observed between lactulose dose and whether ammonia levels were obtained (ie, presence/absence of increased ammonia level did not guide therapy), or between time to OHE resolution and ammonia levels.2 Additionally, there is substantial interlaboratory variability in sample handling and processing, which may affect ammonia measurements.4.

Mass Spectrometry Identifies Taenia solium Proteins in Sera of Patients With and Without Parenchymal Neurocysticercosis.

Neurocysticercosis (NCC), a major cause of global acquired epilepsy, results from Taenia solium larval brain infection. T. solium adult worms release large numbers of infective eggs into the environment contributing to high levels of exposure in endemic areas. This study identifies T. solium proteins in the sera of individuals with and without NCC using mass spectrometry to examine exposure in endemic regions. Forty-seven patients (18-51 years), 24 parenchymal NCC (pNCC), 8 epilepsy of unknown aetiology, 7 glioma, 8 brain tuberculoma, and 7 healthy volunteers were studied. Trypsin digested sera were subject to liquid chromatography-tandem mass spectrometry and spectra of 375-1700 m/z matched against T. solium WormBase ParaSite database with MaxQuant software to identify T. solium proteins. Three hundred and nineteen T. solium proteins were identified in 87.5% of pNCC and 56.6% of non-NCC subjects. Three hundred and four proteins were exclusive to pNCC sera, seven to non-NCC sera and eight in both. Ten percent, exhibiting immune-modulatory properties, originated from the oncosphere and cyst vesicular fluid. In conclusion, in endemic regions, T. solium proteins are detected in sera of individuals with and without pNCC. The immunomodulatory nature of these proteins may influence susceptibility and course of infection.

The U-shaped association between serum osmolality and 28-day mortality in patients with sepsis: a retrospective cohort study.

BACKGROUND: Sepsis is a recognized global health challenge that places a considerable disease burden on countries. Although there has been some progress in the study of sepsis, the mortality rate of sepsis remains high. The relationship between serum osmolality and the prognosis of patients with sepsis is unclear. METHOD: Patients with sepsis who met the criteria in the Medical Information Mart for Intensive Care IV database were included in the study. Hazard ratios (HRs) and 95% confidence intervals (CIs) were determined using multivariable Cox regression. The relationship between serum osmolality and the 28-day mortality risk in patients with sepsis was investigated using curve fitting, and inflection points were calculated. RESULTS: A total of 13,219 patients with sepsis were enrolled in the study; the mean age was 65.1 years, 56.9 % were male, and the 28-day mortality rate was 18.8 %. After adjusting for covariates, the risk of 28-day mortality was elevated by 99% (HR 1.99, 95%CI 1.74-2.28) in the highest quintile of serum osmolality (Q5 >303.21) and by 59% (HR 1.59, 95%CI 1.39-1.83) in the lowest quintile (Q1 ≤285.80), as compared to the reference quintile (Q3 291.38-296.29). The results of the curve fitting showed a U-shaped relationship between serum osmolality and the risk of 28-day mortality, with an inflection point of 286.9 mmol/L. CONCLUSION: There is a U-shaped relationship between serum osmolality and the 28-day mortality risk in patients with sepsis. Higher or lower serum osmolality is associated with an increased risk of mortality in patients with sepsis. Patients with sepsis have a lower risk of mortality when their osmolality is 285.80-296.29 mmol/L.

Fluorinated Liquid-Crystal Monomers in Serum from the General Population and Their Impact on Human Health.

Fluorinated liquid-crystal monomers (FLCMs) are a potential emerging class of persistent, bioaccumulative, and toxic compounds. Humans inevitably ingest FLCMs via food and the environment. However, there are limited studies on internal exposure biomonitoring of FLCMs. Herein, we evaluated the estimated daily intakes (EDIs) of FLCMs in the general population based on serum residue levels. For the first time, 38 FLCMs were detected in 314 serum samples from the general population in Beijing, with a median value of 132.48 ng/g of lipid weight (lw). BDPrB is a predominant FLCM in serum. The median EDI of ∑38FLCMs in the general residents was 37.96 pg/kg bw/day. The residual levels of most FLCMs were higher in urban than in suburban areas (p < 0.05). The concentrations of EFPEB, EDPrB, EDFPBB, and PDTFMTFT in serum showed positive associations with blood glucose (GLU) (r = 0.126-0.275, p < 0.05). Logistic regression analysis showed that FLCMs were significantly positively correlated with dyslipidemia, with an odds ratio of 2.19; BDPrB was significantly positively correlated with hyperglycemia (OR: 2.48). Overall, the present study suggests the occurrence of FLCMs in the nonoccupational population, and the exposure of certain FLCMs may cause abnormal blood glucose and lipid levels.

