Host-Specific Evolutionary and Transmission Dynamics Shape the Functional Diversification of Staphylococcus epidermidis in Human Skin.

Metagenomic inferences of bacterial strain diversity and infectious disease transmission studies largely assume a dominant, within-individual haplotype. We hypothesize that within-individual bacterial population diversity is critical for homeostasis of a healthy microbiome and infection risk. We characterized the evolutionary trajectory and functional distribution of Staphylococcus epidermidis-a keystone skin microbe and opportunistic pathogen. Analyzing 1,482 S. epidermidis genomes from 5 healthy individuals, we found that skin S. epidermidis isolates coalesce into multiple founder lineages rather than a single colonizer. Transmission events, natural selection, and pervasive horizontal gene transfer result in population admixture within skin sites and dissemination of antibiotic resistance genes within-individual. We provide experimental evidence for how admixture can modulate virulence and metabolism. Leveraging data on the contextual microbiome, we assess how interspecies interactions can shape genetic diversity and mobile gene elements. Our study provides insights into how within-individual evolution of human skin microbes shapes their functional diversification.

DART.2: bidirectional synaptic pharmacology with thousandfold cellular specificity.

Precision pharmacology aims to manipulate specific cellular interactions within complex tissues. In this pursuit, we introduce DART.2 (drug acutely restricted by tethering), a second-generation cell-specific pharmacology technology. The core advance is optimized cellular specificity-up to 3,000-fold in 15 min-enabling the targeted delivery of even epileptogenic drugs without off-target effects. Additionally, we introduce brain-wide dosing methods as an alternative to local cannulation and tracer reagents for brain-wide dose quantification. We describe four pharmaceuticals-two that antagonize excitatory and inhibitory postsynaptic receptors, and two that allosterically potentiate these receptors. Their versatility is showcased across multiple mouse-brain regions, including cerebellum, striatum, visual cortex and retina. Finally, in the ventral tegmental area, we find that blocking inhibitory inputs to dopamine neurons accelerates locomotion, contrasting with previous optogenetic and pharmacological findings. Beyond enabling the bidirectional perturbation of chemical synapses, these reagents offer intersectional precision-between genetically defined postsynaptic cells and neurotransmitter-defined presynaptic partners.

Attenuation hotspots in neurotropic human astroviruses.

During the last decade, the detection of neurotropic astroviruses has increased dramatically. The MLB genogroup of astroviruses represents a genetically distinct group of zoonotic astroviruses associated with gastroenteritis and severe neurological complications in young children, the immunocompromised, and the elderly. Using different virus evolution approaches, we identified dispensable regions in the 3' end of the capsid-coding region responsible for attenuation of MLB astroviruses in susceptible cell lines. To create recombinant viruses with identified deletions, MLB reverse genetics (RG) and replicon systems were developed. Recombinant truncated MLB viruses resulted in imbalanced RNA synthesis and strong attenuation in iPSC-derived neuronal cultures confirming the location of neurotropism determinants. This approach can be used for the development of vaccine candidates using attenuated astroviruses that infect humans, livestock animals, and poultry.

Baby food pouches and Baby-Led Weaning: Associations with energy intake, eating behaviour and infant weight status.

Although concern is frequently expressed regarding the potential impact of baby food pouch use and Baby-Led Weaning (BLW) on infant health, research is scarce. Data on pouch use, BLW, energy intake, eating behaviour and body mass index (BMI) were obtained for 625 infants aged 7-10 months in the First Foods New Zealand study. Frequent pouch use was defined as ≥5 times/week during the past month. Traditional spoon-feeding (TSF), "partial" BLW and "full" BLW referred to the relative proportions of spoon-feeding versus infant self-feeding, assessed at 6 months (retrospectively) and current age. Daily energy intake was determined using two 24-h dietary recalls, and caregivers reported on a variety of eating behaviours. Researchers measured infant length and weight, and BMI z-scores were calculated (World Health Organization Child Growth Standards). In total, 28% of infants consumed food from pouches frequently. Frequent pouch use was not significantly related to BMI z-score (mean difference, 0.09; 95% CI -0.09, 0.27) or energy intake (92 kJ/day; -19, 202), but was associated with greater food responsiveness (standardised mean difference, 0.3; 95% CI 0.1, 0.4), food fussiness (0.3; 0.1, 0.4) and selective/restrictive eating (0.3; 0.2, 0.5). Compared to TSF, full BLW was associated with greater daily energy intake (BLW at 6 months: mean difference 150 kJ/day; 95% CI 4, 297; BLW at current age: 180 kJ/day; 62, 299) and with a range of eating behaviours, including greater satiety responsiveness, but not BMI z-score (6 months: 0.06 (-0.18, 0.30); current age: 0.06 (-0.13, 0.26)). In conclusion, neither feeding approach was associated with weight in infants, despite BLW being associated with greater energy intake compared with TSF. However, infants who consumed pouches frequently displayed higher food fussiness and more selective eating.

