Microbial liberation of N-methylserotonin from orange fiber in gnotobiotic mice and humans.

Plant fibers in byproduct streams produced by non-harsh food processing methods represent biorepositories of diverse, naturally occurring, and physiologically active biomolecules. To demonstrate one approach for their characterization, mass spectrometry of intestinal contents from gnotobiotic mice, plus in vitro studies, revealed liberation of N-methylserotonin from orange fibers by human gut microbiota members including Bacteroides ovatus. Functional genomic analyses of B. ovatus strains grown under permissive and non-permissive N-methylserotonin "mining" conditions revealed polysaccharide utilization loci that target pectins whose expression correlate with strain-specific liberation of this compound. N-methylserotonin, orally administered to germ-free mice, reduced adiposity, altered liver glycogenesis, shortened gut transit time, and changed expression of genes that regulate circadian rhythm in the liver and colon. In human studies, dose-dependent, orange-fiber-specific fecal accumulation of N-methylserotonin positively correlated with levels of microbiome genes encoding enzymes that digest pectic glycans. Identifying this type of microbial mining activity has potential therapeutic implications.

Bioactive glycans in a microbiome-directed food for children with malnutrition.

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Here we analyse biospecimens from a randomized, controlled trial of a microbiome-directed complementary food (MDCF-2) that produced superior rates of weight gain compared with a calorically more dense conventional ready-to-use supplementary food in 12-18-month-old Bangladeshi children with moderate acute malnutrition4. We reconstructed 1,000 bacterial genomes (metagenome-assembled genomes (MAGs)) from the faecal microbiomes of trial participants, identified 75 MAGs of which the abundances were positively associated with ponderal growth (change in weight-for-length Z score (WLZ)), characterized changes in MAG gene expression as a function of treatment type and WLZ response, and quantified carbohydrate structures in MDCF-2 and faeces. The results reveal that two Prevotella copri MAGs that are positively associated with WLZ are the principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilizing the component glycans of MDCF-2. The predicted specificities of carbohydrate-active enzymes expressed by their polysaccharide-utilization loci are correlated with (1) the in vitro growth of Bangladeshi P. copri strains, possessing varying degrees of polysaccharide-utilization loci and genomic conservation with these MAGs, in defined medium containing different purified glycans representative of those in MDCF-2, and (2) the levels of faecal carbohydrate structures in the trial participants. These associations suggest that identifying bioactive glycan structures in MDCFs metabolized by growth-associated bacterial taxa will help to guide recommendations about their use in children with acute malnutrition and enable the development of additional formulations.

A Microbiota-Directed Food Intervention for Undernourished Children.

BACKGROUND: More than 30 million children worldwide have moderate acute malnutrition. Current treatments have limited effectiveness, and much remains unknown about the pathogenesis of this condition. Children with moderate acute malnutrition have perturbed development of their gut microbiota. METHODS: In this study, we provided a microbiota-directed complementary food prototype (MDCF-2) or a ready-to-use supplementary food (RUSF) to 123 slum-dwelling Bangladeshi children with moderate acute malnutrition between the ages of 12 months and 18 months. The supplementation was given twice daily for 3 months, followed by 1 month of monitoring. We obtained weight-for-length, weight-for-age, and length-for-age z scores and mid-upper-arm circumference values at baseline and every 2 weeks during the intervention period and at 4 months. We compared the rate of change of these related phenotypes between baseline and 3 months and between baseline and 4 months. We also measured levels of 4977 proteins in plasma and 209 bacterial taxa in fecal samples. RESULTS: A total of 118 children (59 in each study group) completed the intervention. The rates of change in the weight-for-length and weight-for-age z scores are consistent with a benefit of MDCF-2 on growth over the course of the study, including the 1-month follow-up. Receipt of MDCF-2 was linked to the magnitude of change in levels of 70 plasma proteins and of 21 associated bacterial taxa that were positively correlated with the weight-for-length z score (P<0.001 for comparisons of both protein and bacterial taxa). These proteins included mediators of bone growth and neurodevelopment. CONCLUSIONS: These findings provide support for MDCF-2 as a dietary supplement for young children with moderate acute malnutrition and provide insight into mechanisms by which this targeted manipulation of microbiota components may be linked to growth. (Supported by the Bill and Melinda Gates Foundation and the National Institutes of Health; ClinicalTrials.gov number, NCT04015999.).

Versatile volumetric additive manufacturing with 3D ray tracing.