The stability of 65 biochemistry analytes in plasma, serum, and whole blood.

OBJECTIVES: The pre-analytical stability of various biochemical analytes requires careful consideration, as it can lead to the release of erroneous laboratory results. There is currently significant variability in the literature regarding the pre-analytical stability of various analytes. The aim of this study was to determine the pre-analytical stability of 65 analytes in whole blood, serum and plasma using a standardized approach. METHODS: Blood samples were collected from 30 healthy volunteers (10 volunteers per analyte) into five vacutainers; either SST, Li-heparin, K2-EDTA, or Na-fluoride/K-oxalate. Several conditions were tested, including delayed centrifugation with storage of whole blood at room temperature (RT) for 8 h, delayed centrifugation with storage of whole blood at RT or 4 °C for 24 h, and immediate centrifugation with storage of plasma or serum at RT for 24 h. Percent deviation (% PD) from baseline was calculated for each analyte and compared to the maximum permissible instability (MPI) derived from intra- and inter-individual biological variation. RESULTS: The majority of the analytes evaluated remained stable across all vacutainer types, temperatures, and timepoints tested. Glucose, potassium, and aspartate aminotransferase, among others, were significantly impacted by delayed centrifugation, having been found to be unstable in whole blood specimens stored at room temperature for 8 h. CONCLUSIONS: The data presented provides insight into the pre-analytical variables that impact the stability of routine biochemical analytes. This study may help to reduce the frequency of erroneous laboratory results released due to exceeded stability and reduce unnecessary repeat phlebotomy for analytes that remain stable despite delayed processing.

N-terminal modification of an LAH4-derived peptide increases mRNA delivery in the presence of serum.

The recently developed mRNA-based coronavirus SARS-CoV-2 vaccines highlighted the great therapeutic potential of the mRNA technology. Although the lipid nanoparticles used for the delivery of the mRNA are very efficient, they showed, in some cases, the induction of side effects as well as the production of antibodies directed against particle components. Thus, the development of alternative delivery systems is of great interest in the pursuit of more effective mRNA treatments. In the present work, we evaluated the mRNA transfection capacities of a series of cationic histidine-rich amphipathic peptides derived from LAH4. We found that while the LAH4-A1 peptide was an efficient carrier for mRNA, its activity was highly serum sensitive. Interestingly, modification of this cell penetrating peptide at the N-terminus with two tyrosines or with salicylic acid allowed to confer serum resistance to the carrier.

Quantitative analysis of mRNA-lipid nanoparticle stability in human plasma and serum by size-exclusion chromatography coupled with dual-angle light scattering.

Understanding the stability of mRNA loaded lipid nanoparticles (mRNA-LNPs) is imperative for their clinical development. Herein, we propose the use of size-exclusion chromatography coupled with dual-angle light scattering (SEC-MALS) as a new approach to assessing mRNA-LNP stability in pure human serum and plasma. By applying a dual-column configuration to attenuate interference from plasma components, SEC-MALS was able to elucidate the degradation kinetics and physical property changes of mRNA-LNPs, which have not been observed accurately by conventional dynamic light scattering techniques. Interestingly, both serum and plasma had significantly different impacts on the molecular weight and radius of gyration of mRNA-LNPs, suggesting the involvement of clotting factors in desorption of lipids from mRNA-LNPs. We also discovered that a trace impurity (~1 %) in ALC-0315, identified as its O-tert-butyloxycarbonyl-protected form, greatly diminished mRNA-LNP stability in serum. These results demonstrated the potential utility of SEC-MALS for optimization and quality control of LNP formulations.

Serum levels of inflammatory cytokines in mercury mining workers in a precarious situation: A preliminary study.

