Associations of Maternal Breastmilk microRNAs and Infant Obesity Status at 1 Year.

Infant consumption of human milk (HM) is associated with a reduced risk of overweight and obesity, but the reasons for this relationship are not completely understood. There is emerging evidence that micro RNAs (miRNAs) regulate infant development and metabolism, but the associations between HM miRNAs and infant growth remain poorly understood. We examined the relationship between HM miRNA consumption and infant obesity in 163 mother-infant dyads to determine (1) if miRNA profiles differentiate infants with obesity, and (2) if individual miRNAs accurately predicted infant obesity status at one year of age. Infant obesity was categorized as weight-for-length (WFL) Z scores or conditional weight gain (CWG) in the 95th percentile. HM miRNA profile was associated with infant age (r2 = 6.4%, p = 0.001), but not maternal obesity status (r2 = 1.5%, p = 0.87) or infant weight status (WFL Z-score) at birth (r2 = 0.6%, p = 0.4), 1 month (r2 = 0.5%, p = 0.6), or 4 months (r2 = 0.8%, p = 0.2). Nine HM miRNAs were associated with either 12-month CWG or 12-month WFL Z scores. Among these 9 miRNAs, miR-224-5p remained significant in a logistic regression model that accounted for additional demographic factors (estimate = -27.57, p = 0.004). These findings suggest involvement of HM miRNAs and particularly miR-224-5p in infant growth, warranting further investigation. To our knowledge, this is the largest study of HM miRNAs and early-life obesity and contributes to the understanding of the relationship between HM miRNAs and infant growth.

Visual Impairment in Pre-Clinical Models of Mild Traumatic Brain Injury.

Impairment in visual function is common after traumatic brain injury (TBI) in the clinical setting, a phenomenon that translates to pre-clinical animal models as well. In Morris et al. (2021), we reported histological changes following weight-drop-induced TBI in a rodent model including retinal ganglion cell (RGC) loss, decreased electroretinogram (ERG) evoked potential, optic nerve diameter reduction, induced inflammation and gliosis, and loss of myelin accompanied by markedly impaired visual acuity. In this review, we will describe several pre-clinical TBI models that result in injuries to the visual system, indicating that visual function may be impaired following brain injury induced by a number of different injury modalities. This underscores the importance of understanding the role of the visual system and the potential detrimental sequelae to this sensory modality post-TBI. Given that most commonly employed behavioral tests such as the Elevated Plus Maze and Morris Water Maze rely on an intact visual system, interpretation of functional deficits in diffuse models may be confounded by off- target effects on the visual system.

Preclinical characterization of macrophage-adhering gadolinium micropatches for MRI contrast after traumatic brain injury in pigs.

The choroid plexus (ChP) of the brain plays a central role in orchestrating the recruitment of peripheral leukocytes into the central nervous system (CNS) through the blood-cerebrospinal fluid (BCSF) barrier in pathological conditions, thus offering a unique niche to diagnose CNS disorders. We explored whether magnetic resonance imaging of the ChP could be optimized for mild traumatic brain injury (mTBI). mTBI induces subtle, yet influential, changes in the brain and is currently severely underdiagnosed. We hypothesized that mTBI induces sufficient alterations in the ChP to cause infiltration of circulating leukocytes through the BCSF barrier and developed macrophage-adhering gadolinium [Gd(III)]-loaded anisotropic micropatches (GLAMs), specifically designed to image infiltrating immune cells. GLAMs are hydrogel-based discoidal microparticles that adhere to macrophages without phagocytosis. We present a fabrication process to prepare GLAMs at scale and demonstrate their loading with Gd(III) at high relaxivities, a key indicator of their effectiveness in enhancing image contrast and clarity in medical imaging. In vitro experiments with primary murine and porcine macrophages demonstrated that GLAMs adhere to macrophages also under shear stress and did not affect macrophage viability or functions. Studies in a porcine mTBI model confirmed that intravenously administered macrophage-adhering GLAMs provide a differential signal in the ChP and lateral ventricles at Gd(III) doses 500- to 1000-fold lower than those used in the current clinical standard Gadavist. Under the same mTBI conditions, Gadavist did not offer a differential signal at clinically used doses. Our results suggest that macrophage-adhering GLAMs could facilitate mTBI diagnosis.

The Association between Infant Colic and the Multi-Omic Composition of Human Milk.

Infant colic is a common condition with unclear biologic underpinnings and limited treatment options. We hypothesized that complex molecular networks within human milk (i.e., microbes, micro-ribonucleic acids (miRNAs), cytokines) would contribute to colic risk, while controlling for medical, social, and nutritional variables. This hypothesis was tested in a cohort of 182 breastfed infants, assessed with a modified Infant Colic Scale at 1 month. RNA sequencing was used to interrogate microbial and miRNA features. Luminex assays were used to measure growth factors and cytokines. Milk from mothers of infants with colic (n = 28) displayed higher levels of Staphylococcus (adj. p = 0.038, d = 0.30), miR-224-3p (adj. p = 0.023, d = 0.33), miR-125b-5p (adj. p = 0.028, d = 0.29), let-7a-5p (adj. p = 0.028, d = 0.27), and miR-205-5p (adj. p = 0.029, d = 0.26) compared to milk from non-colic mother-infant dyads (n = 154). Colic symptom severity was directly associated with milk hepatocyte growth factor levels (R = 0.21, p = 0.025). A regression model involving let-7a-5p, miR-29a-3p, and Lactobacillus accurately modeled colic risk (X2 = 16.7, p = 0.001). Molecular factors within human milk may impact colic risk, and provide support for a dysbiotic/inflammatory model of colic pathophysiology.