Skin microbiome attributes associate with biophysical skin ageing.

Two major arms of skin ageing are changes in the skin's biophysical conditions and alterations in the skin microbiome. This work partitioned both arms to study their interaction in detail. Leveraging the resolution provided by shotgun metagenomics, we explored how skin microbial species, strains and gene content interact with the biophysical traits of the skin during ageing. With a dataset well-controlled for confounding factors, we found that skin biophysical traits, especially the collagen diffusion coefficient, are associated with the composition and the functional potential of the skin microbiome, including the abundance of bacterial strains found in nosocomial infections and the abundance of antibiotic resistance genes. Our findings reveal important associations between skin biophysical features and ageing-related changes in the skin microbiome and generate testable hypotheses for the mechanisms of such associations.

Selenium intakes and plasma selenium of New Zealand toddlers: secondary analysis of a randomised controlled trial.

Little is known about Se intakes and status in very young New Zealand children. However, Se intakes below recommendations and lower Se status compared with international studies have been reported in New Zealand (particularly South Island) adults. The Baby-Led Introduction to SolidS (BLISS) randomised controlled trial compared a modified version of baby-led weaning (infants feed themselves rather than being spoon-fed), with traditional spoon-feeding (Control). Weighed 3-d diet records were collected and plasma Se concentration measured using inductively coupled plasma mass spectrometry (ICP-MS). In total, 101 (BLISS n 50, Control n 51) 12-month-old toddlers provided complete data. The OR of Se intakes below the estimated average requirement (EAR) was no different between BLISS and Control (OR: 0·89; 95 % CI 0·39, 2·03), and there was no difference in mean plasma Se concentration between groups (0·04 µmol/l; 95 % CI -0·03, 0·11). In an adjusted model, consuming breast milk was associated with lower plasma Se concentrations (-0·12 µmol/l; 95 % CI -0·19, -0·04). Of the food groups other than infant milk (breast milk or infant formula), 'breads and cereals' contributed the most to Se intakes (12 % of intake). In conclusion, Se intakes and plasma Se concentrations of 12-month-old New Zealand toddlers were no different between those who had followed a baby-led approach to complementary feeding and those who followed traditional spoon-feeding. However, more than half of toddlers had Se intakes below the EAR.

Sodium in the New Zealand diet: proposed voluntary food reformulation targets will not meet the WHO goal of a 30% reduction in total sodium intake.

PURPOSE: To simulate the potential impact of the HeartSAFE 2020 programme, a food reformulation initiative by the New Zealand (NZ) Heart Foundation, on sodium intake in the NZ adult population. METHODS: A representative sample of NZ adults aged 15 years and older completed a 24-h diet recall survey, with 25% of participants completing a second diet recall, in the 2008/09 New Zealand Adult Nutrition Survey (n = 4721). These data were used to estimate sodium intakes of participants. The effect of altering the sodium content of 840 foods in 17 categories and 35 sub-categories included in the NZ HeartSAFE 2020 programme was simulated. The simulated sodium intake reductions in each food sub-category for the entire sample were calculated. Using sampling weights, simulated reductions in population sodium intake and by sociodemographic subgroups were also analysed. RESULTS: Sodium intake from foods included in the HeartSAFE 2020 programme was 1307 mg/day (95% CI 1279, 1336) at baseline. After applying the HeartSAFE 2020 targets, potential sodium intake was 1048 mg/day (95% CI 1024, 1027). The absolute sodium reduction was 260 mg/day (95% CI 252, 268), corresponding to 20% sodium reduction for the foods included in the NZ HeartSAFE programme. CONCLUSION: Current sodium targets featured in the NZ HeartSAFE programme will not meet the 30% sodium intake reduction set out by the WHO Global Action Plan. A more comprehensive strategy consistent with the WHO SHAKE Technical Package is needed to advance the goal of sodium intake reduction.

Peanut protein acts as a TH2 adjuvant by inducing RALDH2 in human antigen-presenting cells.