Mercury is a ubiquitous environmental xenobiotic; the primary sources of exposure to this metal are artisanal gold mining and the direct production of mercury. In Mexico, artisanal mercury mining continues to be an important activity in different regions of the country. Exposure to mercury vapors releases can have severe health impacts, including immunotoxic effects such as alterations in cytokine profiling. Therefore, in the present work, we evaluated the inflammatory cytokines profile in the blood serum of miners exposed to mercury. A cross-sectional observational study was performed on 27 mining workers (exposed group) and 20 control subjects (nonexposed group) from central Mexico. The mercury urine concentration (U-Hg) was determined by atomic absorption spectrometry, and IL-2, IL-6, IL-8, IL-10, and TNF-α were measured using a Multiplex Assay. The results showed that the U-Hg in the miners had a median value of 552.70 µg/g creatinine. All cytokines showed a significant increase in the miner group compared with the control group, except for TNF-α. In addition, we observed a positive correlation between U-Hg concentration and cytokine levels. In conclusion, mercury exposure correlated with cytokine levels (considered acute inflammatory marker) in miners; therefore, workers exposed to this metal show an acute systemic inflammation that could lead to alterations in other organs and systems.

Selenium Discrepancies in Fetal Bovine Serum: Impact on Cellular Selenoprotein Expression.

Selenium is an essential trace element in our diet, crucial for the composition of human selenoproteins, which include 25 genes such as glutathione peroxidases and thioredoxin reductases. The regulation of the selenoproteome primarily hinges on the bioavailability of selenium, either from dietary sources or cell culture media. This selenium-dependent control follows a specific hierarchy, with "housekeeping" selenoproteins maintaining constant expression while "stress-regulated" counterparts respond to selenium level fluctuations. This study investigates the variability in fetal bovine serum (FBS) selenium concentrations among commercial batches and its effects on the expression of specific stress-related cellular selenoproteins. Despite the limitations of our study, which exclusively used HEK293 cells and focused on a subset of selenoproteins, our findings highlight the substantial impact of serum selenium levels on selenoprotein expression, particularly for GPX1 and GPX4. The luciferase reporter assay emerged as a sensitive and precise method for evaluating selenium levels in cell culture environments. While not exhaustive, this analysis provides valuable insights into selenium-mediated selenoprotein regulation, emphasizing the importance of serum composition in cellular responses and offering guidance for researchers in the selenoprotein field.

Bone Regenerative Effect of Injectable Hypoxia Preconditioned Serum-Fibrin (HPS-F) in an Ex Vivo Bone Defect Model.

Biofunctionalized hydrogels are widely used in tissue engineering for bone repair. This study examines the bone regenerative effect of the blood-derived growth factor preparation of Hypoxia Preconditioned Serum (HPS) and its fibrin-hydrogel formulation (HPS-F) on drilled defects in embryonic day 19 chick femurs. Measurements of bone-related growth factors in HPS reveal significant elevations of Osteopontin, Osteoprotegerin, and soluble-RANKL compared with normal serum (NS) but no detection of BMP-2/7 or Osteocalcin. Growth factor releases from HPS-F are measurable for at least 7 days. Culturing drilled femurs organotypically on a liquid/gas interface with HPS media supplementation for 10 days demonstrates a 34.6% increase in bone volume and a 52.02% increase in bone mineral density (BMD) within the defect area, which are significantly higher than NS and a basal-media-control, as determined by microcomputed tomography. HPS-F-injected femur defects implanted on a chorioallantoic membrane (CAM) for 7 days exhibit an increase in bone mass of 123.5% and an increase in BMD of 215.2%, which are significantly higher than normal-serum-fibrin (NS-F) and no treatment. Histology reveals calcification, proteoglycan, and collagen fiber deposition in the defect area of HPS-F-treated femurs. Therefore, HPS-F may offer a promising and accessible therapeutic approach to accelerating bone regeneration by a single injection into the bone defect site.

Orbitrap Mass Spectrometry and High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Enable the in-Depth Analysis of Human Serum Proteoforms.

Blood serum and plasma are arguably the most commonly analyzed clinical samples, with dozens of proteins serving as validated biomarkers for various human diseases. Top-down proteomics may provide additional insights into disease etiopathogenesis since this approach focuses on protein forms, or proteoforms, originally circulating in blood, potentially providing access to information about relevant post-translational modifications, truncations, single amino acid substitutions, and many other sources of protein variation. However, the vast majority of proteomic studies on serum and plasma are carried out using peptide-centric, bottom-up approaches that cannot recapitulate the original proteoform content of samples. Clinical laboratories have been slow to adopt top-down analysis, also due to higher sample handling requirements. In this study, we describe a straightforward protocol for intact proteoform sample preparation based on the depletion of albumin and immunoglobulins, followed by simplified protein fractionation via polyacrylamide gel electrophoresis. After molecular weight-based fractionation, we supplemented the traditional liquid chromatography-tandem mass spectrometry (LC-MS2) data acquisition with high-field asymmetric waveform ion mobility spectrometry (FAIMS) to further simplify serum proteoform mixtures. This LC-FAIMS-MS2 method led to the identification of over 1000 serum proteoforms < 30 kDa, outperforming traditional LC-MS2 data acquisition and more than doubling the number of proteoforms identified in previous studies.