Tomographic volumetric additive manufacturing (VAM) is an optical 3D printing technique where an object is formed by photopolymerizing resin via tomographic projections. Currently, these projections are calculated using the Radon transform from computed tomography but it ignores two fundamental properties of real optical projection systems: finite etendue and non-telecentricity. In this work, we introduce 3D ray tracing as a new method of computing projections in tomographic VAM and demonstrate high fidelity printing in non-telecentric and higher etendue systems, leading to a 3x increase in vertical build volume than the standard Radon method. The method introduced here expands the possible tomographic VAM printing configurations, enabling faster, cheaper, and higher fidelity printing.

Stop Waiting for Tomorrow: Disk Diffusion Performed on Early Growth Is an Accurate Method for Antimicrobial Susceptibility Testing with Reduced Turnaround Time.

Disk diffusion is a slow but reliable standard method for measuring the antimicrobial susceptibility of microorganisms. Our objective was to improve the turnaround time for this method by reducing the time that cultures are incubated before setting up disk diffusion testing. For initial method development, clinical isolates (n = 13) and quality control strains (n = 8) of bacteria were inoculated on blood agar and were incubated at 35°C for either 6, 10, or 24 h before performing disk diffusion testing, in triplicate, using a panel of clinically appropriate antimicrobial agents. Disk diffusion zone sizes were interpreted using Clinical and Laboratory Standards Institute (CLSI) guidelines. Compared to standard 24 h of incubation, early 6-h growth had 1.3% major errors (MEs) and 1.9% very major errors (VMEs), whereas 10-h growth yielded 0.7% MEs and no VMEs. Categorical agreement with standard incubation was similar for both 6 h (96.7%) and 10 h (96.7%) growth. Inhibitory zone size from 6 h (r2 = 0.98) and 10 h (r2 = 0.99) growth correlated well with results from standard conditions. Based on these results, we performed disk diffusion under optimized conditions (6 h growth), using 100 additional clinical isolates, demonstrating a high level of categorical agreement (917 of 950 measurements [96.5%]; 95% confidence interval [CI], 95.2 to 97.5%), as well as no VMEs or MEs. Using early growth for disk diffusion testing is a simple and accurate method for susceptibility testing that can reduce time to results by as much as 18 h, compared to standard incubation, with no additional supply costs or equipment/instrumentation.

Evaluation of the BioFire FilmArray Pneumonia Panel for Detection of Viral and Bacterial Pathogens in Lower Respiratory Tract Specimens in the Setting of a Tertiary Care Academic Medical Center.

Our objective was to evaluate the diagnostic yield and accuracy of the BioFire FilmArray pneumonia panel (BFPP) for identification of pathogens in lower respiratory tract specimens (n = 200) from emergency department (ED) and intensive care unit (ICU) patients at a tertiary care academic medical center. Specimens were collected between January and November 2018, from patients ≥18 years of age, and culture was performed as part of standard-of-care testing. The BFPP identified a viral or bacterial target in 117/200 (58.5%) samples, including Staphylococcus aureus in 22% of samples and Haemophilus influenzae in 14%, and both a viral and bacterial target in 4% of samples. The most common viruses detected by BFPP were rhinovirus/enterovirus (4.5%), influenza A virus (3%), and respiratory syncytial virus (RSV) (2%). Overall, there was strong correlation between BFPP and standard methods for detection of viruses (99.2%) and bacteria (96.8%). Most bacteria (60/61 [98.4%]) detected by standard methods were also identified by BFPP, and 92 additional bacteria were identified by BFPP alone, including 22/92 (23.9%) additional S. aureus isolates and 25/92 (27.2%) H. influenzae isolates, which were more frequently discordant when detected at low concentrations (S. aureus, P < 0.001; H. influenzae, P < 0.0001) and in sputum-type specimens (S. aureus, P < 0.05). A potential limitation of the BFPP assay is the absence of fungal targets and Stenotrophomonas maltophilia, which were detected in 26 and 4 of 200 specimens, respectively. Real-time specimen analysis with BFPP has the potential to identify bacterial pathogens and resistance markers 44.2 and 56.3 h faster than culture-based methods. The BFPP is a rapid and accurate method for detection of pathogens from lower respiratory tract infections.

Parameters for Validating a Hospital Pneumatic Tube System.