BACKGROUND: Peanut is a potent inducer of proallergenic TH2 responses in susceptible individuals. Antigen-presenting cells (APCs) including dendritic cells and monocytes instruct naive T cells to differentiate into various effector cells, determining immune responses such as allergy and tolerance. OBJECTIVE: We sought to detect peanut protein (PN)-induced changes in gene expression in human myeloid dendritic cells (mDCs) and monocytes, identify signaling receptors that mediate these changes, and assess how PN-induced genes in mDCs impact their ability to promote T-cell differentiation. METHODS: mDCs, monocytes, and naive CD4+ T cells were isolated from blood bank donors and peanut-allergic patients. APCs were incubated with PN and other stimulants, and gene expression was measured using microarray and RT quantitative PCR. To assess T-cell differentiation, mDCs were cocultured with naive TH cells. RESULTS: PN induced a unique gene expression profile in mDCs, including the gene that encodes retinaldehyde dehydrogenase 2 (RALDH2), a rate-limiting enzyme in the retinoic acid (RA)-producing pathway. Stimulation of mDCs with PN also induced a 7-fold increase in the enzymatic activity of RALDH2. Blocking antibodies against Toll-like receptor (TLR)1/TLR2, as well as small interfering RNA targeting TLR1/TLR2, reduced the expression of RALDH2 in PN-stimulated APCs by 70%. Naive TH cells cocultured with PN-stimulated mDCs showed an RA-dependent 4-fold increase in production of IL-5 and expression of integrin α4ß7. CONCLUSIONS: PN induces RALDH2 in human APCs by signaling through the TLR1/TLR2 heterodimer. This leads to production of RA, which acts on TH cells to induce IL-5 and gut-homing integrin. RALDH2 induction by PN in APCs and RA-promoted TH2 differentiation could be an important factor determining allergic responses to peanut.

Innate Intracellular Antiviral Responses Restrict the Amplification of Defective Virus Genomes of Parainfluenza Virus 5.

During the replication of parainfluenza virus 5 (PIV5), copyback defective virus genomes (DVGs) are erroneously produced and are packaged into "infectious" virus particles. Copyback DVGs are the primary inducers of innate intracellular responses, including the interferon (IFN) response. While DVGs can interfere with the replication of nondefective (ND) virus genomes and activate the IFN-induction cascade before ND PIV5 can block the production of IFN, we demonstrate that the converse is also true, i.e., high levels of ND virus can block the ability of DVGs to activate the IFN-induction cascade. By following the replication and amplification of DVGs in A549 cells that are deficient in a variety of innate intracellular antiviral responses, we show that DVGs induce an uncharacterized IFN-independent innate response(s) that limits their replication. High-throughput sequencing was used to characterize the molecular structure of copyback DVGs. While there appears to be no sequence-specific break or rejoining points for the generation of copyback DVGs, our findings suggest there are region, size, and/or structural preferences selected for during for their amplification.IMPORTANCE Copyback defective virus genomes (DVGs) are powerful inducers of innate immune responses both in vitro and in vivo They impact the outcome of natural infections, may help drive virus-host coevolution, and promote virus persistence. Due to their potent interfering and immunostimulatory properties, DVGs may also be used therapeutically as antivirals and vaccine adjuvants. However, little is known of the host cell restrictions which limit their amplification. We show here that the generation of copyback DVGs readily occurs during parainfluenza virus 5 (PIV5) replication, but that their subsequent amplification is restricted by the induction of innate intracellular responses. Molecular characterization of PIV5 copyback DVGs suggests that while there are no genome sequence-specific breaks or rejoin points for the generation of copyback DVGs, genome region, size, and structural preferences are selected for during their evolution and amplification.

The switch between acute and persistent paramyxovirus infection caused by single amino acid substitutions in the RNA polymerase P subunit.

Paramyxoviruses can establish persistent infections both in vitro and in vivo, some of which lead to chronic disease. However, little is known about the molecular events that contribute to the establishment of persistent infections by RNA viruses. Using parainfluenza virus type 5 (PIV5) as a model we show that phosphorylation of the P protein, which is a key component of the viral RNA polymerase complex, determines whether or not viral transcription and replication becomes repressed at late times after infection. If the virus becomes repressed, persistence is established, but if not, the infected cells die. We found that single amino acid changes at various positions within the P protein switched the infection phenotype from lytic to persistent. Lytic variants replicated to higher titres in mice than persistent variants and caused greater infiltration of immune cells into infected lungs but were cleared more rapidly. We propose that during the acute phases of viral infection in vivo, lytic variants of PIV5 will be selected but, as the adaptive immune response develops, variants in which viral replication can be repressed will be selected, leading to the establishment of prolonged, persistent infections. We suggest that similar selection processes may operate for other RNA viruses.