Infant consumption of microRNA miR-375 in human milk lipids is associated with protection from atopy.

BACKGROUND: Human milk is thought to reduce infant atopy risk. The biologic mechanism for this protective effect is not fully understood. OBJECTIVES: We tested the hypothesis that infant consumption of 4 microRNAs (miR-146b-5p, miR-148b-3p, miR-21-5p, and miR-375-3p) in human milk would be associated with reduced atopy risk. METHODS: The Breast Milk Influence of the Microtranscriptome Profile on Atopy in Children over Time (IMPACT) study involved a cohort of mother-infant dyads who planned to breastfeed beyond 4 mo. Infant consumption of the 4 human milk microRNAs (miRNAs) in the first 6 mo was calculated as the product of milk miRNA concentration and the number of human milk feeds, across 3 lactation stages: early milk (0-4 wk), transitional milk (4-16 wk), and mature milk (16-24 wk). The primary outcome was infant atopy in the first year, defined as atopic dermatitis (AD), food allergies, or wheezing. The final analysis included 432 human milk samples and 7824 wk of longitudinal health data from 163 dyads. RESULTS: Seventy-three infants developed atopy. Forty-one were diagnosed with AD (25%), 33 developed food allergy (20%), and 10 had wheezing (6%). Eleven developed >1 condition (7%). Infants who did not develop atopy consumed higher concentrations of miR-375-3p (d = 0.18, P = 0.022, adj P = 0.044) and miR-148b-3p (d = 0.23, P = 0.007, adj P = 0.028). The consumption of miR-375-3p (X2 = 5.7, P = 0.017, OR: 0.92, 95% CI: 0.86, 0.99) was associated with reduced atopy risk. Concentrations of miR-375-3p increased throughout lactation (r = 0.46, F = 132.3, P = 8.4 × 10-34) and were inversely associated with maternal body mass (r = -0.11, t = -2.1, P = 0.032). CONCLUSIONS: This study provides evidence that infant consumption of miR-375-3p may reduce atopy risk.

Maternal Variants in the MFGE8 Gene are Associated with Perceived Breast Milk Supply.

Background: The World Health Organization recommends exclusive breastfeeding for ≥6 months, but many mothers are unable to meet this goal. A major reason why mothers undergo early, unplanned breastfeeding cessation is perceived inadequate of milk supply (PIMS). We hypothesized that defining genetic polymorphisms associated with PIMS could aid early identification of at-risk mothers, providing an opportunity for targeted lactation support. Materials and Methods: This prospective observational cohort study followed 221 breastfeeding mothers for 12 months, collecting medical, demographic, and breastfeeding characteristics. Eighteen mammary secretory genes were assessed for single-nucleotide polymorphisms in 88 women (45 with PIMS and 43 with perceived adequate milk supply [PAMS]), matched by age/race/parity. Hierarchical regressions were used to assess the ability of genotype to aid PIMS prediction. Results: Mothers with PIMS exclusively breastfed for a shorter period (7 ± 12 weeks; p = 0.001) and reported lower milk production (17.6 ± 13.3 oz/day; p = 0.001), and their infants displayed reduced weight-for-length Z-score gains (0.74 ± 1.4; p = 0.038) relative to mothers with PAMS (22 ± 19 weeks; 27.03 ± 12.2 oz/day; 1.4 ± 1.5). Maternal genotype for the rs2271714 variant within milk fat globule EGF and factor V/VIII domain containing gene (MFGE8) was associated with PIMS status (p = 0.009, adjusted p = 0.09, likelihood ratio = 9.33) and duration of exclusive breastfeeding (p = 0.009, adjusted p = 0.09, χ2 = 9.39). Addition of MFGE8 genotype to a model employing maternal characteristics (age, parity, previous breast-feeding duration, body mass index, education, and depression status) significantly increased predictive accuracy for PIMS status (p = 0.001; χ2 = 13.5; area under the curve = 0.813 versus 0.725). Conclusions: Genotyping one lactogenic gene aided identification of mothers at risk for PIMS. If validated in a larger cohort, such an approach could be used to identify mothers who may benefit from increased lactation support.

Levels of Breast Milk MicroRNAs and Other Non-Coding RNAs Are Impacted by Milk Maturity and Maternal Diet.