BACKGROUND: Pneumatic tube systems (PTSs) provide rapid transport of patient blood samples, but physical stress of PTS transport can damage blood cells and alter test results. Despite this knowledge, there is limited information on how to validate a hospital PTS. METHODS: We compared 2 accelerometers and evaluated multiple PTS routes. Variabilities in PTS forces over the same routes were assessed. Response curves that demonstrate the relationship between the number and magnitude of accelerations on plasma lactate dehydrogenase (LD), hemolysis index, and potassium in PTS-transported blood from volunteers were generated. Extrapolations from these relationships were used to predict PTS routes that may be prone to false laboratory results. Historical data and prospective patient studies were compared with predicted effects. RESULTS: The maximum recorded g-force was 10g for the smartphone and 22g for the data logger. There was considerable day-to-day variation in the magnitude of accelerations (CV, 4%-39%) within a single route. The linear relationship between LD and accelerations within the PTS revealed 2 PTS routes predicted to increase LD by ≥20%. The predicted increase in LD was similar to that observed in patient results when using that PTS route. CONCLUSIONS: Hospital PTSs can be validated by documenting the relationship between the concentrations of analytes in plasma, such as LD, with PTS forces recorded by 3-axis accelerometers. Implementation of this method for PTS validation is relatively inexpensive, simple, and robust.

Ultrafast acoustic phonon scattering in CH3NH3PbI3 revealed by femtosecond four-wave mixing.

Carrier scattering processes are studied in CH3NH3PbI3 using temperature-dependent four-wave mixing experiments. Our results indicate that scattering by ionized impurities limits the interband dephasing time (T2) below 30 K, with strong electron-phonon scattering dominating at higher temperatures (with a time scale of 125 fs at 100 K). Our theoretical simulations provide quantitative agreement with the measured carrier scattering rate and show that the rate of acoustic phonon scattering is enhanced by strong spin-orbit coupling, which modifies the band-edge density of states. The Rashba coefficient extracted from fitting the experimental results (γc = 2 eV Å) is in agreement with calculations of the surface Rashba effect and recent experiments using the photogalvanic effect on thin films.

Developments in our understanding of the genetic basis of birth defects.

Birth defects are a major cause of morbidity and mortality worldwide. There has been much progress in understanding the genetic basis of familial and syndromic forms of birth defects. However, the etiology of nonsydromic birth defects is not well-understood. Although there is still much work to be done, we have many of the tools needed to accomplish the task. Advances in next-generation sequencing have introduced a sea of possibilities, from disease-gene discovery to clinical screening and diagnosis. These advances have been fruitful in identifying a host of candidate disease genes, spanning the spectrum of birth defects. With the advent of CRISPR-Cas9 gene editing, researchers now have a precise tool for characterizing this genetic variation in model systems. Work in model organisms has also illustrated the importance of epigenetics in human development and birth defects etiology. Here we review past and current knowledge in birth defects genetics. We describe genotyping and sequencing methods for the detection and analysis of rare and common variants. We remark on the utility of model organisms and explore epigenetics in the context of structural malformation. We conclude by highlighting approaches that may provide insight into the complex genetics of birth defects.

Vitamin D supplementation does not improve human skeletal muscle contractile properties in insufficient young males.

PURPOSE: Vitamin D may be a regulator of skeletal muscle function, although human trials investigating this hypothesis are limited to predominantly elderly populations. We aimed to assess the effect of oral vitamin D3 in healthy young males upon skeletal muscle function. METHODS: Participants (n = 29) received an oral dose of 10,000 IU day(-1) vitamin D3 (VITD) or a visually identical placebo (PLB) for 3 months. Serum 25[OH]D and intact parathyroid hormone (iPTH) were measured at baseline and at week 4, 8 and 12. Muscle function was assessed in n = 22 participants by isokinetic dynamometry and percutaneous isometric electromyostimulation at baseline and at week 6 and 12. RESULTS: Baseline mean total serum 25[OH]D was 40 ± 17 and 41 ± 20 nmol L(-1) for PLB and VITD, respectively. VITD showed a significant improvement in total 25[OH]D at week 4 (150 ± 31 nmol L(-1)) that remained elevated throughout the trial (P < 0.005). Contrastingly, PLB showed a significant decrease in 25[OH]D at week 12 (25 ± 15 nmol L(-1)) compared with baseline. Despite marked increases in total serum 25[OH]D in VITD and a decrease in PLB, there were no significant changes in any of the muscle function outcome measures at week 6 or 12 for either group (P > 0.05). CONCLUSIONS: Elevating total serum 25[OH]D to concentrations > 120 nmol L(-1) has no effect on skeletal muscle function. We postulate that skeletal muscle function is only perturbed in conditions of severe deficiency (<12.5 nmol L(-1)).