Omic-Scale High-Throughput Quantitative LC-MS/MS Approach for Circulatory Lipid Phenotyping in Clinical Research.

Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera (n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.

Luminescence Sensing of Biomacromolecules Heparin and Protamine in 100% Human Serum and Plasma by Supramolecular Polymeric Assemblies.

Heparin, an anionic biomacromolecule, is routinely used as an anticoagulant during medical surgery to prevent blood clot formation and in the treatment of several heart, lung, and circulatory disorders having a higher risk of blood clotting. We herein report supramolecular polymeric nanoassemblies of cationic pyrene-tagged bis-imidazolium amphiphiles for heparin detection with high sensitivity and selectivity in aqueous buffer, plasma, and serum media. The nano-assemblies exhibited cyan-green excimeric emission in aqueous media, and their multivalent array of positive surface charges allowed them to form co-assemblies with heparin, resulting in significantly enhanced emission. This provided a convenient method for heparin detection in buffer at nanomolar concentrations, and most notably, a ratiometric fluorescence response was obtained even in highly competitive 100% human serum and 100% human plasma in a clinically relevant concentration range. Moreover, using the heparin-based luminescent co-assemblies, protamine sulfate, a clinically administered antidote to heparin, was also detected in 100% human serum and 100% human plasma at sub-micromolar concentrations.

Diagnostic performance of the IMMY cryptococcal antigen lateral flow assay on serum and cerebrospinal fluid for diagnosis of cryptococcosis in HIV-negative patients: a systematic review.

BACKGROUND: The incidence of cryptococcosis amongst HIV-negative persons is increasing. Whilst the excellent performance of the CrAg testing in people living with HIV is well described, the diagnostic performance of the CrAg LFA has not been systematically evaluated in HIV-negative cohorts on serum or cerebrospinal fluid. METHODS: We performed a systematic review to characterise the diagnostic performance of IMMY CrAg® LFA in HIV-negative populations on serum and cerebrospinal fluid. A systematic electronic search was performed using Medline, Embase, Global Health, CENTRAL, WoS Science Citation Index, SCOPUS, Africa-Wide Information, LILACS and WHO Global Health Library. Studies were screened and data extracted from eligible studies by two independent reviewers. A fixed effect meta-analysis was used to estimate the diagnostic sensitivity and specificity. RESULTS: Of 447 records assessed for eligibility, nine studies met our inclusion criteria, including 528 participants overall. Amongst eight studies that evaluated the diagnostic performance of the IMMY CrAg® LFA on serum, the pooled median sensitivity was 96% (95% Credible Interval (CrI) 68-100%) with a pooled specificity estimate of 96% (95%CrI 84-100%). Amongst six studies which evaluated the diagnostic performance of IMMY CrAg® LFA on CSF, the pooled median sensitivity was 99% (95%CrI 95-100%) with a pooled specificity median of 99% (95%CrI 95-100%). CONCLUSIONS: This review demonstrates a high pooled sensitivity and specificity for the IMMY CrAg® LFA in HIV-negative populations, in keeping with findings in HIV-positive individuals. The review was limited by the small number of studies. Further studies using IMMY CrAg® LFA in HIV-negative populations would help to better determine the diagnostic value of this test.

A sensitive and robust method for the simultaneous determination of thirty-three legacy and emerging per- and polyfluoroalkyl substances in human plasma and serum.

Legacy and emerging per- and polyfluoroalkyl substances (PFAS) have attracted growing attention due to their potential adverse effects on humans. We developed a method to simultaneously determine thirty-three PFAS (legacy PFAS, precursors, and alternatives) in human plasma and serum using solid phase extraction coupled to ultra-performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS). The method yielded good linearity (>0.995) and excellent limits of detection (LODs) (0.0005~0.012 ng mL-1 in plasma and 0.002~0.016 ng mL-1 in serum). The relative recoveries ranged from 80.1 to 116%, with intra- and inter-day precision less than 14.3%. The robustness of this method has been tested continuously for 10 months (coefficients of variation <14.9%). Our method was successfully applied to the PFAS analysis of 42 real human plasma and serum samples collected from women. The proposed method is attractive for the biomonitoring of multi-class PFAS in human health risk assessment and epidemiological studies.

A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites.

The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.