Cultivation of common bacterial species and strains from human skin, oral, and gut microbiota.

BACKGROUND: Genomics-driven discoveries of microbial species have provided extraordinary insights into the biodiversity of human microbiota. In addition, a significant portion of genetic variation between microbiota exists at the subspecies, or strain, level. High-resolution genomics to investigate species- and strain-level diversity and mechanistic studies, however, rely on the availability of individual microbes from a complex microbial consortia. High-throughput approaches are needed to acquire and identify the significant species- and strain-level diversity present in the oral, skin, and gut microbiome. Here, we describe and validate a streamlined workflow for cultivating dominant bacterial species and strains from the skin, oral, and gut microbiota, informed by metagenomic sequencing, mass spectrometry, and strain profiling. RESULTS: Of total genera discovered by either metagenomic sequencing or culturomics, our cultivation pipeline recovered between 18.1-44.4% of total genera identified. These represented a high proportion of the community composition reconstructed with metagenomic sequencing, ranging from 66.2-95.8% of the relative abundance of the overall community. Fourier-Transform Infrared spectroscopy (FT-IR) was effective in differentiating genetically distinct strains compared with whole-genome sequencing, but was less effective as a proxy for genetic distance. CONCLUSIONS: Use of a streamlined set of conditions selected for cultivation of skin, oral, and gut microbiota facilitates recovery of dominant microbes and their strain variants from a relatively large sample set. FT-IR spectroscopy allows rapid differentiation of strain variants, but these differences are limited in recapitulating genetic distance. Our data highlights the strength of our cultivation and characterization pipeline, which is in throughput, comparisons with high-resolution genomic data, and rapid identification of strain variation.

Stepwise tailoring and test-retest of reproducibility of an ethnic-specific FFQ to estimate nutrient intakes for South Asians in New Zealand.

OBJECTIVE: To develop and test-retest the reproducibility of an ethnic-specific FFQ to estimate nutrient intakes for South Asians (SA) in New Zealand (NZ). DESIGN: Using culturally appropriate methods, the NZFFQ, a validated dietary assessment tool for NZ adults, was modified to include SA food items by analysing foods consumed by SA participants of the Adult Nutrition Survey, in-person audit of ethnic food stores and a web scan of ethnic food store websites in NZ. This was further refined via three focus group discussions, and the resulting New Zealand South Asian Food Frequency Questionnaire (NZSAFFQ) was tested for reproducibility. SETTING: Auckland and Dunedin, NZ. PARTICIPANTS: Twenty-nine and 110 males and females aged 25-59 years of SA ethnicity participated in the focus group discussions and the test-retest, respectively. RESULTS: The development phase resulted in a SA-specific FFQ comprising of 11 food groups and 180 food items. Test-retest of the NZSAFFQ showed good reproducibility between the two FFQ administrations, 6 months apart. Most reproducibility coefficients were within or higher than the acceptable range of 0·5-0·7. The lowest intraclass correlation coefficients (ICC) were observed for ß-carotene (0·47), vitamin B12 (0·50), fructose (0·55), vitamin C (0·57) and selenium (0·58), and the highest ICC were observed for alcohol (0·81), iodine (0·79) and folate (0·77). The ICC for fat ranged from 0·70 for saturated fats to 0·77 for polyunsaturated fats. The ICC for protein and energy were 0·68 and 0·72, respectively. CONCLUSIONS: The developed FFQ showed good reproducibility to estimate nutrient intakes and warrants the need for validation of the instrument.

Expansion of the CD4+ effector T-cell repertoire characterizes peanut-allergic patients with heightened clinical sensitivity.

