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.

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.

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.

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.

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.

Bioactive glycans in a microbiome-directed food for malnourished children.

Evidence is accumulating that perturbed postnatal development of the gut microbiome contributes to childhood malnutrition1-4. Designing effective microbiome-directed therapeutic foods to repair these perturbations requires knowledge about how food components interact with the microbiome to alter its expressed functions. Here we use biospecimens from a randomized, controlled trial of a microbiome-directed complementary food prototype (MDCF-2) that produced superior rates of weight gain compared to a conventional ready-to-use supplementary food (RUSF) in 12-18-month-old Bangladeshi children with moderate acute malnutrition (MAM)4. We reconstructed 1000 bacterial genomes (metagenome-assembled genomes, MAGs) present in their fecal microbiomes, identified 75 whose abundances were positively associated with weight gain (change in weight-for-length Z score, WLZ), characterized gene expression changes in these MAGs as a function of treatment type and WLZ response, and used mass spectrometry to quantify carbohydrate structures in MDCF-2 and feces. The results reveal treatment-induced changes in expression of carbohydrate metabolic pathways in WLZ-associated MAGs. Comparing participants consuming MDCF-2 versus RUSF, and MDCF-2-treated children in the upper versus lower quartiles of WLZ responses revealed that two Prevotella copri MAGs positively associated with WLZ were principal contributors to MDCF-2-induced expression of metabolic pathways involved in utilization of its component glycans. Moreover, the predicted specificities of carbohydrate active enzymes expressed by polysaccharide utilization loci (PULs) in these two MAGs correlate with the (i) in vitro growth of Bangladeshi P. copri strains, possessing differing degrees of PUL and overall genomic content similarity to these MAGs, cultured in defined medium containing different purified glycans representative of those in MDCF-2, and (ii) levels of carbohydrate structures identified in feces from clinical trial participants. In the accompanying paper5, we use a gnotobiotic mouse model colonized with age- and WLZ-associated bacterial taxa cultured from this study population, and fed diets resembling those consumed by study participants, to directly test the relationship between P. copri, MDCF-2 glycan metabolism, host ponderal growth responses, and intestinal gene expression and metabolism. The ability to identify bioactive glycan structures in MDCFs that are metabolized by growth-associated bacterial taxa will help guide recommendations about use of this MDCF for children with acute malnutrition representing different geographic locales and ages, as well as enable development of bioequivalent, or more efficacious, formulations composed of culturally acceptable and affordable ingredients.

Changes in spirituality partly explain health-related quality of life outcomes after Mindfulness-Based Stress Reduction.

Mindfulness-Based Stress Reduction is a secular behavioral medicine program that has roots in meditative spiritual practices. Thus, spirituality may partly explain Mindfulness-Based Stress Reduction outcomes. Participants (N = 279; M (SD) age = 45(12); 75% women) completed an online survey before and after an 8-week Mindfulness-Based Stress Reduction program. Structural equation modeling was used to test the hypothesis that, following Mindfulness-Based Stress Reduction, the relationship between enhanced mindfulness and improved health-related quality of life is mediated by increased daily spiritual experiences. Changes in both spirituality and mindfulness were significantly related to improvement in mental health. Although the initial mediation hypothesis was not supported, an alternate model suggested that enhanced mindfulness partly mediated the association between increased daily spiritual experiences and improved mental health-related quality of life (indirect effect: ß = 0.07, P = 0.017). Effects on physical health-related quality of life were not significant. Findings suggest a novel mechanism by which increased daily spiritual experiences following Mindfulness-Based Stress Reduction may partially explain improved mental health as a function of greater mindfulness.

Modifiable disease risk, readiness to change, and psychosocial functioning improve with integrative medicine immersion model.

BACKGROUND: Stroke, diabetes, and coronary heart disease (CHD) remain leading causes of death in the United States and are largely attributable to lifestyle behaviors. Integrative medicine can provide a supportive partnership that focuses on improving health by identifying and implementing lifestyle changes based upon personal values and goals. OBJECTIVE: This prospective observational study was designed to assess the effectiveness of an integrative medicine intervention on modifiable disease risk, patient activation, and psychosocial risk factors for stroke, diabetes, and CHD. DESIGN: Sixty-three adults participated in a 3-day comprehensive, multimodal health immersion program at Duke Integrative Medicine, Duke University Medical Center, Durham, North Carolina. Participants received follow-up education, physician support, and telephonic health coaching between the immersion program and the endpoint 7 to 9 months later. PRIMARY OUTCOME MEASURES: Psychosocial functioning, readiness to change health behaviors, and risk of developing diabetes, stroke, and CHD were assessed at baseline and endpoint. RESULTS: Although cardiac risk remained unchanged (P = .19) during the study period, risk of diabetes (P = .02) and stroke (P < .01) decreased significantly. Perceived stress remained unchanged, but improvements were seen in mood (P < .05) and relationship satisfaction (P < .004). Patients became more activated towards self-management of health (P <.001), endorsed greater readiness to change health behaviors (P <.01), and reported increased aerobic exercise (P <.001) and stretching (P = .006) following the intervention. CONCLUSION: An integrative health model can help patients become more engaged in self-management of health and support them in making and maintaining healthy lifestyle changes. These findings provide support for use of an integrative health model in adult disease risk reduction.