There is emerging evidence that non-coding RNAs (ncRNAs) within maternal breast milk (MBM) impart unique metabolic and immunologic effects on developing infants. Most studies examining ncRNAs in MBM have focused on microRNAs. It remains unclear whether microRNA levels are related to other ncRNAs, or whether they are impacted by maternal characteristics. This longitudinal cohort study examined 503 MBM samples from 192 mothers to: 1) identify the most abundant ncRNAs in MBM; 2) examine the impact of milk maturity on ncRNAs; and 3) determine whether maternal characteristics affect ncRNAs. MBM was collected at 0, 1, and 4 months post-delivery. High throughput sequencing quantified ncRNAs within the lipid fraction. There were 3069 ncRNAs and 238 microRNAs with consistent MBM presence (≥10 reads in ≥10% samples). Levels of 17 ncRNAs and 11 microRNAs accounted for 80% of the total RNA content. Most abundant microRNAs displayed relationships ([R]>0.2, adj p< 0.05) with abundant ncRNAs. A large proportion of ncRNAs (1269/3069; 41%) and microRNAs (206/238; 86%) were affected by MBM maturity. The majority of microRNAs (111/206; 54%) increased from 0-4 months. Few ncRNAs and microRNAs were affected (adj p < 0.05) by maternal age, race, parity, body mass index, gestational diabetes, or collection time. However, nearly half of abundant microRNAs (4/11) were impacted by diet. To our knowledge this is the largest study of MBM ncRNAs, and the first to demonstrate a relationship between MBM microRNAs and maternal diet. Such knowledge could guide nutritional interventions aimed at optimizing metabolic and immunologic microRNA profiles within MBM.

WDR68 is essential for the transcriptional activation of the PRC1-AUTS2 complex and neuronal differentiation of mouse embryonic stem cells.

Recent studies on Polycomb repressive complexes (PRC) reveal a surprising role in transcriptional activation, yet the underlying mechanism remains poorly understood. We previously identified a type 1 PRC (PRC1) that contains Autism Susceptibility Candidate 2 (AUTS2), which positively regulates transcription of neuronal genes. However, the mechanism by which the PRC1-AUTS2 complex influences neurodevelopment is unclear. Here we demonstrate that WDR68 is not only an integral component of the PRC1-AUTS2 complex, but it is also required for PRC1-AUTS2-mediated transcription activation. Furthermore, deletion of Wdr68 in mouse embryonic stem cells leads to defects in neuronal differentiation without affecting self-renewal. Through transcriptomic analysis, we found that many genes responsible for neuronal differentiation are down-regulated in Wdr68 deficient neural progenitors. These genes include those targeted by the PRC1-AUTS2 complex. In summary, our studies uncovered a previously unknown but essential component of the active PRC1 complex and evidence of its role in regulating the expression of genes that are important for neuronal differentiation.

Establishing and maintaining primary cell cultures derived from the ctenophore Mnemiopsis leidyi.

We have developed an efficient method for the preparation and maintenance of primary cell cultures isolated from adult Mnemiopsis leidyi, a lobate ctenophore. Our primary cell cultures are derived from tissue explants or enzymatically dissociated cells, and maintained in a complex undefined ctenophore mesogleal serum. These methods can be used to isolate, maintain and visually monitor ctenophore cells to assess proliferation, cellular morphology and cell differentiation in future studies. Exemplar cell types that can be easily isolated from primary cultures include proliferative ectodermal and endodermal cells, motile amebocyte-like cells, and giant smooth muscle cells that exhibit inducible contractile properties. We have also derived 'tissue envelopes' containing sections of endodermal canal surrounded by mesoglea and ectoderm that can be used to monitor targeted cell types in an in vivo context. Access to efficient and reliably generated primary cell cultures will facilitate the analysis of ctenophore development, physiology and morphology from a cell biological perspective.

The Presence of a Functionally Tripartite Through-Gut in Ctenophora Has Implications for Metazoan Character Trait Evolution.

The current paradigm of gut evolution assumes that non-bilaterian metazoan lineages either lack a gut (Porifera and Placozoa) or have a sac-like gut (Ctenophora and Cnidaria) and that a through-gut originated within Bilateria [1-8]. An important group for understanding early metazoan evolution is Ctenophora (comb jellies), which diverged very early from the animal stem lineage [9-13]. The perception that ctenophores possess a sac-like blind gut with only one major opening remains a commonly held misconception [4, 5, 7, 14, 15]. Despite descriptions of the ctenophore digestive system dating to Agassiz [16] that identify two openings of the digestive system opposite of the mouth-called "excretory pores" by Chun [17], referred to as an "anus" by Main [18], and coined "anal pores" by Hyman [19]-contradictory reports, particularly prominent in recent literature, posit that waste products are primarily expelled via the mouth [4, 5, 7, 14, 19-23]. Here we demonstrate that ctenophores possess a unidirectional, functionally tripartite through-gut and provide an updated interpretation for the evolution of the metazoan through-gut. Our results resolve lingering questions regarding the functional anatomy of the ctenophore gut and long-standing misconceptions about waste removal in ctenophores. Moreover, our results present an intriguing evolutionary quandary that stands in stark contrast to the current paradigm of gut evolution: either (1) the through-gut has its origins very early in the metazoan stem lineage or (2) the ctenophore lineage has converged on an arrangement of organs functionally similar to the bilaterian through-gut.