A multi-glycomic platform for the analysis of food carbohydrates.

Carbohydrates comprise the largest fraction of most diets and exert a profound impact on health. Components such as simple sugars and starch supply energy, while indigestible components, deemed dietary fiber, reach the colon to provide food for the tens of trillions of microbes that make up the gut microbiota. The interactions between dietary carbohydrates, our gastrointestinal tracts, the gut microbiome and host health are dictated by their structures. However, current methods for analysis of food glycans lack the sensitivity, specificity and throughput needed to quantify and elucidate these myriad structures. This protocol describes a multi-glycomic approach to food carbohydrate analysis in which the analyte might be any food item or biological material such as fecal and cecal samples. The carbohydrates are extracted by ethanol precipitation, and the resulting samples are subjected to rapid-throughput liquid chromatography (LC)-tandem mass spectrometry (LC-MS/MS) methods. Quantitative analyses of monosaccharides, glycosidic linkages, polysaccharides and alcohol-soluble carbohydrates are performed in 96-well plates at the milligram scale to reduce the biomass of sample required and enhance throughput. Detailed stepwise processes for sample preparation, LC-MS/MS and data analysis are provided. We illustrate the application of the protocol to a diverse set of foods as well as different apple cultivars and various fermented foods. Furthermore, we show the utility of these methods in elucidating glycan-microbe interactions in germ-free and colonized mice. These methods provide a framework for elucidating relationships between dietary fiber, the gut microbiome and human physiology. These structures will further guide nutritional and clinical feeding studies that enhance our understanding of the role of diet in nutrition and health.

A microbiome-directed therapeutic food for children recovering from severe acute malnutrition.

Severe acute malnutrition (SAM), defined anthropometrically as a weight-for-length z-score more than 3 standard deviations below the mean (WLZ<-3), affects 19 million children under 5-years-old worldwide. Complete anthropometric recovery after standard inventions is rare with children often left with moderate acute malnutrition (MAM; WLZ -2 to -3). Here we conduct a randomized controlled trial (RCT), involving 12-18-month-old Bangladeshi children from urban and rural sites, who after hospital-based treatment for SAM received a 3-month intervention with a microbiota-directed complementary food (MDCF-2) or a ready-to-use supplementary food (RUSF) as they transitioned to MAM. The rate of WLZ improvement was significantly greater with MDCF-2 than the more calorically-dense RUSF, as we observed in a previous RCT of Bangladeshi children with MAM without antecedent SAM. A correlated meta-analysis of aptamer-based measurements of 4,520 plasma proteins in this and the prior RCT revealed 215 proteins positively-associated with WLZ (prominently those involved in musculoskeletal and CNS development) and 44 negatively-associated proteins (related to immune activation), with a significant enrichment in levels of the positively WLZ-associated proteins in the MDCF-2 arm. Characterizing changes in 754 bacterial metagenome-assembled genomes in serially collected fecal samples disclosed the effects of acute rehabilitation for SAM on the microbiome, its transition as each child achieves a state of MAM, and how specific strains of Prevotella copri function at the intersection between MDCF-2 glycan metabolism and the rescue of growth faltering. These results provide a rationale for further testing the generalizability of the efficacy of MDCF and identify biomarkers for defining treatment responses.

Prevotella copri and microbiota members mediate the beneficial effects of a therapeutic food for malnutrition.

Microbiota-directed complementary food (MDCF) formulations have been designed to repair the gut communities of malnourished children. A randomized controlled trial demonstrated that one formulation, MDCF-2, improved weight gain in malnourished Bangladeshi children compared to a more calorically dense standard nutritional intervention. Metagenome-assembled genomes from study participants revealed a correlation between ponderal growth and expression of MDCF-2 glycan utilization pathways by Prevotella copri strains. To test this correlation, here we use gnotobiotic mice colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains, with or without P. copri isolates closely matching the metagenome-assembled genomes. Combining gut metagenomics and metatranscriptomics with host single-nucleus RNA sequencing and gut metabolomic analyses, we identify a key role of P. copri in metabolizing MDCF-2 glycans and uncover its interactions with other microbes including Bifidobacterium infantis. P. copri-containing consortia mediated weight gain and modulated energy metabolism within intestinal epithelial cells. Our results reveal structure-function relationships between MDCF-2 and members of the gut microbiota of malnourished children with potential implications for future therapies.