Gene-by-gene interactions associated with the risk of conotruncal heart defects.

BACKGROUND: The development of conotruncal heart defects (CTDs) involves a complex relationship among genetic variants and maternal lifestyle factors. In this article, we focused on the interactions between 13 candidate genes within folate, homocysteine, and transsulfuration pathways for potential association with CTD risk. METHODS: Targeted sequencing was used for 328 case-parental triads enrolled in the National Birth Defects Prevention Study (NBDPS). To evaluate the interaction of two genes, we applied a conditional logistic regression model for all possible SNP pairs within two respective genes by contrasting the affected infants with their pseudo-controls. The findings were replicated in an independent sample of 86 NBDPS case-parental triads genotyped by DNA microarrays. The results of two studies were further integrated by a fixed-effect meta-analysis. RESULTS: One SNP pair (i.e., rs4764267 and rs6556883) located in gene MGST1 and GLRX, respectively, was found to be associated with CTD risk after multiple testing adjustment using simpleM, a modified Bonferroni correction approach (nominal p-value of 4.62e-06; adjusted p-value of .04). Another SNP pair (i.e., rs11892646 and rs56219526) located in gene DNMT3A and MTRR, respectively, achieved marginal significance after multiple testing adjustment (adjusted p-value of .06). CONCLUSION: Further studies with larger sample sizes are needed to confirm and elucidate these potential interactions.

Mindfulness Meditation Targets Transdiagnostic Symptoms Implicated in Stress-Related Disorders: Understanding Relationships between Changes in Mindfulness, Sleep Quality, and Physical Symptoms.

Mindfulness-Based Stress Reduction (MBSR) is an 8-week meditation program known to improve anxiety, depression, and psychological well-being. Other health-related effects, such as sleep quality, are less well established, as are the psychological processes associated with therapeutic change. This prospective, observational study (n = 213) aimed to determine whether perseverative cognition, indicated by rumination and intrusive thoughts, and emotion regulation, measured by avoidance, thought suppression, emotion suppression, and cognitive reappraisal, partly accounted for the hypothesized relationship between changes in mindfulness and two health-related outcomes: sleep quality and stress-related physical symptoms. As expected, increased mindfulness following the MBSR program was directly correlated with decreased sleep disturbance (r = -0.21, p = 0.004) and decreased stress-related physical symptoms (r = -0.38, p < 0.001). Partial correlations revealed that pre-post changes in rumination, unwanted intrusive thoughts, thought suppression, experiential avoidance, emotion suppression, and cognitive reappraisal each uniquely accounted for up to 32% of the correlation between the change in mindfulness and change in sleep disturbance and up to 30% of the correlation between the change in mindfulness and change in stress-related physical symptoms. Results suggest that the stress-reducing effects of MBSR are due, in part, to improvements in perseverative cognition and emotion regulation, two "transdiagnostic" mental processes that cut across stress-related disorders.

Phenolic profile and antioxidant activity of extracts prepared from fermented heat-stabilized defatted rice bran.

Heat-stabilized, defatted rice bran (HDRB) serves as a potential source of phenolic compounds which have numerous purported health benefits. An estimated 70% of phenolics present in rice bran are esterified to the arabinoxylan residues of the cell walls. Release of such compounds could provide a value-added application for HDRB. The objective of this study was to extract and quantify phenolics from HDRB using fermentation technology. Out of 8 organisms selected for rice bran fermentation, Bacillus subtilis subspecies subtilis had the maximum phenolic release of 26.8 mg ferulic acid equivalents (FAE) per gram HDRB. Response surface methodology was used to further optimize the release of rice bran phenolics. An optimum of 28.6 mg FAE/g rice bran was predicted at 168 h, 0.01% inoculation level, and 100 mg HDRB/mL. Fermentation of HDRB for 96 h with B. subtilis subspecies subtilis resulted in a significant increase in phenolic yield, phenolic concentration, and radical scavenging capacity. Fermented rice bran had 4.86 mg gentistic acid, 1.38 mg caffeic acid, 6.03 mg syringic acid, 19.02 mg (-)-epicatechin, 4.08 mg p-courmaric acid, 4.64 mg ferulic acid, 10.04 mg sinapic acid, and 17.59 mg benzoic acid per 100 g fermented extract compared to 0.65 mg p-courmaric acid and 0.36 mg ferulic acid per 100 g nonfermented extract. The high phenolic content and antioxidant activity of fermented HDRB extract indicates that rice bran fermentation under optimized condition is a potential means of meeting the demand for an effective and affordable antioxidant.