BACKGROUND: Individuals with peanut allergy range in clinical sensitivity: some can consume grams of peanut before experiencing any symptoms, whereas others suffer systemic reactions to 10 mg or less. Current diagnostic testing only partially predicts this clinical heterogeneity. OBJECTIVE: We sought to identify characteristics of the peanut-specific CD4+ T-cell response in peanut-allergic patients that correlate with high clinical sensitivity. METHODS: We studied the T-cell receptor ß-chain (TCRß) usage and phenotypes of peanut-activated, CD154+ CD4+ memory T cells using fluorescence-activated cell sorting, TCRß sequencing, and RNA-Seq, in reactive and hyporeactive patients who were stratified by clinical sensitivity. RESULTS: TCRß analysis of the CD154+ and CD154- fractions revealed more than 6000 complementarity determining region 3 sequences and motifs that were significantly enriched in the activated cells and 17% of the sequences were shared between peanut-allergic individuals, suggesting strong convergent selection of peanut-specific clones. These clones were more numerous among the reactive patients, and this expansion was identified within effector, but not regulatory T-cell populations. The transcriptional profile of CD154+ T cells in the reactive group skewed toward a polarized TH2 effector phenotype, and expression of TH2 cytokines strongly correlated with peanut-specific IgE levels. There were, however, also non-TH2-related differences in phenotype. Furthermore, the ratio of peanut-specific clones in the effector versus regulatory T-cell compartment, which distinguished the clinical groups, was independent of specific IgE concentration. CONCLUSIONS: Expansion of the peanut-specific effector T-cell repertoire is correlated with clinical sensitivity, and this observation may be useful to inform our assessment of disease phenotype and to monitor disease longitudinally.

Analysis of Paramyxovirus Transcription and Replication by High-Throughput Sequencing.

We have developed a high-throughput sequencing (HTS) workflow for investigating paramyxovirus transcription and replication. We show that sequencing of oligo(dT)-selected polyadenylated mRNAs, without considering the orientation of the RNAs from which they had been generated, cannot accurately be used to analyze the abundance of viral mRNAs because genomic RNA copurifies with the viral mRNAs. The best method is directional sequencing of infected cell RNA that has physically been depleted of ribosomal and mitochondrial RNA followed by bioinformatic steps to differentiate data originating from genomes from viral mRNAs and antigenomes. This approach has the advantage that the abundance of viral mRNA (and antigenomes) and genomes can be analyzed and quantified from the same data. We investigated the kinetics of viral transcription and replication during infection of A549 cells with parainfluenza virus type 2 (PIV2), PIV3, PIV5, or mumps virus and determined the abundances of individual viral mRNAs and readthrough mRNAs. We found that the mRNA abundance gradients differed significantly between all four viruses but that for each virus the pattern remained relatively stable throughout infection. We suggest that rapid degradation of non-poly(A) mRNAs may be primarily responsible for the shape of the mRNA abundance gradient in parainfluenza virus 3, whereas a combination of this factor and disengagement of RNA polymerase at intergenic sequences, particularly those at the NP:P and P:M gene boundaries, may be responsible in the other viruses.IMPORTANCE High-throughput sequencing (HTS) of virus-infected cells can be used to study in great detail the patterns of virus transcription and replication. For paramyxoviruses, and by analogy for all other negative-strand RNA viruses, we show that directional sequencing must be used to distinguish between genomic RNA and mRNA/antigenomic RNA because significant amounts of genomic RNA copurify with poly(A)-selected mRNA. We found that the best method is directional sequencing of total cell RNA, after the physical removal of rRNA (and mitochondrial RNA), because quantitative information on the abundance of both genomic RNA and mRNA/antigenomes can be simultaneously derived. Using this approach, we revealed new details of the kinetics of virus transcription and replication for parainfluenza virus (PIV) type 2, PIV3, PIV5, and mumps virus, as well as on the relative abundance of the individual viral mRNAs.

Serotonin regulates dynamics of cerebellar granule cell activity by modulating tonic inhibition.

Understanding how afferent information is integrated by cortical structures requires identifying the factors shaping excitation and inhibition within their input layers. The input layer of the cerebellar cortex integrates diverse sensorimotor information to enable learned associations that refine the dynamics of movement. Specifically, mossy fiber afferents relay sensorimotor input into the cerebellum to excite granule cells, whose activity is regulated by inhibitory Golgi cells. To test how this integration can be modulated, we have used an acute brain slice preparation from young adult rats and found that encoding of mossy fiber input in the cerebellar granule cell layer can be regulated by serotonin (5-hydroxytryptamine, 5-HT) via a specific action on Golgi cells. We find that 5-HT depolarizes Golgi cells, likely by activating 5-HT2A receptors, but does not directly act on either granule cells or mossy fibers. As a result of Golgi cell depolarization, 5-HT significantly increases tonic inhibition onto both granule cells and Golgi cells. 5-HT-mediated Golgi cell depolarization is not sufficient, however, to alter the probability or timing of mossy fiber-evoked feed-forward inhibition onto granule cells. Together, increased granule cell tonic inhibition paired with normal feed-forward inhibition acts to reduce granule cell spike probability without altering spike timing. Hence, these data provide a circuit mechanism by which 5-HT can reduce granule cell activity without altering temporal representations of mossy fiber input. Such changes in network integration could enable flexible, state-specific suppression of cerebellar sensorimotor input that should not be learned or enable reversal learning for unwanted associations. NEW & NOTEWORTHY Serotonin (5-hydroxytryptamine, 5-HT) regulates synaptic integration at the input stage of cerebellar processing by increasing tonic inhibition of granule cells. This circuit mechanism reduces the probability of granule cell spiking without altering spike timing, thus suppressing cerebellar input without altering its temporal representation in the granule cell layer.

Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition.

After fertilization, maternal factors direct development and trigger zygotic genome activation (ZGA) at the maternal-to-zygotic transition (MZT). In zebrafish, ZGA is required for gastrulation and clearance of maternal messenger RNAs, which is in part regulated by the conserved microRNA miR-430. However, the factors that activate the zygotic program in vertebrates are unknown. Here we show that Nanog, Pou5f1 (also called Oct4) and SoxB1 regulate zygotic gene activation in zebrafish. We identified several hundred genes directly activated by maternal factors, constituting the first wave of zygotic transcription. Ribosome profiling revealed that nanog, sox19b and pou5f1 are the most highly translated transcription factors pre-MZT. Combined loss of these factors resulted in developmental arrest before gastrulation and a failure to activate >75% of zygotic genes, including miR-430. Our results demonstrate that maternal Nanog, Pou5f1 and SoxB1 are required to initiate the zygotic developmental program and induce clearance of the maternal program by activating miR-430 expression.

Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation.

Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.

Mediation Analysis as a Means of Identifying Dietary Components That Differentially Affect the Fecal Microbiota of Infants Weaned by Modified Baby-Led and Traditional Approaches.

The introduction of "solids" (i.e., complementary foods) to the milk-only diet in early infancy affects the development of the gut microbiota. The aim of this study was to determine whether a "baby-led" approach to complementary feeding that encourages the early introduction of an adult-type diet results in alterations of the gut microbiota composition compared to traditional spoon-feeding. The Baby-Led Introduction to SolidS (BLISS) study randomized 206 infants to BLISS (a modified version of baby-led weaning [BLW], the introduction of solids at 6 months of age, followed by self-feeding of family foods) or control (traditional spoon-feeding of purées) groups. Fecal microbiotas and 3-day weighed-diet records were analyzed for a subset of 74 infants at 7 and 12 months of age. The composition of the microbiota was determined by sequencing of 16S rRNA genes amplified by PCR from bulk DNA extracted from feces. Diet records were used to estimate food and dietary fiber intake. Alpha diversity (number of operational taxonomic units [OTUs]) was significantly lower in BLISS infants at 12 months of age (difference [95% confidence interval {CI}] of 31 OTUs [3.4 to 58.5]; P = 0.028), and while there were no significant differences between control and BLISS infants in relative abundances of Bifidobacteriaceae, Enterobacteriaceae, Veillonellaceae, Bacteroidaceae, Erysipelotrichaceae, Lachnospiraceae, or Ruminococcaceae at 7 or 12 months of age, OTUs representing the genus Roseburia were less prevalent in BLISS microbiotas at 12 months. Mediation models demonstrated that the intake of "fruit and vegetables" and "dietary fiber" explained 29% and 25%, respectively, of the relationship between group (BLISS versus control) and alpha diversity.IMPORTANCE The introduction of solid foods (complementary feeding or weaning) to infants leads to more-complex compositions of microbial communities (microbiota or microbiome) in the gut. In baby-led weaning (BLW), infants are given only finger foods that they can pick up and feed themselves-there is no parental spoon-feeding of puréed baby foods-and infants are encouraged to eat family meals. BLW is a new approach to infant feeding that is increasing in popularity in the United States, New Zealand, the United Kingdom, and Canada. We used mediation modeling, commonly used in health research but not in microbiota studies until now, to identify particular dietary components that affected the development of the infant gut microbiota.

Gaudeamus Igitur at 40-A Tribute and Assessment.

This Arts and Medicine feature discusses the continuing relevance of the 1983 poem Gaudeamus Igitur by John Stone, which offers wisdom and guiding principles about the practice of medicine to newly graduated young physicians.

Notes on Healing After a Missed Diagnosis.

In this narrative medicine essay, a family physician maintains her patient's trust despite missing what could have been a catastrophic diagnosis because she apologized for her oversight.