Gene-Folic Acid Interactions and Risk of Conotruncal Heart Defects: Results from the National Birth Defects Prevention Study.

Conotruncal heart defects (CTDs) are heart malformations that affect the cardiac outflow tract and typically cause significant morbidity and mortality. Evidence from epidemiological studies suggests that maternal folate intake is associated with a reduced risk of heart defects, including CTD. However, it is unclear if folate-related gene variants and maternal folate intake have an interactive effect on the risk of CTDs. In this study, we performed targeted sequencing of folate-related genes on DNA from 436 case families with CTDs who are enrolled in the National Birth Defects Prevention Study and then tested for common and rare variants associated with CTD. We identified risk alleles in maternal MTHFS (ORmeta = 1.34; 95% CI 1.07 to 1.67), maternal NOS2 (ORmeta = 1.34; 95% CI 1.05 to 1.72), fetal MTHFS (ORmeta = 1.35; 95% CI 1.09 to 1.66), and fetal TCN2 (ORmeta = 1.38; 95% CI 1.12 to 1.70) that are associated with an increased risk of CTD among cases without folic acid supplementation. We detected putative de novo mutations in genes from the folate, homocysteine, and transsulfuration pathways and identified a significant association between rare variants in MGST1 and CTD risk. Results suggest that periconceptional folic acid supplementation is associated with decreased risk of CTD among individuals with susceptible genotypes.

Deconvolution volumetric additive manufacturing.

Volumetric additive manufacturing techniques are a promising pathway to ultra-rapid light-based 3D fabrication. Their widespread adoption, however, demands significant improvement in print fidelity. Currently, volumetric additive manufacturing prints suffer from systematic undercuring of fine features, making it impossible to print objects containing a wide range of feature sizes, precluding effective adoption in many applications. Here, we uncover the reason for this limitation: light dose spread in the resin due to chemical diffusion and optical blurring, which becomes significant for features ⪅0.5 mm. We develop a model that quantitatively predicts the variation of print time with feature size and demonstrate a deconvolution method to correct for this error. This enables prints previously beyond the capabilities of volumetric additive manufacturing, such as a complex gyroid structure with variable thickness and a fine-toothed gear. These results position volumetric additive manufacturing as a mature 3D printing method, all but eliminating the gap to industry-standard print fidelity.

Prevotella copri-related effects of a therapeutic food for malnutrition.

Preclinical and clinical studies are providing evidence that the healthy growth of infants and children reflects, in part, healthy development of their gut microbiomes1-5. This process of microbial community assembly and functional maturation is perturbed in children with acute malnutrition. Gnotobiotic animals, colonized with microbial communities from children with severe and moderate acute malnutrition, have been used to develop microbiome-directed complementary food (MDCF) formulations for repairing the microbiomes of these children during the weaning period5. Bangladeshi children with moderate acute malnutrition (MAM) participating in a previously reported 3-month-long randomized controlled clinical study of one such formulation, MDCF-2, exhibited significantly improved weight gain compared to a commonly used nutritional intervention despite the lower caloric density of the MDCF6. Characterizing the 'metagenome assembled genomes' (MAGs) of bacterial strains present in the microbiomes of study participants revealed a significant correlation between accelerated ponderal growth and the expression by two Prevotella copri MAGs of metabolic pathways involved in processing of MDCF-2 glycans1. To provide a direct test of these relationships, we have now performed 'reverse translation' experiments using a gnotobiotic mouse model of mother-to-offspring microbiome transmission. Mice were colonized with defined consortia of age- and ponderal growth-associated gut bacterial strains cultured from Bangladeshi infants/children in the study population, with or without P. copri isolates resembling the MAGs. By combining analyses of microbial community assembly, gene expression and processing of glycan constituents of MDCF-2 with single nucleus RNA-Seq and mass spectrometric analyses of the intestine, we establish a principal role for P. copri in mediating metabolism of MDCF-2 glycans, characterize its interactions with other consortium members including Bifidobacterium longum subsp. infantis, and demonstrate the effects of P. copri-containing consortia in mediating weight gain and modulating the activities of metabolic pathways involved in lipid, amino acid, carbohydrate plus other facets of energy metabolism within epithelial cells positioned at different locations in intestinal crypts and villi. Together, the results provide insights into structure/function relationships between MDCF-2 and members of the gut communities of malnourished children; they also have implications for developing future prebiotic, probiotic and/or synbiotic therapeutics for microbiome restoration in children with already manifest malnutrition, or who are at risk for this pervasive health challenge.